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Sample records for identify eggshell proteins

  1. Gene expression profiling to identify eggshell proteins involved in physical defense of the chicken egg

    PubMed Central

    2010-01-01

    Background As uricoletic animals, chickens produce cleidoic eggs, which are self-contained bacteria-resistant biological packages for extra-uterine development of the chick embryo. The eggshell constitutes a natural physical barrier against bacterial penetration if it forms correctly and remains intact. The eggshell's remarkable mechanical properties are due to interactions among mineral components and the organic matrix proteins. The purpose of our study was to identify novel eggshell proteins by examining the transcriptome of the uterus during calcification of the eggshell. An extensive bioinformatic analysis on genes over-expressed in the uterus allowed us to identify novel eggshell proteins that contribute to the egg's natural defenses. Results Our 14 K Del-Mar Chicken Integrated Systems microarray was used for transcriptional profiling in the hen's uterus during eggshell deposition. A total of 605 transcripts were over-expressed in the uterus compared with the magnum or white isthmus across a wide range of abundance (1.1- to 79.4-fold difference). The 605 highly-expressed uterine transcripts correspond to 469 unique genes, which encode 437 different proteins. Gene Ontology (GO) analysis was used for interpretation of protein function. The most over-represented GO terms are related to genes encoding ion transport proteins, which provide eggshell mineral precursors. Signal peptide sequence was found for 54 putative proteins secreted by the uterus during eggshell formation. Many functional proteins are involved in calcium binding or biomineralization--prerequisites for interacting with the mineral phase during eggshell fabrication. While another large group of proteins could be involved in proper folding of the eggshell matrix. Many secreted uterine proteins possess antibacterial properties, which would protect the egg against microbial invasion. A final group includes proteases and protease inhibitors that regulate protein activity in the acellular uterine fluid

  2. iTRAQ-Based Quantitative Proteomics Identifies Potential Regulatory Proteins Involved in Chicken Eggshell Brownness

    PubMed Central

    Wu, Guiqin; Shi, Fengying; Liu, Aiqiao; Yang, Ning

    2016-01-01

    Brown eggs are popular in many countries and consumers regard eggshell brownness as an important indicator of egg quality. However, the potential regulatory proteins and detailed molecular mechanisms regulating eggshell brownness have yet to be clearly defined. In the present study, we performed quantitative proteomics analysis with iTRAQ technology in the shell gland epithelium of hens laying dark and light brown eggs to investigate the candidate proteins and molecular mechanisms underlying variation in chicken eggshell brownness. The results indicated 147 differentially expressed proteins between these two groups, among which 65 and 82 proteins were significantly up-regulated in the light and dark groups, respectively. Functional analysis indicated that in the light group, the down-regulated iron-sulfur cluster assembly protein (Iba57) would decrease the synthesis of protoporphyrin IX; furthermore, the up-regulated protein solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 5 (SLC25A5) and down-regulated translocator protein (TSPO) would lead to increased amounts of protoporphyrin IX transported into the mitochondria matrix to form heme with iron, which is supplied by ovotransferrin protein (TF). In other words, chickens from the light group produce less protoporphyrin IX, which is mainly used for heme synthesis. Therefore, the exported protoporphyrin IX available for eggshell deposition and brownness is reduced in the light group. The current study provides valuable information to elucidate variation of chicken eggshell brownness, and demonstrates the feasibility and sensitivity of iTRAQ-based quantitative proteomics analysis in providing useful insights into the molecular mechanisms underlying brown eggshell pigmentation. PMID:28006025

  3. iTRAQ-Based Quantitative Proteomics Identifies Potential Regulatory Proteins Involved in Chicken Eggshell Brownness.

    PubMed

    Li, Guangqi; Sun, Congjiao; Wu, Guiqin; Shi, Fengying; Liu, Aiqiao; Yang, Ning

    2016-01-01

    Brown eggs are popular in many countries and consumers regard eggshell brownness as an important indicator of egg quality. However, the potential regulatory proteins and detailed molecular mechanisms regulating eggshell brownness have yet to be clearly defined. In the present study, we performed quantitative proteomics analysis with iTRAQ technology in the shell gland epithelium of hens laying dark and light brown eggs to investigate the candidate proteins and molecular mechanisms underlying variation in chicken eggshell brownness. The results indicated 147 differentially expressed proteins between these two groups, among which 65 and 82 proteins were significantly up-regulated in the light and dark groups, respectively. Functional analysis indicated that in the light group, the down-regulated iron-sulfur cluster assembly protein (Iba57) would decrease the synthesis of protoporphyrin IX; furthermore, the up-regulated protein solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 5 (SLC25A5) and down-regulated translocator protein (TSPO) would lead to increased amounts of protoporphyrin IX transported into the mitochondria matrix to form heme with iron, which is supplied by ovotransferrin protein (TF). In other words, chickens from the light group produce less protoporphyrin IX, which is mainly used for heme synthesis. Therefore, the exported protoporphyrin IX available for eggshell deposition and brownness is reduced in the light group. The current study provides valuable information to elucidate variation of chicken eggshell brownness, and demonstrates the feasibility and sensitivity of iTRAQ-based quantitative proteomics analysis in providing useful insights into the molecular mechanisms underlying brown eggshell pigmentation.

  4. Differential label-free quantitative proteomic analysis of avian eggshell matrix and uterine fluid proteins associated with eggshell mechanical property.

    PubMed

    Sun, Congjiao; Xu, Guiyun; Yang, Ning

    2013-12-01

    Eggshell strength is a crucial economic trait for table egg production. During the process of eggshell formation, uncalcified eggs are bathed in uterine fluid that plays regulatory roles in eggshell calcification. In this study, a label-free MS-based protein quantification technology was used to detect differences in protein abundance between eggshell matrix from strong and weak eggs (shell matrix protein from strong eggshells and shell matrix protein from weak eggshells) and between the corresponding uterine fluids bathing strong and weak eggs (uterine fluid bathing strong eggs and uterine fluid bathing weak eggs) in a chicken population. Here, we reported the first global proteomic analysis of uterine fluid. A total of 577 and 466 proteins were identified in uterine fluid and eggshell matrix, respectively. Of 447 identified proteins in uterine fluid bathing strong eggs, up to 357 (80%) proteins were in common with proteins in uterine fluid bathing weak eggs. Similarly, up to 83% (328/396) of the proteins in shell matrix protein from strong eggshells were in common with the proteins in shell matrix protein from weak eggshells. The large amount of common proteins indicated that the difference in protein abundance should play essential roles in influencing eggshell strength. Ultimately, 15 proteins mainly relating to eggshell matrix specific proteins, calcium binding and transportation, protein folding and sorting, bone development or diseases, and thyroid hormone activity were considered to have closer association with the formation of strong eggshell.

  5. Unique Immunogenic Proteins in Heterodera glycines Eggshells

    PubMed Central

    Kennedy, M. J.; Schoelz, J. E.; Donald, P. A.; Niblack, T. L.

    1997-01-01

    Polyclonal antibodies were raised against Heterodera glycines eggshells to determine the feasibility of developing an immunoassay for H. glycines eggs. An indirect enzyme-linked immunosorbent assay (ELISA) was developed from anfisera collected 10 weeks after the initial injection. From serial dilutions of sonicated eggshells or whole eggs, a sensitivity of detection to 5 ng/ml sonicated eggshells or 1 egg of H. glycines was determined. The method of eggshell preparation had no effect on the antibodies produced; however, the antibodies cross-reacted with sonicated J2 of H. glycines and eggs of Meloidogyne incognita and H. schachtii. Most of the proteins in both life stages of H. glycines and eggs of M. incognita and H. schachtii had similar migration properties when separated on SDS-PAGE gels and stained with Coomassie blue. Western blot analysis, with antisera adsorbed with homogenized J2 of H. glycines, showed proteins that were specifically localized to eggshells of H. glycines. Monoclonal antibodies might provide a useful immunoassay where polyclonal antibodies lack sufficient specificity. PMID:19274159

  6. Possible eggshell protein gene from Schistosoma mansoni.

    PubMed

    Johnson, K S; Taylor, D W; Cordingley, J S

    1987-01-02

    We have identified and sequenced a cDNA clone of a mRNA found only in mature female schistosomes. This mRNA is not detectably synthesized by female worms from single sex infections (unisexual females), by males or by the developing miracidia in the eggs. The clone hybridises to a highly abundant polyadenylated mRNA of approximately 1500 nucleotides. The nucleotide sequence of the clone predicts a polypeptide comprising two repetitive regions. A pentapeptide repeat with the consensus sequence Gly-Tyr-Asp-Lys-Tyr, and a region rich in histidine residues. Hybrid selected mRNA translated in vitro with [3H]tyrosine as labelled amino acid yields a polypeptide of 48 kDa (p48) that corresponds to the major [3H]tyrosine labelled translation product of female worm total mRNA. p48 does not label with [35S]methionine and is absent from the translation products of male and unisexual female mRNAs. The amino acid sequence of p48 has significant homologies to silk moth chorion proteins and we suggest that it is one of the major components of the schistosome eggshell probably accounting for the high level of [3H]tyrosine incorporation into the vitellaria of Schistosoma mansoni. The tyrosine content of the polypeptide suggests that it may play a role in phenol oxidase mediated cross-linking of the schistosome eggshell and in support of this we find that mushroom phenol oxidase will cause the specific cross-linking of p48 in in vitro translation products.

  7. Presclerotized eggshell protein from the liver fluke Fasciola hepatica.

    PubMed

    Waite, J H; Rice-Ficht, A C

    1987-12-01

    Trematode parasites protect their eggs with a tough tanned eggshell. Eggshell precursor proteins are synthesized and stockpiled within the extensive vitellaria of the animal. A major eggshell precursor protein with an apparent molecular weight of 31,000 and pI of 7.4 was isolated from the vitellaria of Fasciola hepatica. This protein, which represents 6-7% of the total protein in mature Fasciola, is unique in containing rather high levels of the amino acid 3,4-dihydroxyphenylalanine (DOPA), i.e., 110 residues per 1000. Other prominent amino acids are glycine, aspartic acid, and lysine. A prominent DOPA-containing tryptic peptide derived from eggshell precursor protein has the sequence Gly-Gly-Gly-DOPA-Gly-Gly-DOPA-Gly-Lys. DOPA residues disappear during the maturation of the eggshell and by treatment in vitro with mushroom polyphenol oxidase. This disappearance may be related to the formation of cross-links in the eggshell protein.

  8. Proteins of insoluble matrix of avian (gallus gallus) eggshell.

    PubMed

    Miksík, Ivan; Eckhardt, Adam; Sedláková, Pavla; Mikulikova, Katerina

    2007-01-01

    The protein composition of the insoluble avian eggshell matrix was studied. The determination of these proteins insoluble in water (EDTA-insoluble) was carried out using enzymatic cleavage followed by a high-performance liquid chromatography-mass spectrometry method. The influence of various enzymes on the protein splitting also was studied. The distribution of proteins depends on the type of layer (localization within the eggshell): ovocalyxin-32 was found mainly in the outer layer (the cuticle); ovocleidin-116 and 17 and ovocalyxin-36 were found throughout the whole eggshell, whereas ovalbumin was only found in the inner layer, the mammillary. The pigment (protoporphyrin IX) was mainly found in the cuticle and is incorporated into the protein network.

  9. Quantitative proteomics and bioinformatic analysis provide new insight into protein function during avian eggshell biomineralization.

    PubMed

    Marie, Pauline; Labas, Valérie; Brionne, Aurélien; Harichaux, Grégoire; Hennequet-Antier, Christelle; Nys, Yves; Gautron, Joël

    2015-01-15

    Gallus gallus eggshell is a bioceramic composed of 95% calcium carbonate in calcitic form and 3.5% extracellular organic matrix. The calcification process occurs in the uterine fluid where biomineralization follows a temporal sequence corresponding to the initiation, growth and termination stages of crystal growth. Eggshell texture and its ultrastructure are regulated by organic matrix proteins, which control mineralization process and influence the eggshell biomechanical properties. We performed proteomic qualitative analyses and identified 308 uterine fluid proteins. Quantitative analysis showed differential abundances at the three stages of shell biomineralization for 64 of them. Cluster analysis revealed a first group of proteins related to mineralization and mainly present at the onset of calcification including OVOT, OVAL, OC-17, and two novel calcium binding proteins (EDIL3, MFGE8). A second group of proteins mainly present at the initiation and termination of shell formation was potentially involved in the regulation of the activity of the uterine fluid proteins (e.g. molecular chaperones, folding proteins, proteases and protease inhibitors). OCX21, a protein highly concentrated in the fluid and the shell, belongs to this group. A third group equally represented at all stages of shell mineralization corresponded to antibacterial proteins that could protect the forming egg against microbial invasion. The calcitic avian eggshell protects the developing embryo and, moreover, ensures that the nutritious table egg remains free of pathogens. The eggshell is formed by nucleation upon a fibrous scaffold (the eggshell membranes) followed by an interaction between the growing mineral crystals and the shell organic matrix. This interaction leads to a highly ordered shell microstructure and texture which contribute to its exceptional mechanical properties. Shell mineralization occurs in three distinct phases of calcification (initiation, growth and termination), which

  10. Integrating Transcriptome and Genome Re-Sequencing Data to Identify Key Genes and Mutations Affecting Chicken Eggshell Qualities

    PubMed Central

    Liu, Long; Zheng, Chuan Wei; Wang, De He; Hou, Zhuo Cheng; Ning, Zhong Hua

    2015-01-01

    Eggshell damages lead to economic losses in the egg production industry and are a threat to human health. We examined 49-wk-old Rhode Island White hens (Gallus gallus) that laid eggs having shells with significantly different strengths and thicknesses. We used HiSeq 2000 (Illumina) sequencing to characterize the chicken transcriptome and whole genome to identify the key genes and genetic mutations associated with eggshell calcification. We identified a total of 14,234 genes expressed in the chicken uterus, representing 89% of all annotated chicken genes. A total of 889 differentially expressed genes were identified by comparing low eggshell strength (LES) and normal eggshell strength (NES) genomes. The DEGs are enriched in calcification-related processes, including calcium ion transport and calcium signaling pathways as reveled by gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. Some important matrix proteins, such as OC-116, LTF and SPP1, were also expressed differentially between two groups. A total of 3,671,919 single-nucleotide polymorphisms (SNPs) and 508,035 Indels were detected in protein coding genes by whole-genome re-sequencing, including 1775 non-synonymous variations and 19 frame-shift Indels in DEGs. SNPs and Indels found in this study could be further investigated for eggshell traits. This is the first report to integrate the transcriptome and genome re-sequencing to target the genetic variations which decreased the eggshell qualities. These findings further advance our understanding of eggshell calcification in the chicken uterus. PMID:25974068

  11. Changes in eggshell mechanical properties, crystallographic texture and in matrix proteins induced by moult in hens.

    PubMed

    Ahmed, A M H; Rodriguez-Navarro, A B; Vidal, M L; Gautron, J; García-Ruiz, J M; Nys, Y

    2005-06-01

    The effect of moult on eggshell mechanical properties, on composition and concentrations of organic matrix components and on eggshell microstructure was investigated. The observed changes were studied to understand the role of organic matrix and eggshell microstructure in eggshell strength. Moult was induced by zinc oxide (20 g zinc/kg diet) in 53 ISA Brown laying hens at 78 weeks of age. No difference was observed for egg or eggshell weights after moult. In contrast, moult improved the shell breaking strength (28.09 vs 33.71 N). After moult, there was a decrease in the average size of calcite crystals composing the eggshell and in their heterogeneity, whereas crystal orientation remained basically the same. After moulting, the total protein concentration in eggshell increased slightly. The comparisons of SDS-PAGE profiles of the organic matrix constituents extracted before and after moulting showed changes in staining intensity of certain bands. After moult, bands associated with main proteins specific to eggshell formation (OC-116 and OC-17) showed higher staining intensity, while the intensity of the egg white proteins (ovotransferrin, ovalbumin and lysozyme) decreased. ELISA confirmed the decrease in ovotransferrin after moult. Its concentration was inversely correlated with breaking strength before moult. These observations suggest that changes in eggshell crystal size could be due to changes in organic matrix composition. These changes may provide a mechanism for the improvement in shell solidity after moulting.

  12. Fabrication of a biocomposite reinforced with hydrophilic eggshell proteins

    NASA Astrophysics Data System (ADS)

    Kim, Geun Hyung; Min, Taijin; Park, Su A.; Doo Kim, Wan; Koh, Young Ho

    2007-12-01

    Soluble eggshell proteins were used as a reinforcing material of electrospun micro/nanofibers for tissue engineering. A biocomposite composed of poly(ɛ-caprolactone) (PCL) micro/nanofibers and soluble eggshell protein was fabricated with a two-step fabrication method, which is an electrospinning process followed by an air-spraying process. To achieve a stable electrospinning process, we used an auxiliary cylindrical electrode connected with a spinning nozzle. PCL biocomposite was characterized in water contact angle and mechanical properties as well as cell proliferation for its application as a tissue engineering material. It showed an improved hydrophilic characteristic compared with that of a micro/nanofiber web generated from a pure PCL solution using a typical electrospinning process. Moreover, the fabricated biocomposite had good mechanical properties compared to a typical electrospun micro/nanofiber mat. The fabricated biocomposite made human dermal fibroblasts grow better than pure PCL. From the results, the reinforced polymeric micro/nanofiber scaffold can be easily achieved with these modified processes.

  13. Fabrication of a biocomposite reinforced with hydrophilic eggshell proteins.

    PubMed

    Kim, GeunHyung; Min, Taijin; Park, Su A; Kim, Wan Doo; Koh, Young Ho

    2007-12-01

    Soluble eggshell proteins were used as a reinforcing material of electrospun micro/nanofibers for tissue engineering. A biocomposite composed of poly(epsilon-caprolactone) (PCL) micro/nanofibers and soluble eggshell protein was fabricated with a two-step fabrication method, which is an electrospinning process followed by an air-spraying process. To achieve a stable electrospinning process, we used an auxiliary cylindrical electrode connected with a spinning nozzle. PCL biocomposite was characterized in water contact angle and mechanical properties as well as cell proliferation for its application as a tissue engineering material. It showed an improved hydrophilic characteristic compared with that of a micro/nanofiber web generated from a pure PCL solution using a typical electrospinning process. Moreover, the fabricated biocomposite had good mechanical properties compared to a typical electrospun micro/nanofiber mat. The fabricated biocomposite made human dermal fibroblasts grow better than pure PCL. From the results, the reinforced polymeric micro/nanofiber scaffold can be easily achieved with these modified processes.

  14. Structure of struthiocalcin-1, an intramineral protein from Struthio camelus eggshell, in two crystal forms.

    PubMed

    Ruiz-Arellano, Rayana R; Medrano, Francisco J; Moreno, Abel; Romero, Antonio

    2015-04-01

    Biomineralization is the process by which living organisms produce minerals. One remarkable example is the formation of eggshells in birds. Struthiocalcins present in the ostrich (Struthio camellus) eggshell matrix act as biosensors of calcite growth during eggshell formation. Here, the crystal structure of struthiocalcin-1 (SCA-1) is reported in two different crystal forms. The structure is a compact single domain with an α/β fold characteristic of the C-type lectin family. In contrast to the related avian ovocleidin OC17, the electrostatic potential on the molecular surface is dominated by an acidic patch. Scanning electron microscopy combined with Raman spectroscopy indicates that these intramineral proteins (SCA-1 and SCA-2) induce calcium carbonate precipitation, leading to the formation of a stable form of calcite in the mature eggshell. Finally, the implications of these two intramineral proteins SCA-1 and SCA-2 in the nucleation of calcite during the formation of eggshells in ratite birds are discussed.

  15. The proteome of the insoluble Schistosoma mansoni eggshell skeleton.

    PubMed

    Dewalick, Saskia; Bexkens, Michiel L; van Balkom, Bas W M; Wu, Ya-Ping; Smit, Cornelis H; Hokke, Cornelis H; de Groot, Philip G; Heck, Albert J R; Tielens, Aloysius G M; van Hellemond, Jaap J

    2011-04-01

    In schistosomiasis, the majority of symptoms of the disease is caused by the eggs that are trapped in the liver. These eggs elicit an immune reaction that leads to the formation of granulomas. The eggshell, which is a rigid insoluble structure built from cross-linked proteins, is the site of direct interaction between the egg and the immune system. However, the exact protein composition of the insoluble eggshell was previously unknown. To identify the proteins of the eggshell of Schistosoma mansoni we performed LC-MS/MS analysis, immunostaining and amino acid analysis on eggshell fragments. For this, eggshell protein skeleton was prepared by thoroughly cleaning eggshells in a four-step stripping procedure of increasing strength including urea and SDS to remove all material that is not covalently linked to the eggshell itself, but is part of the inside of the egg, such as Reynold's layer, von Lichtenberg's envelope and the miracidium. We identified 45 proteins of which the majority are non-structural proteins and non-specific for eggs, but are house-keeping proteins that are present in large quantities in worms and miracidia. Some of these proteins are known to be immunogenic, such as HSP70, GST and enolase. In addition, a number of schistosome-specific proteins with unknown function and no homology to any known annotated protein were found to be incorporated in the eggshell. Schistosome-specific glycoconjugates were also shown to be present on the eggshell protein skeleton. This study also confirmed that the putative eggshell protein p14 contributes largely to the eggshell. Together, these results give new insights into eggshell composition as well as eggshell formation. Those proteins that are present at the site and time of eggshell formation are incorporated in the cross-linked eggshell and this cross-linking does no longer occur when the miracidium starts secreting proteins. Copyright © 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd

  16. The Effect of Ultrasound on the Alkali Extraction of Proteins from Eggshell Membranes.

    PubMed

    Marcet, Ismael; Salvadores, Marina; Rendueles, Manuel; Díaz, Mario

    2017-09-01

    Eggshell contains two layers formed by a dense network of fibrous proteins. These proteins are highly insoluble in a broad variety of solvents, but their composition makes them suitable for a broad range of applications. In this study, in order to extract and solubilise these proteins, the eggshell membranes were treated in an alkali solution. A Box-Behnken design was employed to determine the influence of the treatment variables on the amount of protein solubilised. Furthermore, the effect of ultrasound on the protein recovery yield was also evaluated and compared with the unmodified process. A solubilised protein yield close to 100% of the total eggshell membrane protein was obtained. The optimal conditions could be set at 70 °C in a 1.0 M NaOH solution for 60 minutes. However, when ultrasound was applied, it was possible to decrease the time of reaction by half. In the two processes, the temperature was found to be the most important independent variable evaluated. Finally, the antioxidant properties of the proteins obtained in each case were similar. Ultrasound favours the detachment of big clumps of proteins from the eggshell membrane, facilitating the solubilisation of its compounds. The ultrasound had no effect on the protein properties tested in this study. This article is protected by copyright. All rights reserved.

  17. Soluble eggshell membrane protein: preparation, characterization and biocompatibility.

    PubMed

    Yi, Feng; Guo, Zhao-Xia; Zhang, Li-Xia; Yu, Jian; Li, Qiang

    2004-08-01

    The preparation, characterization and biocompatibility of soluble eggshell membrane (SEP) are reported. The dissolution process, which is the key step of the preparation of SEP, has been followed by scanning electron microscopy (SEM) to observe the changes of the surfaces and thickness of the eggshell membrane (ESM). The composition of SEP has been investigated by amino acid analysis and elemental analysis. Based on the fact that SEP losses significantly cystine, and that SEP has a higher content of sulfur, an assumption involving combination with 3-mercaptopropionic acid (the reagent used for reductive cleavage of disulfide bonds) following the cleavage of the original disulfide bonds has been proposed, which explains the solubility of SEP. The thermal and surface properties have been studied by thermogravimetric analysis (TGA) and contact angle measurement. The biocompatibility of SEP, as demonstrated by cell culture of NIH3T3, is comparable to collagen type I and superior to raw ESM either inside or outside surface.

  18. Quantitative expression of candidate genes affecting eggshell color.

    PubMed

    Zheng, Chuanwei; Li, Zesheng; Yang, Ning; Ning, Zhonghua

    2014-05-01

    There are three pigments that affect the color of an eggshell: protoporphyrin, biliverdin and biliverdin-zinc chelate. Protoporphyrin is the main pigment in brown and light-brown eggshells, whereas very little protoporphyrin is found in white eggshells. Eggshell protoporphyrin is derived from the heme formation in birds. Coproporphyrinogen III oxidase (CPOX) and ferrochelatase (FECH) represent rate-limiting enzymes for the heme-biosynthetic pathway. Breast cancer resistance protein (BCRP), feline leukemia virus receptor (FLVCR), and heme-responsive gene-1 (HRG1) serve as primary transporters for both protoporphyrinogen and heme. Finally, four organic anion transporting polypeptide family members (including solute carrier organic anion transporter family, SLCO1C1, SLCO1A2, SLCO1B3 and LOC418189) may affect pigment transport within eggshells. Here we measured gene expression levels in key tissues of egg-producing hens. We analyzed three different types of hens that generated distinct eggshell colors: white, pink or brown. Our data revealed three ways in which eggshell color was genetically influenced. First, high-level expression of CPOX generated more protoporphyrinogen and a brown eggshell color. In contrast, high expression of FECH likely converted more protoporphyrinogen into heme, reduced protoporphyrinogen levels within the eggshell and generated a light color. Second, heme transporters also affected eggshell color. High-level expression of BCRP, HRG1 and FLVCR were associated with brown, white and generally lighter eggshell colors, respectively. Finally, protoporphyrin precipitation also affected eggshell color, as high expression of both SLCO1A2 and SLCO1C1 were associated with brown eggshell color. As such, we have identified seven genes in which expression levels in different tissues were associated with eggshell color. © 2014 Japanese Society of Animal Science.

  19. The proteome of the calcified layer organic matrix of turkey (Meleagris gallopavo) eggshell.

    PubMed

    Mann, Karlheinz; Mann, Matthias

    2013-08-27

    Chicken eggshell mineralization is a prominent model for biomineralization not only because of its importance for avian reproduction but also because of the commercial interest associated with eggshell quality. An analysis and comparison of the protein constituents of eggshells of several species would contribute to a better understanding of the shell mineralization process. The recent publication of the turkey genome sequence now provides a basis for the in-depth analysis of the turkey eggshell proteome. Proteomic analysis of turkey acid-soluble and acid-insoluble organic eggshell matrix yielded 697 identified proteins/protein groups. However, intensity-based absolute quantification (iBAQ) results indicated that the 47 most abundant identified proteins already constituted 95% of the total turkey eggshell matrix proteome. Forty-four of these proteins were also identified in chicken eggshell matrix previously. Despite these similarities there were important and unexpected differences. While ovocleidin-116 and ovocalyxin-36 were major proteins constituting approximately 37% of the identified proteome, other members of the group of so-called eggshell-specific proteins were not identified. Thus ovocalyxin-21 and ovocalyxin-32 were missing among matrix proteins. Conversely, major turkey eggshell proteins were not detected in chicken, such as the bone protein periostin, the mammalian counterpart of which is involved in many aspects of bone metabolism and which represented 10-11% of the total identified proteome. Even members of the same avian family show important differences in eggshell matrix composition and more studies on the proteome and the transcriptome level will be necessary to identify a common toolkit of eggshell mineralization and to work out species differences among functional eggshell protein sets and their role in eggshell production.

  20. The proteome of the calcified layer organic matrix of turkey (Meleagris gallopavo) eggshell

    PubMed Central

    2013-01-01

    Background Chicken eggshell mineralization is a prominent model for biomineralization not only because of its importance for avian reproduction but also because of the commercial interest associated with eggshell quality. An analysis and comparison of the protein constituents of eggshells of several species would contribute to a better understanding of the shell mineralization process. The recent publication of the turkey genome sequence now provides a basis for the in-depth analysis of the turkey eggshell proteome. Results Proteomic analysis of turkey acid-soluble and acid-insoluble organic eggshell matrix yielded 697 identified proteins/protein groups. However, intensity-based absolute quantification (iBAQ) results indicated that the 47 most abundant identified proteins already constituted 95% of the total turkey eggshell matrix proteome. Forty-four of these proteins were also identified in chicken eggshell matrix previously. Despite these similarities there were important and unexpected differences. While ovocleidin-116 and ovocalyxin-36 were major proteins constituting approximately 37% of the identified proteome, other members of the group of so-called eggshell-specific proteins were not identified. Thus ovocalyxin-21 and ovocalyxin-32 were missing among matrix proteins. Conversely, major turkey eggshell proteins were not detected in chicken, such as the bone protein periostin, the mammalian counterpart of which is involved in many aspects of bone metabolism and which represented 10-11% of the total identified proteome. Conclusions Even members of the same avian family show important differences in eggshell matrix composition and more studies on the proteome and the transcriptome level will be necessary to identify a common toolkit of eggshell mineralization and to work out species differences among functional eggshell protein sets and their role in eggshell production. PMID:23981693

  1. Ovocalyxin-36 is a pattern recognition protein in chicken eggshell membranes.

    PubMed

    Cordeiro, Cristianne M M; Esmaili, Hamed; Ansah, George; Hincke, Maxwell T

    2013-01-01

    The avian eggshell membranes are essential elements in the fabrication of the calcified shell as a defense against bacterial penetration. Ovocalyxin-36 (OCX-36) is an abundant avian eggshell membrane protein, which shares protein sequence homology to bactericidal permeability-increasing protein (BPI), lipopolysaccharide-binding protein (LBP) and palate, lung and nasal epithelium clone (PLUNC) proteins. We have developed an efficient method to extract OCX-36 from chicken eggshell membranes for purification with cation and anion exchange chromatographies. Purified OCX-36 protein exhibited lipopolysaccharide (LPS) binding activity and bound lipopolysaccharide (LPS) from Escherichia coli O111:B4 in a dose-dependent manner. OCX-36 showed inhibitory activity against growth of Staphylococcus aureus ATCC 6538. OCX-36 single nucleotide polymorphisms (SNPs) were verified at cDNA 211 position and the corresponding proteins proline-71 (Pro-71) or serine-71 (Ser-71) were purified from eggs collected from genotyped hens. A significant difference between Pro-71 and Ser-71 OCX-36 for S. aureus lipoteichoic acid (LTA) binding activity was detected. The current study is a starting point to understand the innate immune role that OCX-36 may play in protection against bacterial invasion of both embryonated eggs (relevant to avian reproductive success) and unfertilized table eggs (relevant to food safety).

  2. Solubilization and identification of hen eggshell membrane proteins during different times of chicken embryo development using the proteomic approach.

    PubMed

    Kaweewong, Kritsda; Garnjanagoonchorn, Wunwiboon; Jirapakkul, Wannee; Roytrakul, Sittiruk

    2013-04-01

    A fertilized chicken egg is a unit of life. During hatching, transport of nutrients, including calcium, have been reported from the egg components to the developing embryo. Calcium is mobilized from the eggshell with the involvement of Ca(2+)-binding proteins. In addition, other unknown proteins may also play some important roles during embryo developing process. Therefore identification and prediction of biological functions of eggshell membrane (ESM) proteins during chick embryo development was conducted by proteome analysis. Comparison of different lysis solutions indicated that the highest ability to extract ESM proteins could be obtained with 1 % sodium dodecyl sulfate in 5 mM Tris-HCl buffer pH 8.8 containing 0.1 % 2-mercaptoethanol. In this study fertilized Cornish chicken eggs were incubated at 37 °C in humidified incubators for up to 21 days. At selected times (days 1, 9, 15 and 21), samples were taken and the ESMs were carefully separated by hand, washed with distilled water, and air-dried at room temperature. The ESM proteins were then solubilized and analyzed by proteome analysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis combined with high performance liquid chromatography and mass spectrometry revealed 62 proteins in the ESM; only keratin is known ESM protein, 8 of which are egg white proteins and related while 53 others have not previously been reported. Some differences in the types of proteins and their molecular functions were noted in ESM at different incubation times. One protein which was present only at days 15 and 21 of egg incubation was identified as a calcium binding protein i.e. EGF like repeats and discoidin I like domain 3 (EDIL3 homologous protein).

  3. Small gene family encoding an eggshell (chorion) protein of the human parasite Schistosoma mansoni

    SciTech Connect

    Bobek, L.A.; Rekosh, D.M.; Lo Verde, P.T.

    1988-08-01

    The authors isolated six independent genomic clones encoding schistosome chorion or eggshell proteins from a Schistosoma mansoni genomic library. A linkage map of five of the clones spanning 35 kilobase pairs (kbp) of the S. mansoni genome was constructed. The region contained two eggshell protein genes closely linked, separated by 7.5 kbp of intergenic DNA. The two genes of the cluster were arranged in the same orientation, that is, they were transcribed from the same strand. The sixth clone probably represents a third copy of the eggshell gene that is not contained within the 35-kbp region. The 5- end of the mRNA transcribed from these genes was defined by primer extension directly off the RNA. The ATCAT cap site sequence was homologous to a silkmoth chorion PuTCATT cap site sequence, where Pu indicates any purine. DNA sequence analysis showed that there were no introns in these genes. The DNA sequences of the three genes were very homologous to each other and to a cDNA clone, pSMf61-46, differing only in three or four nucleotices. A multiple TATA box was located at positions -23 to -31, and a CAAAT sequence was located at -52 upstream of the eggshell transcription unit. Comparison of sequences in regions further upstream with silkmoth and Drosophila sequences revealed very short elements that were shared. One such element, TCACGT, recently shown to be an essential cis-regulatory element for silkmoth chorion gene promoter function, was found at a similar position in all three organisms.

  4. Accumulation of the Drosophila Torso-like protein at the blastoderm plasma membrane suggests that it translocates from the eggshell.

    PubMed

    Mineo, Alessandro; Furriols, Marc; Casanova, Jordi

    2015-04-01

    The eggshell serves as a depository for proteins that play an important role in early embryonic development. In particular, the Drosophila eggshell is responsible for transferring asymmetries from the egg chamber to specify the regions at both ends of the embryo through the uneven activation of the Torso (Tor) receptor in its membrane. This process relies on the restricted expression of the gene torso-like (tsl) in subpopulations of follicle cells during oogenesis and its protein accumulation at both poles of the eggshell, but it is not known how this signal is transmitted to the embryo. Here, we show that Tsl accumulates at the embryonic plasma membrane, even in the absence of the Tor receptor. However, during oogenesis, we detected Tsl accumulation only at the eggshell. These results suggest that there is a two-step mechanism to transfer the asymmetric positional cues from the egg chamber into the early embryo: initial anchoring of Tsl at the eggshell as it is secreted, followed by its later translocation to the egg plasma membrane, where it enables Tor receptor activation. Translocation of anchored determinants from the eggshell might then regulate the spatial and temporal control of early embryonic developmental processes. © 2015. Published by The Company of Biologists Ltd.

  5. Hen uterine gene expression profiling during eggshell formation reveals putative proteins involved in the supply of minerals or in the shell mineralization process

    PubMed Central

    2014-01-01

    Background The chicken eggshell is a natural mechanical barrier to protect egg components from physical damage and microbial penetration. Its integrity and strength is critical for the development of the embryo or to ensure for consumers a table egg free of pathogens. This study compared global gene expression in laying hen uterus in the presence or absence of shell calcification in order to characterize gene products involved in the supply of minerals and / or the shell biomineralization process. Results Microarrays were used to identify a repertoire of 302 over-expressed genes during shell calcification. GO terms enrichment was performed to provide a global interpretation of the functions of the over-expressed genes, and revealed that the most over-represented proteins are related to reproductive functions. Our analysis identified 16 gene products encoding proteins involved in mineral supply, and allowed updating of the general model describing uterine ion transporters during eggshell calcification. A list of 57 proteins potentially secreted into the uterine fluid to be active in the mineralization process was also established. They were classified according to their potential functions (biomineralization, proteoglycans, molecular chaperone, antimicrobials and proteases/antiproteases). Conclusions Our study provides detailed descriptions of genes and corresponding proteins over-expressed when the shell is mineralizing. Some of these proteins involved in the supply of minerals and influencing the shell fabric to protect the egg contents are potentially useful biological markers for the genetic improvement of eggshell quality. PMID:24649854

  6. Integrated proteomic and transcriptomic analysis of the Aedes aegypti eggshell

    PubMed Central

    2014-01-01

    Background Mosquito eggshells show remarkable diversity in physical properties and structure consistent with adaptations to the wide variety of environments exploited by these insects. We applied proteomic, transcriptomic, and hybridization in situ techniques to identify gene products and pathways that participate in the assembly of the Aedes aegypti eggshell. Aedes aegypti population density is low during cold and dry seasons and increases immediately after rainfall. The survival of embryos through unfavorable periods is a key factor in the persistence of their populations. The work described here supports integrated vector control approaches that target eggshell formation and result in Ae. aegypti drought-intolerant phenotypes for public health initiatives directed to reduce mosquito-borne diseases. Results A total of 130 proteins were identified from the combined mass spectrometric analyses of eggshell preparations. Conclusions Classification of proteins according to their known and putative functions revealed the complexity of the eggshell structure. Three novel Ae. aegypti vitelline membrane proteins were discovered. Odorant-binding and cysteine-rich proteins that may be structural components of the eggshell were identified. Enzymes with peroxidase, laccase and phenoloxidase activities also were identified, and their likely involvements in cross-linking reactions that stabilize the eggshell structure are discussed. PMID:24707823

  7. Data set for the proteomic inventory and quantitative analysis of chicken eggshell matrix proteins during the primary events of eggshell mineralization and the active growth phase of calcification

    PubMed Central

    Marie, Pauline; Labas, Valérie; Brionne, Aurélien; Harichaux, Grégoire; Hennequet-Antier, Christelle; Rodriguez-Navarro, Alejandro B.; Nys, Yves; Gautron, Joël

    2015-01-01

    Chicken eggshell is a biomineral composed of 95% calcite calcium carbonate mineral and of 3.5% organic matrix proteins. The assembly of mineral and its structural organization is controlled by its organic matrix. In a recent study [1], we have used quantitative proteomic, bioinformatic and functional analyses to explore the distribution of 216 eggshell matrix proteins at four key stages of shell mineralization defined as: (1) widespread deposition of amorphous calcium carbonate (ACC), (2) ACC transformation into crystalline calcite aggregates, (3) formation of larger calcite crystal units and (4) rapid growth of calcite as columnar structure with preferential crystal orientation. The current article detailed the quantitative analysis performed at the four stages of shell mineralization to determine the proteins which are the most abundant. Additionally, we reported the enriched GO terms and described the presence of 35 antimicrobial proteins equally distributed at all stages to keep the egg free of bacteria and of 81 proteins, the function of which could not be ascribed. PMID:26306314

  8. Data set for the proteomic inventory and quantitative analysis of chicken eggshell matrix proteins during the primary events of eggshell mineralization and the active growth phase of calcification.

    PubMed

    Marie, Pauline; Labas, Valérie; Brionne, Aurélien; Harichaux, Grégoire; Hennequet-Antier, Christelle; Rodriguez-Navarro, Alejandro B; Nys, Yves; Gautron, Joël

    2015-09-01

    Chicken eggshell is a biomineral composed of 95% calcite calcium carbonate mineral and of 3.5% organic matrix proteins. The assembly of mineral and its structural organization is controlled by its organic matrix. In a recent study [1], we have used quantitative proteomic, bioinformatic and functional analyses to explore the distribution of 216 eggshell matrix proteins at four key stages of shell mineralization defined as: (1) widespread deposition of amorphous calcium carbonate (ACC), (2) ACC transformation into crystalline calcite aggregates, (3) formation of larger calcite crystal units and (4) rapid growth of calcite as columnar structure with preferential crystal orientation. The current article detailed the quantitative analysis performed at the four stages of shell mineralization to determine the proteins which are the most abundant. Additionally, we reported the enriched GO terms and described the presence of 35 antimicrobial proteins equally distributed at all stages to keep the egg free of bacteria and of 81 proteins, the function of which could not be ascribed.

  9. Intraclutch eggshell colour variation in birds: are females able to identify their eggs individually?

    PubMed Central

    Poláček, Miroslav; Bartíková, Michaela

    2017-01-01

    Background One possibility suggested regarding female post-mating strategies is differential allocation into offspring investment. Female birds produce not only the largest, but also most colourful eggs of all oviparous taxa. Larger eggs provide space for bigger embryos, or more nutrition for their development, but the question why eggs are more colourful and why there is variation in eggshell colouration remains. In this context, the focus of interest has been to explain inter-clutch variation but in many bird species, eggshell colouration also varies within a clutch. Surprisingly, less attention has been paid to this phenomenon. Therefore, we propose the “female egg recognition” hypothesis, suggesting that mothers use colour characteristics to interpret egg attributes and allocate further investment into each egg accordingly. To evaluate the feasibility of the hypothesis, we tested several underlying predictions and examined their suitability using a dataset from our tree sparrow (Passer montanus) study. We predict (i) substantial within-clutch variation in eggshell colouration which, (ii) should be related to laying sequence, (iii) reflect egg quality and, (iv) should stimulate a female response. Methods Eggshell coloration data were obtained via digital photography under standardized conditions, taken after clutch completion. Lightness (L*), representing the achromatic properties of an egg has been chosen as the most important predictor in dark cavities and was related to egg quality and position in the nest. Results In our tree sparrows, first and mainly last eggs were less pigmented, providing information about laying order. Egg volume, which predicts chick quality, positively correlates with eggshell coloration. Finally, we could show that female tree sparrows placed darker, but not bigger, eggs into more central incubation positions. Discussion All basic prerequisites for the “female egg recognition” hypothesis are fulfilled. In this context

  10. Synthesis of Stable Microcapsules from Trematode Eggshell Components

    DTIC Science & Technology

    1990-06-30

    NO Arlington, VA 22217-5000 61153N RR4106 11 TITLE (Include Security Classification) (u) Synthesis of Stable Microcapsules from Trematode Eggshell...Continue on reverse if necessary and identify by block number) The trematode Fasciola hepatica produces a unique protein eggshell or microcapsule the...proteins to produce a hard quinone tanned microcapsule with unusual properties. The focus of this project is to i) characterize the protein components

  11. Outer eggshell membrane as delivery vehicle for polysaccharide/protein microcapsules incorporated with vitamin E.

    PubMed

    Chai, Zhi; Li, Yuanyuan; Liu, Fei; Du, Bingjian; Jiao, Tong; Zhang, Chunyue; Leng, Xiaojing

    2013-01-23

    This study investigates the features of a new type of delivery system prepared by combining a natural outer eggshell membrane (OESM) with emulsified microcapsules. The loading efficiency, controlled release properties, and forming mechanisms of the prepared system were studied. The polysaccharide/protein microcapsules incorporated with vitamin E can be attached to highly cross-linked protein fiber networks of OESM. This attachment could be reinforced more than 2-fold using glutaraldehyde as a cross-linking agent. The combined OESM/microcapsule delivery system significantly exhibited better controlled release properties than the microcapsules alone because of the steric blocking effect. Moreover, the OESM delivery system incorporated with microcapsules formed by pectin/protein as wall material showed more resistance against enzymatic attacks because of the formation of compact aggregates promoted by electrostatic effects.

  12. Fibrous scaffolds made by co-electrospinning soluble eggshell membrane protein with biodegradable synthetic polymers.

    PubMed

    Xiong, Xi; Li, Qiang; Lu, Jian-Wei; Guo, Zhao-Xia; Sun, Zhao-Hui; Yu, Jian

    2012-01-01

    Soluble eggshell membrane protein (SEP), isolated from natural eggshell membrane, was co-electrospun with biodegradable synthetic polymers poly(propylene carbonate) (PPC) and poly(lactic acid) (PLA) in various proportions from 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) solutions in order to prepare fibrous scaffolds having simultaneously good mechanical properties and biocompatibility. The fiber morphology was observed by field emission scanning electron microscopy, showing uniform fibers with diameter of 1.2-1.0 and 1.3-0.7 um for PPC/SEP and PLA/SEP blend fibers, respectively. Transmission electron microscopy observation shows that the blend fibers have domain-matrix phase morphology with fiber-like SEP domains in the PPC or PLA matrix, indicating the occurrence of phase separation, although interaction exists between PPC (or PLA) and SEP, as revealed by attenuated total reflectance Fourier transform infrared spectroscopy. The mechanical properties were evaluated by uniaxial tensile tests and showed that both the tensile strength and elongation at break increase with increasing incorporation of PPC (or PLA). The surface composition was investigated by X-ray photoelectron spectroscopy and SEP was found on the fiber surfaces, and as a result the surfaces of the fibrous scaffolds are superhydrophilic. NIH3T3 cell culture tests demonstrate that the PPC/SEP and PLA/SEP blend fibrous scaffolds have a much improved biocompatibility compared to pure PPC or PLA fibrous scaffolds.

  13. Synthesis of Stable Microcapsules from Trematode Eggshell Components.

    DTIC Science & Technology

    1988-04-29

    8217 Arlington, VA 22217-5000 61153N RR4106 71,E ’Include Security Classification) (u) Synthesis of Stable Microcapsules from Trematode Eggshell Components 12...necessary and Identify by block number) 3RO~P SUB-P~iA Microcapsule , Dopa-proteins, trematode, crosslinks, eggshell 79 ABSTRAC7 ,Continue an reverse If...All other editions are obsolete. S ,.O 0 V po r t -A’X I T!L~-r ....7 KIT X - ~.W.:,iili Synthesis of Stable Microcapsules from Trematode Eggshell

  14. Structure of synthetic peptide analogues of an eggshell protein of Schistosoma mansoni.

    PubMed Central

    Middaugh, C. R.; Thomson, J. A.; Burke, C. J.; Mach, H.; Naylor, A. M.; Bogusky, M. J.; Ryan, J. A.; Pitzenberger, S. M.; Ji, H.; Cordingley, J. S.

    1993-01-01

    The peptide (Gly-L-Tyr-L-Asp-L-Lys-L-Tyr)6, referred to as F4-6, was synthesized as a model for a schistosome eggshell protein in which the Gly-Tyr-Asp-Lys-Tyr consensus sequence is repeated over 40 times. Analysis by CD, Fourier transform infrared spectroscopy, potentiometric and spectrophotomertric titrations, NMR, and molecular modeling suggests that F4-6 forms some type of left-handed structure. A likely possibility appears to be a left-handed alpha-helix stabilized by Lysi-Aspi +4 salt bridges and possibly Aspi-Tyri +4 hydrogen bonding and Tyr-Tyr interactions. Spectroscopic studies of a number of F4-6 analogues support this conclusion. For example, substitution of D-Ala for Gly produces a peptide with enhanced left-handed helical spectral characteristics, whereas an L-Ala substitution results in a peptide with minimal structure. These studies suggest that the F4 protein from Schistosoma mansoni may be the first example of a naturally occurring protein devoid of proline and carbohydrate that forms a left-handed helix composed of L-amino acids, although alternative forms of other left-handed structures have yet to be rigorously excluded. PMID:8318895

  15. Correlation between hammerhead ribozyme-mediated eggshell protein gene cleavage and reproduction inhibition of Schistosoma japonicum

    PubMed Central

    LIANG, YU; ZHOU, YUELAN; YIN, WEIGUO; LI, YINGJU; YANG, QIULIN; GAO, YUAN; ZHANG, YUKUAI; YANG, YAOFEI; PENG, LI; XIAO, JIANHUA

    2012-01-01

    Schistosoma japonicum (S. japonicum) is an extremely harmful pathogen, which infects humans and causes severe public health problems. To date, no effective therapeutic drugs for this pathogen are available. In this study, we designed and constructed three hammerhead ribozymes targeting the eggshell protein gene of S. japonicum (SjESG). The cleavage activities of these three ribozymes were determined using cleavage experiments. The in vitro cleavage results showed that among the three synthesized ribozymes (Rz1, Rz2 and Rz3), Rz1 and Rz3 cleaved their target RNAs effectively. However, Rz2 did not cleave its target RNA detectably. The putative therapeutic roles of these three ribozymes to inhibit the reproduction of S. japonicum in mice were studied in vivo. Compared with the negative controls, Rz1 and Rz3 treatments resulted in increased levels of IFN-γ but decreased levels of IL-4 in mice. Rz2 affected levels of IFN-γ and IL-4 to degrees similar with those caused by the vector controls. In addition, Rz1 and Rz3 reduced the amounts of adult worms and eggs in the livers of mice more extensively than Rz2 and the vector controls. Altogether, these results suggest a correlation between the in vitro cleavage abilities of Rz1 and Rz3 and their roles in reproduction inhibition of S. japonicum. PMID:22246067

  16. Transfer of Dorsoventral and Terminal Information from the Ovary to the Embryo by a Common Group of Eggshell Proteins in Drosophila.

    PubMed

    Mineo, Alessandro; Furriols, Marc; Casanova, Jordi

    2017-04-01

    The Drosophila eggshell is an extracellular matrix that confers protection to the egg and also plays a role in transferring positional information from the ovary to pattern the embryo. Among the constituents of the Drosophila eggshell, Nasrat, Polehole, and Closca form a group of proteins related by sequence, secreted by the oocyte, and mutually required for their incorporation into the eggshell. Besides their role in eggshell integrity, Nasrat, Polehole, and Closca are also required for embryonic terminal patterning by anchoring or stabilizing Torso-like at the eggshell. Here, we show that they are also required for dorsoventral patterning, thereby unveiling that the dorsoventral and terminal systems, hitherto considered independent, share a common extracellular step. Furthermore, we show that Nasrat, Polehole, and Closca are required for proper Nudel activity, a protease acting both in embryonic dorsoventral patterning and eggshell integrity, thus providing a means to account for the role of Nasrat, Polehole, and Closca. We propose that a Nasrat/Polehole/Closca complex acts as a multifunctional hub to anchor various proteins synthesized at oogenesis, ensuring their spatial and temporal restricted function. Copyright © 2017 by the Genetics Society of America.

  17. A novel cDNA clone of Schistosoma japonicum encoding the 34,000 Dalton eggshell precursor protein.

    PubMed

    Sugiyama, H; Kawanaka, M; Kameoka, Y; Nakamura, M

    1997-07-01

    A cDNA clone encoding the 34 kDa eggshell protein of Schistosoma japonicum was isolated from an adult female cDNA library with a rabbit antiserum raised against the 34 kDa female worm fraction. A 230 bp-insert of this clone (Sj23A) was introduced in frame into the expression plasmid vector, pMAL-c2, and the recombinant fusion protein of the Sj23A transiation product was induced in Escherichia coli. The antiserum raised against the recombinant protein reacted only with the native 34 kDa protein of mature female worms, which localized in the vitelline cells of the vitelline glands. By reverse transcription-polymerase chain reaction (RT-PCR) analysis, it was found that the gene corresponding to the Sj23A was expressed exclusively in mature female worms. The clone Sj23A showed a high degree of homology to the genes for the eggshell precursor proteins of Fasciola hepatica. At the deduced polypeptide level, the Sj23A also had similarities with the F. hepatica-protein sequence, the amino acid composition [high glycine (16%), lysine (12%) and tyrosine (11%)] and the presence of tyrosine residues flanked by glycine. The clone Sj23A also shared an extensive sequence homology with 3 S. mansoni expression sequence tags (ESTs). The present results suggest that the protein encoded by the female-specific Sj23A gene of S. japonicum is widely conserved in trematodes and plays a significant role as a precursor involved in eggshell formation.

  18. A Novel Disulfide-Rich Protein Motif from Avian Eggshell Membranes

    PubMed Central

    Kodali, Vamsi K.; Gannon, Shawn A.; Paramasivam, Sivakumar; Raje, Sonali; Polenova, Tatyana; Thorpe, Colin

    2011-01-01

    Under the shell of a chicken egg are two opposed proteinaceous disulfide-rich membranes. They are fabricated in the avian oviduct using fibers formed from proteins that are extensively coupled by irreversible lysine-derived crosslinks. The intractability of these eggshell membranes (ESM) has slowed their characterization and their protein composition remains uncertain. In this work, reductive alkylation of ESM followed by proteolytic digestion led to the identification of a cysteine rich ESM protein (abbreviated CREMP) that was similar to spore coat protein SP75 from cellular slime molds. Analysis of the cysteine repeats in partial sequences of CREMP reveals runs of remarkably repetitive patterns. Module a contains a C-X4-C-X5-C-X8-C-X6 pattern (where X represents intervening non-cysteine residues). These inter-cysteine amino acid residues are also strikingly conserved. The evolutionarily-related module b has the same cysteine spacing as a, but has 11 amino acid residues at its C-terminus. Different stretches of CREMP sequences in chicken genomic DNA fragments show diverse repeat patterns: e.g. all a modules; an alternation of a-b modules; or an a-b-b arrangement. Comparable CREMP proteins are found in contigs of the zebra finch (Taeniopygia guttata) and in the oviparous green anole lizard (Anolis carolinensis). In all these cases the long runs of highly conserved modular repeats have evidently led to difficulties in the assembly of full length DNA sequences. Hence the number, and the amino acid lengths, of CREMP proteins are currently unknown. A 118 amino acid fragment (representing an a-b-a-b pattern) from a chicken oviduct EST library expressed in Escherichia coli is a well folded, highly anisotropic, protein with a large chemical shift dispersion in 2D solution NMR spectra. Structure is completely lost on reduction of the 8 disulfide bonds of this protein fragment. Finally, solid state NMR spectra suggest a surprising degree of order in intact ESM fibers. PMID

  19. A novel disulfide-rich protein motif from avian eggshell membranes.

    PubMed

    Kodali, Vamsi K; Gannon, Shawn A; Paramasivam, Sivakumar; Raje, Sonali; Polenova, Tatyana; Thorpe, Colin

    2011-03-30

    Under the shell of a chicken egg are two opposed proteinaceous disulfide-rich membranes. They are fabricated in the avian oviduct using fibers formed from proteins that are extensively coupled by irreversible lysine-derived crosslinks. The intractability of these eggshell membranes (ESM) has slowed their characterization and their protein composition remains uncertain. In this work, reductive alkylation of ESM followed by proteolytic digestion led to the identification of a cysteine rich ESM protein (abbreviated CREMP) that was similar to spore coat protein SP75 from cellular slime molds. Analysis of the cysteine repeats in partial sequences of CREMP reveals runs of remarkably repetitive patterns. Module a contains a C-X(4)-C-X(5)-C-X(8)-C-X(6) pattern (where X represents intervening non-cysteine residues). These inter-cysteine amino acid residues are also strikingly conserved. The evolutionarily-related module b has the same cysteine spacing as a, but has 11 amino acid residues at its C-terminus. Different stretches of CREMP sequences in chicken genomic DNA fragments show diverse repeat patterns: e.g. all a modules; an alternation of a-b modules; or an a-b-b arrangement. Comparable CREMP proteins are found in contigs of the zebra finch (Taeniopygia guttata) and in the oviparous green anole lizard (Anolis carolinensis). In all these cases the long runs of highly conserved modular repeats have evidently led to difficulties in the assembly of full length DNA sequences. Hence the number, and the amino acid lengths, of CREMP proteins are currently unknown. A 118 amino acid fragment (representing an a-b-a-b pattern) from a chicken oviduct EST library expressed in Escherichia coli is a well folded, highly anisotropic, protein with a large chemical shift dispersion in 2D solution NMR spectra. Structure is completely lost on reduction of the 8 disulfide bonds of this protein fragment. Finally, solid state NMR spectra suggest a surprising degree of order in intact ESM

  20. Analysing avian eggshell pigments with Raman spectroscopy.

    PubMed

    Thomas, Daniel B; Hauber, Mark E; Hanley, Daniel; Waterhouse, Geoffrey I N; Fraser, Sara; Gordon, Keith C

    2015-09-01

    Avian eggshells are variable in appearance, including coloration. Here, we demonstrate that Raman spectroscopy can provide accurate diagnostic information about major eggshell constituents, including the pigments biliverdin and protoporphyrin IX. Eggshells pigmented with biliverdin showed a series of pigment-diagnostic Raman peaks under 785 nm excitation. Eggshells pigmented with protoporphyrin IX showed strong emission under 1064 nm and 785 nm excitation, whereas resonance Raman spectra (351 nm excitation) showed a set of protoporphyrin IX informative peaks characteristic of protoporphyrin IX. As representative examples, we identified biliverdin in the olive green eggshells of elegant crested tinamous (Eudromia elegans) and in the blue eggshells of extinct upland moa (Megalapteryx didinus). This study encourages the wider use of Raman spectroscopy in pigment and coloration research and highlights the value of this technique for non-destructive analyses of museum eggshell specimens. © 2015. Published by The Company of Biologists Ltd.

  1. An Efficient Method for Co-purification of Eggshell Matrix Proteins OC-17, OC-116, and OCX-36

    PubMed Central

    2016-01-01

    In this study, we improved the eggshell-membrane separation process by separating the shell and membrane with EDTA solution, evaluating effects of three different extraction solutions (acetic acid, EDTA, and phosphate solution), and co-purifying multiple eggshell proteins with two successive ion-exchange chromatography procedures (CM Sepharose Fast Flow and DEAE Sepharose Fast Flow). The recovery and residual rates of eggshell and membrane separated by the modified method with added EDTA solution were 93.88%, 91.15% and 1.01%, 2.87%, respectively. Ovocleidin-116 (OC-116) and ovocalyxin-36 (OCX-36) were obtained by loading 50 mM Na-Hepes, pH 7.5, 2 mM DTT and 350 mM NaCl buffer onto the DEAE-FF column at a flow rate of 1 mL/min, ovocleidin-17 (OC-17) was obtained by loading 100 mM NaCl, 50 mM Tris, pH 8.0 on the CM-FF column at a flow rate of 0.5 mL/min. The purities of OCX-36, OC-17 and OC-116 were 96.82%, 80.15% and 73.22%, and the recovery rates were 55.27%, 53.38% and 36.34%, respectively. Antibacterial activity test suggested that phosphate solution extract exhibited significantly higher activity against the tested bacterial strains than the acetic acid or EDTA extract, probably due to more types of proteins in the extract. These results demonstrate that this separation method is feasible and efficient. PMID:28115888

  2. closca, a new gene required for both Torso RTK activation and vitelline membrane integrity. Germline proteins contribute to Drosophila eggshell composition.

    PubMed

    Ventura, Gemma; Furriols, Marc; Martín, Nicolás; Barbosa, Vitor; Casanova, Jordi

    2010-08-01

    The Drosophila eggshell is a specialised extracellular matrix (ECM) that surrounds and protects the oocyte and the embryo until its eclosion. In addition, the vitelline membrane, the innermost layer of the eggshell, holds the local determinant required to activate the Torso RTK pathway, which establishes the embryonic terminal regions. Here we report the identification and characterisation of closca, a gene encoding a new member of a group of proteins that act non-redundantly in vitelline membrane biogenesis and in Torso signalling. We also show that the Nasrat protein, another member of this group, is incorporated into the vitelline membrane, thereby indicating that the eggshell is a shared ECM that receives contributions from both follicle cells and the germline. This observation also provides a new scenario that accounts for the long known contribution of germline products to vitelline membrane biogenesis and to the follicle cell-dependent activation of the Torso receptor.

  3. Removal of heavy metals using waste eggshell.

    PubMed

    Park, Heung Jai; Jeong, Seong Wook; Yang, Jae Kyu; Kim, Boo Gil; Lee, Seung Mok

    2007-01-01

    The removal capacity of toxic heavy metals by the reused eggshell was studied. As a pretreatment process for the preparation of reused material from waste eggshell, calcination was performed in the furnace at 800 degrees C for 2 h after crushing the dried waste eggshell. Calcination behavior, qualitative and quantitative elemental information, mineral type and surface characteristics before and after calcination of eggshell were examined by thermal gravimetric analysis (TGA), X-ray fluorescence (XRF), X-ray diffraction (XRD) and scanning electron microscopy (SEM), respectively. After calcination, the major inorganic composition was identified as Ca (lime, 99.63%) and K, P and Sr were identified as minor components. When calcined eggshell was applied in the treatment of synthetic wastewater containing heavy metals, a complete removal of Cd as well as above 99% removal of Cr was observed after 10 min. Although the natural eggshell had some removal capacity of Cd and Cr, a complete removal was not accomplished even after 60 min due to quite slower removal rate. However, in contrast to Cd and Cr, an efficient removal of Pb was observed with the natural eggshell rather than the calcined eggshell. From the application of the calcined eggshell in the treatment of real electroplating wastewater, the calcined eggshell showed a promising removal capacity of heavy metal ions as well as had a good neutralization capacity in the treatment of strong acidic wastewater.

  4. Influence of eggshell matrix proteins on the precipitation of calcium carbonate (CaCO 3)

    NASA Astrophysics Data System (ADS)

    Hernández-Hernández, A.; Vidal, M. L.; Gómez-Morales, J.; Rodríguez-Navarro, A. B.; Labas, V.; Gautron, J.; Nys, Y.; García Ruiz, J. M.

    2008-04-01

    To understand the role of eggshell organic matrix on the biomineralization process, we have tested the influence of different purified fractions of the eggshell organic matrix on calcium carbonate (CaCO 3) precipitation. Purification was carried out after successive anion-exchange chromatography, hydrophobic interaction chromatography and gel filtration chromatography of two different prepurified eggshell extracts (A) and (B); the purified fractions (named g, h, n and r) and ( c', g', i', k') respectively were diluted to 50 μg/ml before being tested in vitro and analysed by the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) procedure and mass spectrometry. The precipitation experiments were carried out by the method of vapour diffusion on crystallization mushrooms. Each purified fraction showed a different effect on CaCO 3 precipitation. Some of them exhibited a strong inhibitory effect on nucleation, thus suppressing the precipitation of CaCO 3 almost totally while the others did not produce any notable effect. However, all fractions favoured the precipitation of calcite over the other CaCO 3 polymorphs. Additionally, all fractions modified in a different manner the size and morphology of the precipitated calcite crystals.

  5. Characterization and adsorption properties of eggshells and eggshell membrane.

    PubMed

    Tsai, W T; Yang, J M; Lai, C W; Cheng, Y H; Lin, C C; Yeh, C W

    2006-02-01

    The objective of this work was to study the chemical and physical characterization of eggshell and eggshell membrane particles prepared from the hen eggshell waste. Under the characterization measurements investigated, it was found that the pore structures of the two biomaterials belong to a typical Type II, indicating that they should be basically characteristic of nonporous materials or materials with macropores or open voids. Further, the chemical composition of the resulting eggshell particle was strongly associated with the presence of carbonate minerals from the Fourier transform infrared (FTIR) spectra. In contrast to the resulting eggshell membrane particle, the presence of functional groups of amines and amides was observable because of its chemical composition of fibrous proteins. From the isotherm data of methylene blue at 25 degrees C, the Freundlich model yielded a somewhat better fit than the Langmuir model. The adsorption isotherms revealed the eggshell biosorbents could only uptake the basic dye of less than 1.0mg/g in aqueous medium, which was attributed to their poor pore properties.

  6. Soluble eggshell membrane: A natural protein to improve the properties of biomaterials used for tissue engineering applications.

    PubMed

    Sah, Mahesh Kumar; Rath, Subha Narayan

    2016-10-01

    Extracellular matrix (ECM) acts as an instructing template for the cells contained in tissues. It plays a vital role in regulating cellular behavior by holding and interacting with various growth factors and signaling molecules. The ECM materials are either directly derived from a natural origin, or synthesized mimicking the natural ECM. In this review, we have addressed the ECM derived from eggshell membrane (ESM). The development of porous structures from natural biopolymers, such as ESM holds a number of advantages for tissue engineering applications. By using ESM in tissue engineering application, the cells attach and function to make a required tissue. Thereafter, the scaffold provides mechanical support as well as a platform for cellular interaction, hence, forming a fully functional tissue. The present review summarizes the structure-function relationship of ESM and advancement in its processing methods; the contribution of its soluble form (soluble eggshell membrane protein, SEP) in the development of promising hybrid biomaterials; and the recent advancement of their applications. In addition, this comprehensive review highlights the use of ESM for guided tissue regeneration; promising future applications of SEP in tissue engineering and regenerative medicine. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Features of eggshell formation in guinea fowl: kinetics of shell deposition, uterine protein secretion and uterine histology.

    PubMed

    Panhéleux, M; Kälin, O; Gautron, J; Nys, Y

    1999-12-01

    1. Rate of calcium carbonate deposition, duration of eggshell formation, organic composition of the uterine fluid, morphology of the egg shells and histochemistry of the uterus were studied in guinea fowl to analyse the origin of such thick, strong egg shells. 2. The egg shell was linearly deposited from 6.4 h to 21.8 h after the oviposition of the previous egg. The rate of egg shell deposition was similar to that in laying hens. However, the duration of linear shell deposition was increased by 2.1 h relative to that in hens. This explained the increased egg shell weight observed in the guinea fowl. 3. Intervals between oviposition of intra-clutch eggs were 24 h throughout the laying period. Ovulation occurred just after oviposition of the previous egg in the guinea fowl, as previously observed in hens but the duration of egg white protein deposition, of plumping and of initiation of shell mineralisation were all 1.5 h shorter than in domestic hen. 4. Uterine fluid can only be collected during the growth and terminal phase of shell formation. The electrophoretic profiles of the uterine fluid differed between phases and were somewhat different from those previously observed in the hen. Ovalbumin and ovocleidin-17 were both present in the uterine fluid and also in egg shell extract. Ovocleidin-17 was predominant during the growth phase. 5. The histology of the uterus differed slightly in guinea fowl compared to hens. Ovocleidin and ovalbumin are both secreted by the tubular glands. 6. Examination of radial ultrathin sections of eggshell showed, above the mammillary layer, intricate interlacing of adjacent exospherite in guinea fowl in contrast to the continuous columnar microstructure in hens. 7. The kinetics of egg shell deposition largely explains the increased egg shell weight of guinea fowl. The organic matrix proteins may be associated with the contrast between the structural organisation of the guinea fowl egg shell and that of the hen egg shell.

  8. Pleistocene geochronology and palaeothermometry from protein diagenesis in ostrich eggshells: implications for the evolution of modern humans.

    PubMed

    Miller, G H; Beaumont, P B; Jull, A J; Johnson, B

    1992-08-29

    Proteinaceous residues incorporated within the crystal structure of ostrich eggshells (OES) are retained without loss over geological time exceeding 10 million years. Degradation of the polypeptides, including hydrolysis to smaller peptide fragments and eventual release of free amino acids, decomposition, and racemization and epimerization occur at regular, predictable rates dependent on ambient temperature. The extent of isoleucine epimerization (aIle/Ile ratio) in OES follows linear first-order reversible kinetics in controlled-temperature laboratory simulations of time up to an aIle/Ile ratio in excess of 1.0. The hydrolysis of leucine also follows a predictable pattern, but deviates from first-order kinetics. A nonlinear mathematical model has been developed that adequately describes the pattern of leucine hydrolysis through a wide temperature range. Arrhenius parameters were derived from laboratory experiments combined with rate constant values found for 14C-dated OES from stratified caves in southern Africa. These parameters for isoleucine epimerization and leucine hydrolysis differ by ca. 10%, allowing the simultaneous solution of the two equations for temperature, independent of sample age. Although the uncertainty of the simultaneous temperature is relatively high (+/- 10 degrees C), it provides an effective means of identifying burned samples. If sample age is known, palaeotemperatures (the integrated thermal history experienced by an eggshell as opposed to an 'instantaneous' temperature) can be calculated with a precision of better than +/- 1 degrees C. The ages of levels at Border Cave, South Africa, from which anatomically modern human skeletal remains have been recovered, are dated by the extent of isoleucine epimerization in associated OES.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Eggshells as an index of aedine mosquito production. 2: Relationship of Aedes taeniorhynchus eggshell density to larval production.

    PubMed

    Addison, D S; Ritchie, S A; Webber, L A; Van Essen, F

    1992-03-01

    To test if eggshell density could be used as an index of aedine mosquito production, we compared eggshell density with the larval production of Aedes taeniorhynchus in Florida mangrove basin forests. Quantitative (n = 7) and categorical (n = 34) estimates of annual larval production were regressed against the number of eggshells per cc of soil. Significant regressions were obtained in both instances. Larval production was concentrated in zones with the highest eggshell density. We suggest that eggshell density and distribution can be used to identify oviposition sites and the sequence of larval appearance.

  10. Eggshells as an index of aedine mosquito production. 1: Distribution, movement and sampling of Aedes taeniorhynchus eggshells.

    PubMed

    Ritchie, S A; Addison, D S; van Essen, F

    1992-03-01

    The distribution of Aedes taeniorhynchus eggshells in Florida mangrove basin forests was determined and used to design a sampling plan. Eggshells were found in 10/11 sites (91%), with a mean +/- SE density of 1.45 +/- 0.75/cc; density did not change significantly year to year. Highest densities were located on the sloping banks of hummocks, ponds and potholes. Eggshells were less clumped in distribution than eggs and larvae and thus required a smaller sample size for a given precision level. While eggshells were flushed from compact soil that was subject to runoff during heavy rain, mangrove peat, the dominant soil of eggshell-bearing sites, was less dense and had little runoff or eggshell flushing. We suggest that eggshell surveys could be used to identify Ae. taeniorhynchus oviposition sites and oviposition patterns.

  11. Antimicrobial activity of the Anseriform outer eggshell and cuticle.

    PubMed

    Wellman-Labadie, Olivier; Picman, Jaroslav; Hincke, Maxwell T

    2008-04-01

    The avian eggshell is a complex, multifunctional biomineral composed of a calcium carbonate mineral phase and an organic phase of lipids and proteins. The outermost layer of the eggshell, the eggshell cuticle, is an organic layer of variable thickness composed of polysaccharides, hydroxyapatite crystals, lipids and glycoprotein. In addition to regulating gas exchanges, the eggshell cuticle may contain antimicrobial elements. In this study, we investigated the antimicrobial activity of eggshell cuticle and outer eggshell protein extracts from four Anseriform species: wood duck (Aix sponsa), hooded merganser (Lophodytes cucullatus), Canada goose (Branta canadensis) and mute swan (Cygnus olor). Cuticle and outer eggshell protein was extracted by urea or HCl treatment of eggs. C-type lysozyme, ovotransferrin and an ovocalyxin-32-like protein were detected in all extracts. Cuticle and outer eggshell protein extracts inhibited the growth of Staphylococcus aureus, Escherichia coli D31, Pseudomonas aeruginosa and Bacillus subtilis. The presence of active antimicrobial proteins within the avian cuticle and outer eggshell suggests a role in antimicrobial defense. Protein extracts from the cavity nesting hooded merganser were especially potent. The unique environmental pressures exerted on cavity-nesting species may have led to the evolution of potent antimicrobial defenses.

  12. Engineering Tough Materials: Biomimetic Eggshell

    DTIC Science & Technology

    2016-08-29

    formation, including a polymer -induced liquid precursor (PILP) mineral- ization. The second examines the interesting role of the eggshell membrane in shell...strategy is to use well-known polymers to mimic the role of proteins in biominerals. This method allows us to form a solution reminiscent of the...biomineralization en- vironment, which includes a liquid phase separation, and is hence called polymer - induced liquid precursor (PILP) process. The additive

  13. Cross-linking of DOPA-proteins in the eggshell of Bdelloura candida, a marine worm: A PIXE study

    NASA Astrophysics Data System (ADS)

    Swann, C. P.; Waite, J. H.; Huggins, L. G.

    1996-04-01

    Mature and hatched eggshells of Bdelloura candida, a member of a family of marine worms, have been examined to determine their structure, composition and mechanism of formation. This report concentrates on the metal content with the purpose of establishing whether or not metals that can form DOPA chelates are present, and, thereby, provide a means of cross-linking. Elevated levels of Fe, Cu and Zn were observed, all of which can form such chelates.

  14. The C. elegans eggshell

    PubMed Central

    Stein, Kathryn K.; Golden, Andy

    2017-01-01

    In all animals, oocytes are surrounded by an extracellular matrix upon fertilization. This matrix serves similar purposes in each animal. It functions to mediate sperm binding, to prevent polyspermy, to control the chemical environment of the embryo, and to provide physical protection to the embryo as it developes. The synthesis of the C. elegans matrix, or eggshell, begins when the oocyte enters the spermatheca and is fertilized by a single sperm. The process of eggshell synthesis is thought to take place during the completion of the maternal meiotic divisions such that the multi-layered eggshell is completed by anaphase II. The synthesis of the eggshell occurs in a hierarchical pattern such that the outermost layers are synthesized first in order to capture and retain the innermost layers as they form. Recent studies have revealed that the lipid-rich permeability barrier is distinct from the outer trilaminar eggshell. These new findings alter our previous understanding of the eggshell. This chapter aims to define each of the eggshell layers and the molecules that are known to play significant roles in their formation. PMID:26715360

  15. The eggshell: structure, composition and mineralization.

    PubMed

    Hincke, Maxwell T; Nys, Yves; Gautron, Joel; Mann, Karlheinz; Rodriguez-Navarro, Alejandro B; McKee, Marc D

    2012-01-01

    The calcareous egg is produced by all birds and most reptiles. Current understanding of eggshell formation and mineralization is mainly based on intensive studies of one species - the domesticated chicken Gallus gallus. The majority of constituents of the chicken eggshell have been identified. In this article we review eggshell microstructure and ultrastructure, and the results of recent genomic, transcriptomic and proteomic analyses of the chicken eggshell matrix to draw attention to areas of current uncertainty such as the potential role of amorphous calcium carbonate and the specific nature of the molecules that initiate (nucleate) mammillary cone formation and terminate palisade layer calcification. Comparative avian genomics and proteomics have only recently become possible with the publication of the Taeniopygia guttata (zebra finch) genome. Further rapid progress is highly anticipated with the soon-to-be-released genomes of turkey (Meleagris gallopavo) and duck (Anas platyrhynchos). These resources will allow rapid advances in comparative studies of the organic constituents of avian eggshell and their functional implications.

  16. Quantitative proteomics provides new insights into chicken eggshell matrix protein functions during the primary events of mineralisation and the active calcification phase.

    PubMed

    Marie, Pauline; Labas, Valérie; Brionne, Aurélien; Harichaux, Grégoire; Hennequet-Antier, Christelle; Rodriguez-Navarro, Alejandro B; Nys, Yves; Gautron, Joël

    2015-08-03

    Eggshell is a bioceramic composed of 95% calcium carbonate mineral and 3.5% organic matrix. Its structural organisation is controlled by its organic matrix. We have used quantitative proteomics to study four key stages of shell mineralisation: 1) widespread deposition of amorphous calcium carbonate (ACC), 2) ACC transformation into crystalline calcite aggregates, 3) formation of larger calcite crystal units and 4) development of a columnar structure with preferential calcite crystal orientation. This approach explored the distribution of 216 shell matrix proteins found at the four stages. Variations in abundance according to these calcification events were observed for 175 proteins. A putative function related to the mineralisation process was predicted by bioinformatics for 77 of them and was further characterised. We confirmed the important role of lysozyme, ovotransferrin, ovocleidin-17 and ovocleidin-116 for shell calcification process, characterised major calcium binding proteins (EDIL3, ALB, MFGE8, NUCB2), and described novel proteoglycans core proteins (GPC4, HAPLN3). We suggest that OVAL and OC-17 play a role in the stabilisation of ACC. Finally, we report proteins involved in the regulation of proteins driving the mineralisation. They correspond to numerous molecular chaperones including CLU, PPIB and OCX21, protease and protease inhibitors including OVM and CST3, and regulators of phosphorylation. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Organic matrix composition and ultrastructure of eggshell: a comparative study.

    PubMed

    Panheleux, M; Bain, M; Fernandez, M S; Morales, I; Gautron, J; Arias, J L; Solomon, S E; Hincke, M; Nys, Y

    1999-05-01

    1. The avian eggshell is a biomineralised composite ceramic consisting of calcium carbonate embedded in an organic matrix. Matrix components are supposed to be involved in the control of mineralisation, crystallographic texture and biomechanical properties of eggshell. 2. The structure and eggshell matrix composition of various domesticated bird species were compared to gain insight into the universality of the eggshell mineralisation process. 3. The SDS-PAGE profiles of soluble eggshell matrix were specific within groups of birds (a: laying hen, breeder hen, quail, pheasant and possibly turkey; b: guinea fowl; c: duck and goose) but some of the protein bands were common to all groups. 4. Analogies between species were confirmed by Western blotting using hen protein antibodies. Ovocleidin-17 (OC-17) and ovalbumin were revealed in all species (except quail for OC-17). Lysozyme was present only in hen eggshell. Another egg white protein: ovotransferrin showed a positive signal in hens, turkey and quail. Osteopontin was observed in laying and breeder hens and quail. 5. Different proteoglycans were localised to discrete regions within the eggshell. Dermatan sulphate was observed within the matrix of the calcified shell of all species except quail which contained chondroitin-6-sulfate. Keratan sulphate was observed in mammillary bodies of breeder and laying hen, quail, pheasant and turkey while chondroitin sulphate was also present in guinea fowl and duck. 6. The general structural organisation of the different avian eggshells was similar but specific differences were observed in the ultrastructure of the mammillary layer. Species of the same taxonomic family could be grouped according to their structural analogies: breeder hen, turkey and pheasant resembled that of the domestic fowl. Guinea fowl was unique. Goose and duck were quite similar with large and confluent mammillary bodies. 7. Some matrix components are therefore common to eggshells of various species but

  18. Amorphous calcium carbonate controls avian eggshell mineralization: A new paradigm for understanding rapid eggshell calcification.

    PubMed

    Rodríguez-Navarro, Alejandro B; Marie, Pauline; Nys, Yves; Hincke, Maxwell T; Gautron, Joel

    2015-06-01

    Avian eggshell mineralization is the fastest biogenic calcification process known in nature. How this is achieved while producing a highly crystalline material composed of large calcite columnar single crystals remains largely unknown. Here we report that eggshell mineral originates from the accumulation of flat disk-shaped amorphous calcium carbonate (ACC) particles on specific organic sites on the eggshell membrane, which are rich in proteins and sulfated proteoglycans. These structures known as mammillary cores promote the nucleation and stabilization of a amorphous calcium carbonate with calcitic short range order which predetermine the calcite composition of the mature eggshell. The amorphous nature of the precursor phase was confirmed by the diffuse scattering of X-rays and electrons. The nascent calcitic short-range order of this transient mineral phase was revealed by infrared spectroscopy and HRTEM. The ACC mineral deposited around the mammillary core sites progressively transforms directly into calcite crystals without the occurrence of any intermediate phase. Ionic speciation data suggest that the uterine fluid is equilibrated with amorphous calcium carbonate, throughout the duration of eggshell mineralization process, supporting that this mineral phase is constantly forming at the shell mineralization front. On the other hand, the transient amorphous calcium carbonate mineral deposits, as well as the calcite crystals into which they are converted, form by the ordered aggregation of nanoparticles that support the rapid mineralization of the eggshell. The results of this study alter our current understanding of avian eggshell calcification and provide new insights into the genesis and formation of calcium carbonate biominerals in vertebrates. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Eggshell Cutter Using Ultrasonic Vibration

    NASA Astrophysics Data System (ADS)

    Miura, Hikaru

    2003-05-01

    An eggshell cutting apparatus which utilizes ultrasonic vibration was developed, replacing the conventional apparatus which uses an air cutter, to cut eggshells at the blunt end of eggs. Two ultrasonic vibration sources were used: one with longitudinal vibration only and the other with torsional vibration plus longitudinal vibration. Eggshell cutting experiments using these vibration sources were conducted. The eggshell cutting time sharply decreased with increasing longitudinal vibration amplitude as well as increasing input power. When the source with torsional vibration plus longitudinal vibration was used and the amplitude of longitudinal vibration was 12 μm or less, the torsional vibration was effective for cutting eggshells. Furthermore, at the same input power, the eggshell cutting time by the source with longitudinal vibration only was shorter than that by the source with torsional vibration plus longitudinal vibration. When an egg was cut using the apparatus, there was essentially no cutting noise and the cut surface was smooth.

  20. Polymorphisms in Ion Transport Genes Are Associated with Eggshell Mechanical Property

    PubMed Central

    Sun, Congjiao; Shi, Fengying; Wu, Guiqin; Liu, Aiqiao; Xu, Guiyun; Yang, Ning

    2015-01-01

    Eggshell mechanical property traits such as eggshell breaking strength (ESS), eggshell thickness (EST) and eggshell weight (ESW) are most common and important indexes to evaluate eggshell quality in poultry industry. Uterine ion transporters involve in eggshell formation and might be associated with eggshell mechanical property traits. In this study, 99 SNPs in 15 ion transport genes were selected to genotype 976 pedigreed hens of Rhode Island Red. ESS, EST and ESW were measured for each bird at 55 weeks of age. The association study showed that 14 SNPs in 8 genes were significantly related (p < 0.05) with at least one trait, and their contributions to phenotypic variance ranged from 0.23% to 4.14%. Both ATP2A3 and SLC4A5 had a significant effect on all the three traits. Strong linkage disequilibrium (LD) was detected among SNPs in four genes: ATP2A3, ITPR1, SLC8A3, SCNN1a. The significant effects of those diplotypes on eggshell mechanical property traits were found, and their contributions to phenotypic variance ranged from 0.50% to 0.70%. It was concluded that the identified SNPs and diplotypes in this study were potential markers influencing the eggshell mechanical properties, which could contribute to the genetic improvement of eggshell quality. PMID:26106883

  1. Purification, crystallization and preliminary X-ray analysis of struthiocalcin 1 from ostrich (Struthio camelus) eggshell

    SciTech Connect

    Reyes-Grajeda, Juan Pablo; Marín-García, Liliana; Stojanoff, Vivian; Moreno, Abel

    2007-11-01

    The purification, crystallization and preliminary X-ray diffraction data of the protein struthiocalcin 1 isolated from ostrich eggshell are reported. The purification, crystallization and preliminary X-ray analysis of struthiocalcin 1 (SCA-1), a protein obtained from the intramineral part of ostrich (Struthio camelus) eggshell, is reported.

  2. The eggshell in the C. elegans oocyte-to-embryo transition.

    PubMed

    Johnston, Wendy L; Dennis, James W

    2012-04-01

    In egg-laying animals, embryonic development takes place within the highly specialized environment provided by the eggshell and its underlying extracellular matrix. Far from being simply a passive physical support, the eggshell is a key player in many early developmental events. Herein, we review current understanding of eggshell structure, biosynthesis, and function in zygotic development of the nematode, C. elegans. Beginning at sperm contact or entry, eggshell layers are produced sequentially. The earlier outer layers are required for secretion or organization of inner layers, and layers differ in composition and function. Developmental events that depend on the eggshell include polyspermy barrier generation, high fidelity meiotic chromosome segregation, osmotic barrier synthesis, polar body extrusion, anterior-posterior polarization, and organization of membrane and cortical proteins. The C. elegans eggshell is proving to be an excellent, tractable system to study the molecular cues of the extracellular matrix that instruct cell polarity and early development. Copyright © 2011 Wiley Periodicals, Inc.

  3. The avian eggshell as a model of biomineralization

    SciTech Connect

    Arias, J.L.; Fernandez, M.S. ); Laraia, V.J.; Janicki, J.; Heuer, A.H.; Caplan, A.I. )

    1990-11-01

    The avian eggshell is one of the most rapidly mineralizing biological systems known. By understanding the key components and steps in this process, we hope to provide relevant information for fabrication of ceramic composites. The calcification of the eggshell occurs in three main steps: (1) fabrication of an organic matrix, (2) nucleation of an inorganic phase on the organic matrix, and (3) space-filling growth of the calcite phase. The different layers of an eggshell can be separately isolated and studied. In this preliminary communication, the organization of the shell matrix and membranes and their association with the crystal phase, the immunohistochemical occurrence and distribution of types I and X collagen, and of different proteoglycans are reviewed. Also the preliminary findings of the remineralization of the intact or modified eggshell are presented. These experiments allow us to identify the essential steps in forming a natural composite ceramic. 47 refs., 6 figs.

  4. BMP-dependent gene repression cascade in Drosophila eggshell patterning

    PubMed Central

    Charbonnier, Enrica; Fuchs, Alisa; Cheung, Lily S.; Chayengia, Mrinal; Veikkolainen, Ville; Seyfferth, Janine; Shvartsman, Stanislav Y.; Pyrowolakis, George

    2015-01-01

    Bone Morphogenetic Proteins (BMPs) signal by activating Smad transcription factors to control a number of decisions during animal development. In Drosophila, signaling by the BMP ligand Decapentaplegic (Dpp) involves the activity of brinker (brk) which, in most contexts, is repressed by Dpp. Brk encodes a transcription factor which represses BMP signaling output by antagonizing Smad-dependent target gene activation. Here, we study BMP-dependent gene regulation during Drosophila oogenesis by following the signal transmission from Dpp to its target broad (br), a gene with a crucial function in eggshell patterning. We identify regulatory sequences that account for expression of both brk and br, and connect these to the transcription factors of the pathway. We show that Dpp directly regulates brk transcription through Smad- and Schnurri (Shn)-dependent repression. Brk is epistatic to Dpp in br expression and activates br indirectly, through removal of a repressor, which is yet to be identified. Our work provides first cis-regulatory insights into transcriptional interpretation of BMP signaling in eggshell morphogenesis and defines a transcriptional cascade that connects Dpp to target gene regulation. PMID:25704512

  5. A long-term increase in eggshell thickness of Greenlandic Peregrine Falcons Falco peregrinus tundrius.

    PubMed

    Falk, Knud; Møller, Søren; Mattox, William G

    2006-02-15

    Thickness of eggshell fragments and whole eggs from the Peregrine Falcon Falco peregrinus collected in South and West Greenland between 1972 and 2003 was measured and compared to shell thickness of pre-DDT eggs, also collected in Greenland. Linear regression yields a significant increase in the average thickness of eggshells over the period of 0.19% per year, corresponding to a change in eggshell thinning from 13.9% in 1972 to 7.8% in 2003. Backwards extrapolation of the data, suggests that the Greenlandic Peregrine population probably was never critically affected by DDT-induced eggshell thinning. By sampling eggshell fragments in many nests the spatial and temporal sample distribution was enlarged, allowing the detection of a significant long-term decrease in pollutant-induced eggshell thinning--a trend that could not have been identified if only the rarer whole, addled eggs had been sampled.

  6. Eggshell thickness in mallards fed methylmercury

    SciTech Connect

    Heinz, G.H.

    1980-09-01

    Eggshell thinning has been linked to impaired reproduction in many wild birds. Previous work of my own and others led me to believe that methylmercury may cause some eggshell thinning in birds. The present study was designed to determine whether methylmercury in the diet of mallards would thin their eggshells and whether it would add to eggshell thinning caused by DDE.

  7. Chemical Proteomic Platform To Identify Citrullinated Proteins

    PubMed Central

    2015-01-01

    Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA) and are routinely used for disease diagnosis. Protein citrullination is also increased in cancer and other autoimmune disorders, suggesting that citrullinated proteins may serve as biomarkers for diseases beyond RA. To identify these citrullinated proteins, we developed biotin-conjugated phenylglyoxal (biotin-PG). Using this probe and our platform technology, we identified >50 intracellular citrullinated proteins. More than 20 of these are involved in RNA splicing, suggesting, for the first time, that citrullination modulates RNA biology. Overall, this chemical proteomic platform will play a key role in furthering our understanding of protein citrullination in rheumatoid arthritis and potentially a wider spectrum of inflammatory diseases. PMID:26360112

  8. Identifying Protein-Calorie Malnutrition Workshop.

    ERIC Educational Resources Information Center

    Walker, Susan S.; Barker, Ellen M.

    Instructional materials are provided for a workshop to enable participants to assist in identifying patients at risk with protein-calorie malnutrition and in corrrecting this nutritional deficiency. Representative topics are nutrients; protein, mineral, and vitamin sources, functions, and deficiency symptoms; malnutrition; nutritional deficiency…

  9. Identifying Protein-Calorie Malnutrition Workshop.

    ERIC Educational Resources Information Center

    Walker, Susan S.; Barker, Ellen M.

    Instructional materials are provided for a workshop to enable participants to assist in identifying patients at risk with protein-calorie malnutrition and in corrrecting this nutritional deficiency. Representative topics are nutrients; protein, mineral, and vitamin sources, functions, and deficiency symptoms; malnutrition; nutritional deficiency…

  10. Identifying tandem Ankyrin repeats in protein structures.

    PubMed

    Chakrabarty, Broto; Parekh, Nita

    2014-12-30

    Tandem repetition of structural motifs in proteins is frequently observed across all forms of life. Topology of repeating unit and its frequency of occurrence are associated to a wide range of structural and functional roles in diverse proteins, and defects in repeat proteins have been associated with a number of diseases. It is thus desirable to accurately identify specific repeat type and its copy number. Weak evolutionary constraints on repeat units and insertions/deletions between them make their identification difficult at the sequence level and structure based approaches are desired. The proposed graph spectral approach is based on protein structure represented as a graph for detecting one of the most frequently observed structural repeats, Ankyrin repeat. It has been shown in a large number of studies that 3-dimensional topology of a protein structure is well captured by a graph, making it possible to analyze a complex protein structure as a mathematical entity. In this study we show that eigen spectra profile of a protein structure graph exhibits a unique repetitive profile for contiguous repeating units enabling the detection of the repeat region and the repeat type. The proposed approach uses a non-redundant set of 58 Ankyrin proteins to define rules for the detection of Ankyrin repeat motifs. It is evaluated on a set of 370 proteins comprising 125 known Ankyrin proteins and remaining non-solenoid proteins and the prediction compared with UniProt annotation, sequence-based approach, RADAR, and structure-based approach, ConSole. To show the efficacy of the approach, we analyzed the complete PDB structural database and identified 641 previously unrecognized Ankyrin repeat proteins. We observe a unique eigen spectra profile for different repeat types and show that the method can be easily extended to detect other repeat types. It is implemented as a web server, AnkPred. It is freely available at 'bioinf.iiit.ac.in/AnkPred'. AnkPred provides an elegant and

  11. Eggshell Types and Their Evolutionary Correlation with Life-History Strategies in Squamates.

    PubMed

    Hallmann, Konstantin; Griebeler, Eva Maria

    2015-01-01

    The eggshell is an important physiological structure for the embryo. It enables gas exchange, physical protection and is a calcium reserve. Most squamates (lizards, snakes, worm lizards) lay parchment-shelled eggs, whereas only some gekkotan species, a subgroup of lizards, have strongly calcified eggshells. In viviparous (live-bearing) squamates the eggshell is reduced or completely missing (hereafter "shell-less"). Recent studies showed that life-history strategies of gekkotan species differ between species with parchment- and rigid-shelled eggshells. Here we test if the three different eggshell types found in the squamates are also associated with different life-history strategies. We first investigated the influence of the phylogeny on the trait "eggshell type" and on six life-history traits of 32 squamate species. Phylogenetic principal component analysis (pPCA) was then conducted to identify an association between life-history strategies and eggshell types. Finally, we also considered adult weight in the pPCA to examine its potential effect on this association. Eggshell types in squamates show a strong phylogenetic signal at a low taxonomical level. Four out of the six life-history traits showed also a phylogenetic signal (birth size, clutch size, clutches per year and age at female maturity), while two had none (incubation time, maximum longevity). The pPCA suggested an association of life-history strategies and eggshell types, which disappeared when adult weight was included in the analysis. We conclude that the variability seen in eggshell types of squamates is weakly influenced by phylogeny. Eggshell types correlate with different life-history strategies, and mainly reflect differences in adult weights of species.

  12. Eggshell Types and Their Evolutionary Correlation with Life-History Strategies in Squamates

    PubMed Central

    Hallmann, Konstantin; Griebeler, Eva Maria

    2015-01-01

    The eggshell is an important physiological structure for the embryo. It enables gas exchange, physical protection and is a calcium reserve. Most squamates (lizards, snakes, worm lizards) lay parchment-shelled eggs, whereas only some gekkotan species, a subgroup of lizards, have strongly calcified eggshells. In viviparous (live-bearing) squamates the eggshell is reduced or completely missing (hereafter “shell-less”). Recent studies showed that life-history strategies of gekkotan species differ between species with parchment- and rigid-shelled eggshells. Here we test if the three different eggshell types found in the squamates are also associated with different life-history strategies. We first investigated the influence of the phylogeny on the trait “eggshell type” and on six life-history traits of 32 squamate species. Phylogenetic principal component analysis (pPCA) was then conducted to identify an association between life-history strategies and eggshell types. Finally, we also considered adult weight in the pPCA to examine its potential effect on this association. Eggshell types in squamates show a strong phylogenetic signal at a low taxonomical level. Four out of the six life-history traits showed also a phylogenetic signal (birth size, clutch size, clutches per year and age at female maturity), while two had none (incubation time, maximum longevity). The pPCA suggested an association of life-history strategies and eggshell types, which disappeared when adult weight was included in the analysis. We conclude that the variability seen in eggshell types of squamates is weakly influenced by phylogeny. Eggshell types correlate with different life-history strategies, and mainly reflect differences in adult weights of species. PMID:26393343

  13. Uterine and eggshell structure and histochemistry in a lizard with prolonged uterine egg retention (Lacertilia, Scincidae, Saiphos).

    PubMed

    Stewart, James R; Mathieson, Ashley N; Ecay, Tom W; Herbert, Jacquie F; Parker, Scott L; Thompson, Michael B

    2010-11-01

    The eggshell of lizards is a complex structure composed of organic and inorganic molecules secreted by the oviduct, which protects the embryo by providing a barrier to the external environment and also allows the exchange of respiratory gases and water for life support. Calcium deposited on the surface of the eggshell provides an important nutrient source for the embryo. Variation in physical conditions encountered by eggs results in a tradeoff among these functions and influences eggshell structure. Evolution of prolonged uterine egg retention results in a significant change in the incubation environment, notably reduction in efficiency of gas exchange, and selection should favor a concomitant reduction in eggshell thickness. This model is supported by studies that demonstrate an inverse correlation between eggshell thickness and length of uterine egg retention. One mechanism leading to thinning of the eggshell is reduction in size of uterine shell glands. Saiphos equalis is an Australian scincid lizard with an unusual pattern of geographic variation in reproductive mode. All populations retain eggs in the uterus beyond the embryonic stage at oviposition typical for lizards, and some are viviparous. We compared structure and histochemistry of the uterus and eggshell of two populations of S. equalis, prolonged egg retention, and viviparous to test the hypotheses: 1) eggshell thickness is inversely correlated with length of egg retention and 2) eggshell thickness is positively correlated with size of shell glands. We found support for the first hypothesis but also found that eggshells of both populations are surprisingly thick compared with other lizards. Our histochemical data support prior conclusions that uterine shell glands are the source of protein fiber matrix of the eggshell, but we did not find a correlation between size of shell glands and eggshell thickness. Eggshell thickness is likely determined by density of uterine shell glands in this species.

  14. The thin eggshell problem

    USGS Publications Warehouse

    Stickel, L.F.; Rhodes, L.I.; Gillett, J.W.

    1970-01-01

    It has long been known that DDT and related chemicals can impair the reproduction of birds. In early years of organochlorine pesticide use, widespread mortality occurred immediately following heavy applications of these chemicals, and survivors contained substantial amounts of toxicant in their tissues. Repopulation from untreated areas tended to conceal the extent of the effects. DDT and dieldrin have become ubiquitous and the original source of the chemicals producing bird deaths often cannot be traced. The extent of sublethal effects cannot be fully appraised, although laboratory experiments continually reveal new and potentially deleterious physiological reactions. Thin eggshells have become prevalent among certain declining species of predatory birds. Shell thinning and associated reproductive effects have been produced experimentally in mallard ducks and in sparrow hawks. Coturnix quail fed dietary dosages of p,p'-DDT produced fewer eggs than did untreated birds and the eggs had thinner shells. Hatchability was not significantly altered. Comparisons between these results and those obtained in other studies indicate significant species differences.

  15. Quantification of Japanese quail eggshell colour by image analysis.

    PubMed

    Sezer, Metin; Tekelioglu, Oguz

    2009-01-01

    The Japanese quail lays eggs with colourful and patterned shells which make the eggshell colour difficult to classify. In this study, the method of measuring colour of patchy eggs using image analyses and its power to discriminate among individual variation were established. Estimated repeatability for egg colour and proportion of patterned areas was high (>0.58), suggesting intermedíate or high heritability of eggshell colour characteristics. Three components have been identified as significant in discriminant function analysis. These three components explained 91.4% of the total variance in egg colour characteristics. In cluster analysis, 78.3% of the eggs that were collected from 15 females were correctly classified. This study indicates that eggshell colour characteristics can be reliably studied by image analyses and that this method can provide a unified character list for future examinations and interpretations of quail egg characteristics.

  16. Identifying protein complexes based on brainstorming strategy.

    PubMed

    Shen, Xianjun; Zhou, Jin; Yi, Li; Hu, Xiaohua; He, Tingting; Yang, Jincai

    2016-11-01

    Protein complexes comprising of interacting proteins in protein-protein interaction network (PPI network) play a central role in driving biological processes within cells. Recently, more and more swarm intelligence based algorithms to detect protein complexes have been emerging, which have become the research hotspot in proteomics field. In this paper, we propose a novel algorithm for identifying protein complexes based on brainstorming strategy (IPC-BSS), which is integrated into the main idea of swarm intelligence optimization and the improved K-means algorithm. Distance between the nodes in PPI network is defined by combining the network topology and gene ontology (GO) information. Inspired by human brainstorming process, IPC-BSS algorithm firstly selects the clustering center nodes, and then they are separately consolidated with the other nodes with short distance to form initial clusters. Finally, we put forward two ways of updating the initial clusters to search optimal results. Experimental results show that our IPC-BSS algorithm outperforms the other classic algorithms on yeast and human PPI networks, and it obtains many predicted protein complexes with biological significance. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Eggshell apex abnormalities associated with Mycoplasma synoviae infection in layers

    PubMed Central

    Jeon, Eun-Ok; Kim, Jong-Nyeo; Lee, Hae-Rim; Koo, Bon-Sang; Min, Kyeong-Cheol; Han, Moo-Sung; Lee, Seung-Baek; Bae, Yeon-Ji; Mo, Jong-Suk; Cho, Sun-Hyung; Lee, Chang-Hee

    2014-01-01

    Eggs exhibiting eggshell apex abnormalities (EAA) were evaluated for changes in shell characteristics such as strength, thickness, and ultrastructure. Mycoplasma synoviae (MS) infection was confirmed by serological assay along with isolation of MS from the trachea and oviduct. Changes in eggshell quality were shown to be statistically significant (p < 0.01). We also identified ultrastructural changes in the mammillary knob layer by Scanning Electron Microscopy. While eggs may seem to be structurally sound, ultrastructural evaluation showed that affected eggs do not regain their former quality. In our knowledge, this is the first report describing the occurrence of EAA in Korea. PMID:24962418

  18. Galactosaminoglycan composition in chicken eggshell.

    PubMed

    Nakano, T; Ikawa, N; Ozimek, L

    2002-05-01

    Galactosaminoglycans, isolated from decalcified chicken eggshell by papain digestion and ion-exchange chromatography, were fractionated by selective precipitation at varying concentrations of ethanol and characterized by chemical and enzymatic methods. The eggshell contained 0.15 microg galactosaminoglycan uronic acid/mg dry weight. Most (to approximately 87% of total) galactosaminoglycans were found to be chondroitin sulfate-dermatan sulfate copolymers with iduronic acid contents being approximately 20 to 30% of uronic acid. The remaining (to approximately 12% of total) galactosaminoglycans were chondroitin sulfate-dermatan sulfate copolymers with higher iduronic acid contents averaging 59% of uronic acid. Results of chondroitinase-ABC digestion demonstrated 4-sulfated disaccharides to be the major repeating units in the chicken eggshell galactosaminoglycans.

  19. The avian eggshell extracellular matrix as a model for biomineralization.

    PubMed

    Carrino, D A; Dennis, J E; Wu, T M; Arias, J L; Fernandez, M S; Rodriguez, J P; Fink, D J; Heuer, A H; Caplan, A I

    1996-01-01

    The avian eggshell is a complex, extracellularly assembled structure which contains both mineralized and non-mineralized regions. The composition of the hen eggshell organic matrix was examined by immunohistochemistry with antibodies to different extracellular matrix molecules. Type I collagen is found in the shell membranes, but only after treatment of the tissue sections with pepsin. When incomplete eggshells are removed from the oviduct and immunostained, type I collagen can be detected in the shell membranes without pepsin treatment. The shell membranes, which are non-mineralized, also contain type X collagen, and this immunostaining does not require pepsin treatment. The occurrence of type X collagen in the shell membranes is surprising, since this collagen has not been found in any tissue other than hypertrophic cartilage. Immunostaining for various glycosaminoglycans shows the presence of keratan sulfate and dermatan sulfate. Several different antibodies to keratan sulfate stain different regions of the eggshell; one keratan sulfate epitope is prominent in the calcium reserve assemblies. Dermatan sulfate staining is very intense in the palisade region. Demineralized matrix from the palisade region was extracted with guanidine and fractionated by ion exchange chromatography. A approximately 200-kDa dermatan sulfate proteoglycan is found in these extracts, along with a number of protein components. This preparation was tested for its ability to affect calcium carbonate crystal formation in vitro. Pieces of demineralized shell membranes were used as a substrate for crystal formation and various amounts of the palisade matrix dermatan sulfate proteoglycan preparation were added to the solution from which the crystals were formed. This material causes a concentration-dependent change in crystal morphology to one in which the crystals are smaller and more rounded, which more closely approximates the crystals normally observed in eggshells. These results suggest

  20. Engineering Tough Materials: Biomimetic Eggshell

    DTIC Science & Technology

    2015-01-30

    larger-­‐scale  production  of  eggshell-­‐like  organic-­‐ inorganic   composite  materials.       Report Documentation Page Form...production of eggshell-?????like organic-????? inorganic composite materials. 15. SUBJECT TERMS 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF...mineral  compounds  in  nature.    Biomaterials  are  almost  always  the   outcome  of  an  organic-­‐ inorganic

  1. Laser Cleaning of Avian Eggshell

    NASA Astrophysics Data System (ADS)

    Cornish, L.; Ball, A.; Russell, D.

    A low vacuum SEM was used to evaluate the effect of using an Nd:YAG laser as a non-contact technique for cleaning avian eggshells. The technique shows potential, since there are no obvious deleterious effects from cleaning, but further study is required to understand how the laser is interacting with the sample surface.

  2. Novel identification of matrix proteins involved in calcitic biomineralization.

    PubMed

    Rose-Martel, Megan; Smiley, Sandy; Hincke, Maxwell T

    2015-02-26

    Calcitic biomineralization is essential for otoconia formation in vertebrates. This process is characterized by protein-crystal interactions that modulate crystal growth on an extracellular matrix. An excellent model for the study of calcitic biomineralization is the avian eggshell, the fastest known biomineralization process. The objective of this study is to identify and characterize matrix proteins associated with the eggshell mammillary cones, which are hypothesized to regulate the earliest stage of eggshell calcification. Mammillary cones were isolated from 2 models, fertilized and unfertilized, and the released proteins were identified by RP-nanoLC and ES-MS/MS proteomics. Proteomics analysis identified 49 proteins associated with the eggshell membrane fibers and, importantly, 18 mammillary cone-specific proteins with an additional 18 proteins identified as enriched in the mammillary cones. Among the most promising candidates for modulating protein-crystal interactions were extracellular matrix proteins, including ABI family member 3 (NESH) binding protein (ABI3BP), tiarin-like, hyaluronan and proteoglycan link protein 3 (HAPLN3), collagen alpha-1(X), collagen alpha-1(II) and fibronectin, in addition to the calcium binding proteins calumenin, EGF-like repeats and discoidin 1-like domains 3 (EDIL3), nucleobindin-2 and SPARC. In conclusion, we identified several cone-resident proteins that are candidates to regulate initiation of eggshell calcification. Further study of these proteins will determine their roles in modulating calcitic biomineralization and lead to insight into the process of otoconia formation/regeneration. Biomineralization is essential for the development of hard tissues in vertebrates, which includes both calcium phosphate and calcium carbonate structures. Calcitic mineralization by calcium carbonate is an important process in the formation of otoconia, which are gravity receptor organs located in the inner ear and are responsible for balance

  3. Reducing the surface roughness of dental acrylic resins by using an eggshell abrasive material.

    PubMed

    Onwubu, Stanley C; Vahed, Anisa; Singh, Shalini; Kanny, Krishnan M

    2017-02-01

    Excessive surface roughness of denture base resins adversely impacts oral health. The purpose of this in vitro study was to examine the abrasive potential of eggshell powder in reducing the surface roughness of denture base resins. Thirty poly(methyl methacrylate) specimens were fabricated and polished with eggshell powders of different particle sizes and with pumice. The average surface roughness (Ra) after polishing was measured with a profilometer. Scanning electron microscope and optical electron microscope techniques were used to assess the surface roughness morphology of the specimens. ANOVA was used to analyze the Ra values. The Tukey honest significant differences and Bonferroni tests were used to identify differences between the 2 abrasive materials (α=.05). Significant differences in the Ra values were observed between the fine and medium eggshell powder abrasives (P<.05). Similarly, significant differences were found between pumice and the fine eggshell powder abrasives (P<.001). No significant differences were found between pumice and the medium eggshell powder abrasive (P>.05). Specimens polished with pumice had the highest Ra values, whereas specimens polished with the fine eggshell powder abrasive had the lowest Ra values. By connecting the Ra values to the threshold limit value of 0.2 μm, eggshell powder abrasive finished denture acrylic resin surfaces better than pumice. Copyright © 2016 Editorial Council for the Journal of Prosthetic Dentistry. Published by Elsevier Inc. All rights reserved.

  4. Embryonic sex steroid hormones accumulate in the eggshell of loggerhead sea turtle (Caretta caretta).

    PubMed

    Kobayashi, Shohei; Saito, Yoshimichi; Osawa, Akihisa; Katsumata, Etsuko; Karaki, Isuke; Nagaoka, Kentaro; Taya, Kazuyoshi; Watanabe, Gen

    2015-12-01

    Steroids hormones such as estradiol-17β (E2) and testosterone (T) are involved in gonadal differentiation of oviparous animals with temperature-dependent sex determination (TSD), and are greatly distributed. This hypothesizes that these embryonic steroid hormones probably accumulate in the eggshell throughout blood or/and chorioallantoic fluid in sea turtle species with TSD, producing females at higher temperature. To demonstrate this hypothesis, concentrations of E2 and T in the blood plasma from the hatchling loggerhead sea turtle (Caretta caretta) and in their eggshells were measured by radioimmunoassay. In the present study we propose that both concentrations of E2 and T in the blood plasma are correlated with amounts of these sex steroids in the eggshell. Moreover, contents of E2 in the eggshell showed a significant positive correlation with mean incubation temperatures during a thermosensitive period in the experimental nests, whereas T contents in the eggshell did not. Taken together, these findings indicated that embryonic E2 and T that accumulated in the eggshell can be extracted and measured. Furthermore, the present study suggested that contents of E2 in the eggshell may differ between male and female, and monitoring of these steroids is a useful method to identify the sex of loggerhead sea turtle hatchling. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Microwave irradiation of nanohydroxyapatite from chicken eggshells and duck eggshells.

    PubMed

    Sajahan, Nor Adzliana; Wan Ibrahim, Wan Mohd Azhar

    2014-01-01

    Due to similarity in composition to the mineral component of bones and human hard tissues, hydroxyapatite with chemical formula Ca10(PO4)6(OH)2 has been widely used in medical field. Both chicken and duck eggshells are mainly composed of calcium carbonate. An attempt has been made to fabricate nanohydroxyapatite (nHA) by chicken (CES) and duck eggshells (DES) as calcium carbonate source (CaCO3). CES and DES were reacted with diammonium hydrogen [(NH4)2HPO4] solution and subjected to microwave heating at 15 mins. Under the effect of microwave irradiation, nHA was produced directly in the solution and involved in crystallographic transformation. Sample characterization was done using by X-ray diffraction (XRD), fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM).

  6. Microwave Irradiation of Nanohydroxyapatite from Chicken Eggshells and Duck Eggshells

    PubMed Central

    Sajahan, Nor Adzliana; Wan Ibrahim, Wan Mohd Azhar

    2014-01-01

    Due to similarity in composition to the mineral component of bones and human hard tissues, hydroxyapatite with chemical formula Ca10(PO4)6(OH)2 has been widely used in medical field. Both chicken and duck eggshells are mainly composed of calcium carbonate. An attempt has been made to fabricate nanohydroxyapatite (nHA) by chicken (CES) and duck eggshells (DES) as calcium carbonate source (CaCO3). CES and DES were reacted with diammonium hydrogen [(NH4)2HPO4] solution and subjected to microwave heating at 15 mins. Under the effect of microwave irradiation, nHA was produced directly in the solution and involved in crystallographic transformation. Sample characterization was done using by X-ray diffraction (XRD), fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). PMID:25383364

  7. The evolution of eggshell cuticle in relation to nesting ecology

    PubMed Central

    Hauber, Mark E.

    2016-01-01

    Avian eggs are at risk of microbial infection prior to and during incubation. A large number of defence mechanisms have evolved in response to the severe costs imposed by these infections. The eggshell's cuticle is an important component of antimicrobial defence, and its role in preventing contamination by microorganisms in domestic chickens is well known. Nanometer-scale cuticular spheres that reduce microbial attachment and penetration have recently been identified on eggs of several wild avian species. However, whether these spheres have evolved specifically for antimicrobial defence is unknown. Here, we use comparative data on eggshell cuticular structure and nesting ecology to test the hypothesis that birds nesting in habitats with higher risk of infection (e.g. wetter and warmer) are more likely to evolve cuticular nanospheres on their eggshells than those nesting in less risky habitats. We found that nanostructuring, present in 54 of 296 analysed species, is the ancestral condition of avian eggshells and has been retained more often in taxa that nest in humid infection-prone environments, suggesting that they serve critical roles in antimicrobial egg defence. PMID:27488648

  8. Fossil avian eggshell preserves ancient DNA

    PubMed Central

    Oskam, Charlotte L.; Haile, James; McLay, Emma; Rigby, Paul; Allentoft, Morten E.; Olsen, Maia E.; Bengtsson, Camilla; Miller, Gifford H.; Schwenninger, Jean-Luc; Jacomb, Chris; Walter, Richard; Baynes, Alexander; Dortch, Joe; Parker-Pearson, Michael; Gilbert, M. Thomas P.; Holdaway, Richard N.; Willerslev, Eske; Bunce, Michael

    2010-01-01

    Owing to exceptional biomolecule preservation, fossil avian eggshell has been used extensively in geochronology and palaeodietary studies. Here, we show, to our knowledge, for the first time that fossil eggshell is a previously unrecognized source of ancient DNA (aDNA). We describe the successful isolation and amplification of DNA from fossil eggshell up to 19 ka old. aDNA was successfully characterized from eggshell obtained from New Zealand (extinct moa and ducks), Madagascar (extinct elephant birds) and Australia (emu and owl). Our data demonstrate excellent preservation of the nucleic acids, evidenced by retrieval of both mitochondrial and nuclear DNA from many of the samples. Using confocal microscopy and quantitative PCR, this study critically evaluates approaches to maximize DNA recovery from powdered eggshell. Our quantitative PCR experiments also demonstrate that moa eggshell has approximately 125 times lower bacterial load than bone, making it a highly suitable substrate for high-throughput sequencing approaches. Importantly, the preservation of DNA in Pleistocene eggshell from Australia and Holocene deposits from Madagascar indicates that eggshell is an excellent substrate for the long-term preservation of DNA in warmer climates. The successful recovery of DNA from this substrate has implications in a number of scientific disciplines; most notably archaeology and palaeontology, where genotypes and/or DNA-based species identifications can add significantly to our understanding of diets, environments, past biodiversity and evolutionary processes. PMID:20219731

  9. Fossil avian eggshell preserves ancient DNA.

    PubMed

    Oskam, Charlotte L; Haile, James; McLay, Emma; Rigby, Paul; Allentoft, Morten E; Olsen, Maia E; Bengtsson, Camilla; Miller, Gifford H; Schwenninger, Jean-Luc; Jacomb, Chris; Walter, Richard; Baynes, Alexander; Dortch, Joe; Parker-Pearson, Michael; Gilbert, M Thomas P; Holdaway, Richard N; Willerslev, Eske; Bunce, Michael

    2010-07-07

    Owing to exceptional biomolecule preservation, fossil avian eggshell has been used extensively in geochronology and palaeodietary studies. Here, we show, to our knowledge, for the first time that fossil eggshell is a previously unrecognized source of ancient DNA (aDNA). We describe the successful isolation and amplification of DNA from fossil eggshell up to 19 ka old. aDNA was successfully characterized from eggshell obtained from New Zealand (extinct moa and ducks), Madagascar (extinct elephant birds) and Australia (emu and owl). Our data demonstrate excellent preservation of the nucleic acids, evidenced by retrieval of both mitochondrial and nuclear DNA from many of the samples. Using confocal microscopy and quantitative PCR, this study critically evaluates approaches to maximize DNA recovery from powdered eggshell. Our quantitative PCR experiments also demonstrate that moa eggshell has approximately 125 times lower bacterial load than bone, making it a highly suitable substrate for high-throughput sequencing approaches. Importantly, the preservation of DNA in Pleistocene eggshell from Australia and Holocene deposits from Madagascar indicates that eggshell is an excellent substrate for the long-term preservation of DNA in warmer climates. The successful recovery of DNA from this substrate has implications in a number of scientific disciplines; most notably archaeology and palaeontology, where genotypes and/or DNA-based species identifications can add significantly to our understanding of diets, environments, past biodiversity and evolutionary processes.

  10. Association between ovocalyxin-32 gene haplotypes and eggshell quality traits in an F2 intercross between two chicken lines divergently selected for eggshell strength.

    PubMed

    Takahashi, H; Sasaki, O; Nirasawa, K; Furukawa, T

    2010-10-01

    Broken and cracked eggshells contribute significantly to economic losses in the egg production industry. We previously identified ovocalyxin-32 as a potential gene influencing eggshell traits, by analysing an intercross between two parent lines developed from the same founder population by a two-way selection for eggshell strength with non-destructive deformation (DEF) conducted over 14 generations. We determined the nucleotide sequences of six ovocalyxin-32 exons in the parent individuals and analysed the association between ovocalyxin-32 and eggshell traits in the F2 individuals. We identified three haplotypes (W, M and S) of ovocalyxin-32 in the parent individuals. A mismatch amplification mutation assay was performed to distinguish six diplotype individuals (WW, MM, SS, WM, MS and WS) inthe F2 population. The egg weight (EW) of SS-diplotype individuals was significantly higher than that of WW-, WM- and WS-diplotypes. Short length of the egg (SLE) of SS-diplotype individuals was significantly higher than that of WW-, WM- and MS-diplotypes. Long length of the egg (LLE) of SS-diplotype individuals was significantly higher than that of WM and WS-diplotypes. DEF of WW-diplotype individuals was significantly higher than that ofSS-, WM, MS and WM-diplotypes. Haplotypic effect analyses showed significant differences between the W-haplotype and the S-haplotypes in the EW, SLE, LLE and DEF. The DEF of M-haplotype was significantly lower than that of W- and S-haplotypes. These results suggest that S- and M-haplotypes are critical for high quality of eggshells in the F2 population. In conclusion, ovocalyxin-32 is a useful marker of eggshell traits and can be used to develop strategies for improving eggshell traits in commercial layer houses.

  11. Extraction of glycosaminoglycans from chicken eggshell.

    PubMed

    Nakano, T; Ikawa, N; Ozimek, L

    2001-05-01

    The objectives of this study were to analyze glycosaminoglycan (GAG) and mineral composition in the chicken eggshell. Eggshells were decalcified with acetic acid, and GAG was extracted from the decalcified shell by digestion with papain. The eggshell contained an average of 0.024% of its dry weight as uronic acid, a carbohydrate moiety of GAG. The eggshell GAG consisted of approximately 48% hyaluronic acid and and 52% galactosaminoglycan. In the latter, chondroitin sulfate-dermatan sulfate copolymers were the major galactosaminoglycans with dermatan sulfate disaccharide as a relatively minor component. The inorganic material recovered after decalcification accounted for approximately 140% of dry weight of the eggshell and contained 24.11% calcium, 0.04% phosphorous, and 0.23% magnesium, with an undetectable amount of nitrogen.

  12. Eggshell composition of squamate reptiles: relationship between eggshell permeability and amino acid distribution.

    PubMed

    Sexton, Owen J; Bramble, Judith E; Heisler, I Lorraine; Phillips, Christopher A; Cox, David L

    2005-10-01

    Most snakes and lizards produce eggs with flexible shells that interact with the environment to maintain water balance. Geckos produce rigid eggshells that are independent of an external source of water and can be oviposited in more open, dryer locations. In this study, we analyzed and compared the amino acid composition of 24 lizard species, six snake species, and four outgroups (including avian and reptilian elastin and chicken eggshell). Rigid Gecko eggshells had significantly lower levels of seven of the 17 amino acids evaluated. Multivariate analysis showed that proline was the most important amino acid in distinguishing between these two groups of eggshells, occurring at significantly higher levels in flexible eggshells. High levels of proline have also been observed in the eggshells of other species. Proline and other amino acids are associated with the alleviation of water and salt stress in plants.

  13. Identifying structural domains of proteins using clustering

    PubMed Central

    2012-01-01

    Background Protein structures are comprised of modular elements known as domains. These units are used and re-used over and over in nature, and usually serve some particular function in the structure. Thus it is useful to be able to break up a protein of interest into its component domains, prior to similarity searching for example. Numerous computational methods exist for doing so, but most operate only on a single protein chain and many are limited to making a series of cuts to the sequence, while domains can and do span multiple chains. Results This study presents a novel clustering-based approach to domain identification, which works equally well on individual chains or entire complexes. The method is simple and fast, taking only a few milliseconds to run, and works by clustering either vectors representing secondary structure elements, or buried alpha-carbon positions, using average-linkage clustering. Each resulting cluster corresponds to a domain of the structure. The method is competitive with others, achieving 70% agreement with SCOP on a large non-redundant data set, and 80% on a set more heavily weighted in multi-domain proteins on which both SCOP and CATH agree. Conclusions It is encouraging that a basic method such as this performs nearly as well or better than some far more complex approaches. This suggests that protein domains are indeed for the most part simply compact regions of structure with a higher density of buried contacts within themselves than between each other. By representing the structure as a set of points or vectors in space, it allows us to break free of any artificial limitations that other approaches may depend upon. PMID:23116496

  14. Variability in Avian Eggshell Colour: A Comparative Study of Museum Eggshells

    PubMed Central

    Cassey, Phillip; Portugal, Steven J.; Maurer, Golo; Ewen, John G.; Boulton, Rebecca L.; Hauber, Mark E.; Blackburn, Tim M.

    2010-01-01

    Background The exceptional diversity of coloration found in avian eggshells has long fascinated biologists and inspired a broad range of adaptive hypotheses to explain its evolution. Three main impediments to understanding the variability of eggshell appearance are: (1) the reliable quantification of the variation in eggshell colours; (2) its perception by birds themselves, and (3) its relation to avian phylogeny. Here we use an extensive museum collection to address these problems directly, and to test how diversity in eggshell coloration is distributed among different phylogenetic levels of the class Aves. Methodology and Results Spectrophotometric data on eggshell coloration were collected from a taxonomically representative sample of 251 bird species to determine the change in reflectance across different wavelengths and the taxonomic level where the variation resides. As many hypotheses for the evolution of eggshell coloration assume that egg colours provide a communication signal for an avian receiver, we also modelled reflectance spectra of shell coloration for the avian visual system. We found that a majority of species have eggs with similar background colour (long wavelengths) but that striking differences are just as likely to occur between congeners as between members of different families. The region of greatest variability in eggshell colour among closely related species coincided with the medium-wavelength sensitive region around 500 nm. Conclusions The majority of bird species share similar background eggshell colours, while the greatest variability among species aligns with differences along a red-brown to blue axis that most likely corresponds with variation in the presence and concentration of two tetrapyrrole pigments responsible for eggshell coloration. Additionally, our results confirm previous findings of temporal changes in museum collections, and this will be of particular concern for studies testing intraspecific hypotheses relating

  15. Variability in avian eggshell colour: a comparative study of museum eggshells.

    PubMed

    Cassey, Phillip; Portugal, Steven J; Maurer, Golo; Ewen, John G; Boulton, Rebecca L; Hauber, Mark E; Blackburn, Tim M

    2010-08-09

    The exceptional diversity of coloration found in avian eggshells has long fascinated biologists and inspired a broad range of adaptive hypotheses to explain its evolution. Three main impediments to understanding the variability of eggshell appearance are: (1) the reliable quantification of the variation in eggshell colours; (2) its perception by birds themselves, and (3) its relation to avian phylogeny. Here we use an extensive museum collection to address these problems directly, and to test how diversity in eggshell coloration is distributed among different phylogenetic levels of the class Aves. Spectrophotometric data on eggshell coloration were collected from a taxonomically representative sample of 251 bird species to determine the change in reflectance across different wavelengths and the taxonomic level where the variation resides. As many hypotheses for the evolution of eggshell coloration assume that egg colours provide a communication signal for an avian receiver, we also modelled reflectance spectra of shell coloration for the avian visual system. We found that a majority of species have eggs with similar background colour (long wavelengths) but that striking differences are just as likely to occur between congeners as between members of different families. The region of greatest variability in eggshell colour among closely related species coincided with the medium-wavelength sensitive region around 500 nm. The majority of bird species share similar background eggshell colours, while the greatest variability among species aligns with differences along a red-brown to blue axis that most likely corresponds with variation in the presence and concentration of two tetrapyrrole pigments responsible for eggshell coloration. Additionally, our results confirm previous findings of temporal changes in museum collections, and this will be of particular concern for studies testing intraspecific hypotheses relating temporal patterns to adaptation of eggshell colour

  16. Global variation and uniformity of eggshell thickness for chicken eggs.

    PubMed

    Sun, C J; Chen, S R; Xu, G Y; Liu, X M; Yang, N

    2012-10-01

    Damaged eggshells result in losses of eggs. Numerous efforts have been carried out to improve eggshell quality, which may lead to increased eggshell thickness. The conventional way of enhancing eggshell strength with thicker eggshell on average may be replaced by a new strategy to improve eggshell uniformity without increasing eggshell thickness. To achieve this, it is necessary to investigate global variation of eggshell thickness. In this study, we used 100 fresh eggs from 52-wk-old layers of a commercial brown-egg variety. To determine the global variation of eggshell thickness, 42 points for each egg along both longitudinal and latitudinal axes were selected to measure thickness using an eggshell thickness gauge. The eggshell thickness from blunt to sharp end varied significantly (P < 0.05). The area surrounding the blunt end was the thinnest (0.341 ± 0.025 mm), whereas the area surrounding the sharp end was the thickest (0.367 ± 0.023 mm). It was found that the thickness of the sharp end was the closest to the average thickness of the whole eggshell and could be considered as a valid measurement of eggshell thickness. A new parameter, eggshell thickness uniformity, was defined as the reciprocal of the coefficient of variation (1/CV) of eggshell thickness from 42 points of each egg and can be used to evaluate the eggshell quality. Eggshell thickness uniformity was positively correlated with breaking strength (r = 0.341; P < 0.01), suggesting that the parameter may be used as a potential selection criterion in breeding program to improve eggshell quality without increasing eggshell thickness.

  17. Subtleties of biomineralisation revealed by manipulation of the eggshell membrane.

    PubMed

    Li, Nan; Niu, Li-na; Qi, Yi-pin; Yiu, Cynthia K Y; Ryou, Heonjune; Arola, Dwayne D; Chen, Ji-hua; Pashley, David H; Tay, Franklin R

    2011-12-01

    Biocalcification of collagen matrices with calcium phosphate and biosilicification of diatom frustules with amorphous silica are two discrete processes that have intrigued biologists and materials scientists for decades. Recent advancements in the understanding of the mechanisms involved in these two biomineralisation processes have resulted in the use of biomimetic strategies to replicate these processes separately using polyanionic, polycationic or zwitterionic analogues of extracellular matrix proteins to stabilise amorphous mineral precursor phases. To date, there is a lack of a universal model that enables the subtleties of these two apparently dissimilar biomineralisation processes to be studied together. Here, we utilise the eggshell membrane as a universal model for differential biomimetic calcification and silicification. By manipulating the eggshell membrane to render it permeable to stabilised mineral precursors, it is possible to introduce nanostructured calcium phosphate or silica into eggshell membrane fibre cores or mantles. We provide a model for infiltrating the two compartmental niches of a biopolymer membrane with different intrafibre minerals to obtain materials with potentially improved structure-property relationships.

  18. A nanostructural basis for gloss of avian eggshells

    PubMed Central

    Igic, Branislav; Fecheyr-Lippens, Daphne; Xiao, Ming; Chan, Andrew; Hanley, Daniel; Brennan, Patricia R. L.; Grim, Tomas; Waterhouse, Geoffrey I. N.; Hauber, Mark E.; Shawkey, Matthew D.

    2015-01-01

    The role of pigments in generating the colour and maculation of birds' eggs is well characterized, whereas the effects of the eggshell's nanostructure on the visual appearance of eggs are little studied. Here, we examined the nanostructural basis of glossiness of tinamou eggs. Tinamou eggs are well known for their glossy appearance, but the underlying mechanism responsible for this optical effect is unclear. Using experimental manipulations in conjunction with angle-resolved spectrophotometry, scanning electron microscopy, atomic force microscopy and chemical analyses, we show that the glossy appearance of tinamou eggshells is produced by an extremely smooth cuticle, composed of calcium carbonate, calcium phosphate and, potentially, organic compounds such as proteins and pigments. Optical calculations corroborate surface smoothness as the main factor producing gloss. Furthermore, we reveal the presence of weak iridescence on eggs of the great tinamou (Tinamus major), an optical effect never previously documented for bird eggs. These data highlight the need for further exploration into the nanostructural mechanisms for the production of colour and other optical effects of avian eggshells. PMID:25505139

  19. A nanostructural basis for gloss of avian eggshells.

    PubMed

    Igic, Branislav; Fecheyr-Lippens, Daphne; Xiao, Ming; Chan, Andrew; Hanley, Daniel; Brennan, Patricia R L; Grim, Tomas; Waterhouse, Geoffrey I N; Hauber, Mark E; Shawkey, Matthew D

    2015-02-06

    The role of pigments in generating the colour and maculation of birds' eggs is well characterized, whereas the effects of the eggshell's nanostructure on the visual appearance of eggs are little studied. Here, we examined the nanostructural basis of glossiness of tinamou eggs. Tinamou eggs are well known for their glossy appearance, but the underlying mechanism responsible for this optical effect is unclear. Using experimental manipulations in conjunction with angle-resolved spectrophotometry, scanning electron microscopy, atomic force microscopy and chemical analyses, we show that the glossy appearance of tinamou eggshells is produced by an extremely smooth cuticle, composed of calcium carbonate, calcium phosphate and, potentially, organic compounds such as proteins and pigments. Optical calculations corroborate surface smoothness as the main factor producing gloss. Furthermore, we reveal the presence of weak iridescence on eggs of the great tinamou (Tinamus major), an optical effect never previously documented for bird eggs. These data highlight the need for further exploration into the nanostructural mechanisms for the production of colour and other optical effects of avian eggshells. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

  20. Subtleties of biomineralisation revealed by manipulation of the eggshell membrane

    PubMed Central

    Li, Nan; Niu, Li-na; Qi, Yi-pin; Yiu, Cynthia K.Y.; Ryou, Heonjune; Arola, Dwayne D.; Chen, Ji-hua; Pashley, David H.; Tay, Franklin R.

    2011-01-01

    Biocalcification of collagen matrices with calcium phosphate and biosilicification of diatom frustules with amorphous silica are two discrete processes that have intrigued biologists and materials scientists for decades. Recent advancements in the understanding of the mechanisms involved in these two biomineralisation processes have resulted in the use of biomimetic strategies to replicate these processes separately using polyanionic, polycationic or zwitterionic analogues of extracellular matrix proteins to stabilise amorphous mineral precursor phases. To date, there is a lack of a universal model that enables the subtleties of these two apparently dissimilar biomineralisation processes to be studied together. Here, we utilise the eggshell membrane as a universal model for differential biomimetic calcification and silicification. By manipulating the eggshell membrane to render it permeable to stabilised mineral precursors, it is possible to introduce nanostructured calcium phosphate or silica into eggshell membrane fibre cores or mantles. We provide a model for infiltrating the two compartmental niches of a biopolymer membrane with different intrafibre minerals to obtain materials with potentially improved structure-property relationships. PMID:21864897

  1. [Progresses in inheritance of genes for avian eggshell color].

    PubMed

    Yuan, Qing-Yan; Lu, Li-Zhi

    2007-03-01

    Eggshell has three colors: white, blue and brown. Chicken and duck eggs with blue eggshell have superior market for its better appearance, delicious taste, abundant nutrition and higher eggshell thickness and strength compared to those with white eggshell. However, error was often made when breeding blue-eggshell chicken or duck lines based on phenotypes. Studies on the forming and controlling mechanism of eggshell color had important theoretic and practical value. This review mainly discussed the types of eggshell color, its pigment composition and synthesis. Inheritance and heritability, genetic model, the number of genes, and the dominant-recessive relationship between genes for eggshell color were also reviewed. Information described in this review is useful for understanding the forming mechanism of eggshell color.

  2. Identification of uterine ion transporters for mineralisation precursors of the avian eggshell

    PubMed Central

    2012-01-01

    Background In Gallus gallus, eggshell formation takes place daily in the hen uterus and requires large amounts of the ionic precursors for calcium carbonate (CaCO3). Both elements (Ca2+, HCO3-) are supplied by the blood via trans-epithelial transport. Our aims were to identify genes coding for ion transporters that are upregulated in the uterine portion of the oviduct during eggshell calcification, compared to other tissues and other physiological states, and incorporate these proteins into a general model for mineral transfer across the tubular gland cells during eggshell formation. Results A total of 37 candidate ion transport genes were selected from our database of overexpressed uterine genes associated with eggshell calcification, and by analogy with mammalian transporters. Their uterine expression was compared by qRTPCR in the presence and absence of eggshell formation, and with relative expression levels in magnum (low Ca2+/HCO3- movement) and duodenum (high rates of Ca2+/HCO3- trans-epithelial transfer). We identified overexpression of eleven genes related to calcium movement: the TRPV6 Ca2+ channel (basolateral uptake of Ca2+), 28 kDa calbindin (intracellular Ca2+ buffering), the endoplasmic reticulum type 2 and 3 Ca2+ pumps (ER uptake), and the inositol trisphosphate receptors type 1, 2 and 3 (ER release). Ca2+ movement across the apical membrane likely involves membrane Ca2+ pumps and Ca2+/Na+ exchangers. Our data suggests that Na+ transport involved the SCNN1 channel and the Na+/Ca2+ exchangers SLC8A1, 3 for cell uptake, the Na+/K+ ATPase for cell output. K+ uptake resulted from the Na+/K+ ATPase, and its output from the K+ channels (KCNJ2, 15, 16 and KCNMA1). We propose that the HCO3- is mainly produced from CO2 by the carbonic anhydrase 2 (CA2) and that HCO3- is secreted through the HCO3-/Cl- exchanger SLC26A9. HCO3- synthesis and precipitation with Ca2+ produce two H+. Protons are absorbed via the membrane’s Ca2+ pumps ATP2B1, 2 in the apical

  3. Productive performance, eggshell quality, and eggshell ultrastructure of laying hens fed diets supplemented with organic trace minerals.

    PubMed

    Stefanello, C; Santos, T C; Murakami, A E; Martins, E N; Carneiro, T C

    2014-01-01

    This study was carried out with the purpose of evaluating the effect of supplementing hens' diets with trace minerals from inorganic or organic sources on the productive performance, eggshell quality, and eggshell ultrastructure of laying hens. Three hundred sixty Hy-Line W36 laying hens between 47 to 62 wk of age were used and distributed in a completely randomized experimental design with 9 treatments, 5 replicates, and 8 birds for each experimental unit. The treatments consisted of a control diet without supplementation of the trace minerals Mn, Zn, and Cu; 4 supplementation levels of these trace minerals from an inorganic source; and the same levels of supplementation from an organic source (proteinates). The supplementation levels in milligrams per kilogram for Mn, Zn, and Cu, were, respectively, 35-30-05, 65-60-10, 95-90-15, and 125-120-20. There was no effect of supplementation of trace minerals on the rate of posture, feed intake, feed conversion, specific weight, and Haugh unit of eggs. However, there was a quadratic effect (P < 0.05) of the levels of trace mineral supplementation on average egg weight and egg mass; the results did not differ regarding the source used. The increase in the levels of supplementation of Mn, Zn, and Cu provided a linear increase (P < 0.05) in the breaking strength and the percentage of eggshell. There was a linear decrease (P < 0.05) in the egg loss and the number of mammillary buttons in the shell. The best results were obtained using diets supplemented with trace minerals from an organic source because these diets provided lower egg loss, higher thickness, and increased strength of the shell. Structurally, organic Mn, Zn, and Cu provided higher thickness of the palisade layer and lower mammillary density. The trace mineral supplementation improved the structural characteristics and the quality of the eggshells.

  4. Isolation of Salmonella enterica in laying-hen flocks and assessment of eggshell contamination in France.

    PubMed

    Chemaly, Marianne; Huneau-Salaün, Adeline; Labbe, Annie; Houdayer, Catherine; Petetin, Isabelle; Fravalo, Philippe

    2009-10-01

    The present investigation was conducted in conjunction with the European Union baseline study for the estimation of Salmonella prevalence in laying-hen flocks. It aimed at evaluating eggshell contamination in farms positive for Salmonella, characterizing the genetic patterns of Salmonella strains and identifying the factors associated with Salmonella contamination of eggshells. For this purpose, a total of 4,200 eggs were collected from 28 positive flocks and analyzed according to draft Annex D of International Organization for Standardization Method 6579. Molecular characterization of the Salmonella strains was obtained by the pulsed-field gel electrophoresis method with two restriction enzymes, XbaI and BlnI. The relationship between the presence of Salmonella on eggshells and rearing practices was studied by using multiple correspondence analysis. Results showed that 39.3% of the positive flocks had at least one positive eggshell, with a total of 1.05% of eggshells testing positive for Salmonella. We detected the same serovars on samples taken from the farm and from eggshells within a given flock, with isolates sharing the same genetic pattern in 7 of 11 flocks. Eggshells tested positive for Salmonella in flocks (i) located where delivery trucks pass near air entrances of the poultry house, (ii) with high holding capacity (>30,000 laying hens), and (iii) with more than five positive samples coming from the farm environment, as well as in cases of flocks with a maximum egg-laying rate of >96% and in cases where farmers worked in other animal production. This study provided valuable information that could be used for risk management and risk assessment studies.

  5. Not so colourful after all: eggshell pigments constrain avian eggshell colour space

    PubMed Central

    Hanley, Daniel; Grim, Tomáš; Cassey, Phillip; Hauber, Mark E.

    2015-01-01

    Birds' eggshells are renowned for their striking colours and varied patterns. Although often considered exceptionally diverse, we report that avian eggshell coloration, sampled here across the full phylogenetic diversity of birds, occupies only 0.08–0.10% of the avian perceivable colour space. The concentrations of the two known tetrapyrrole eggshell pigments (protoporphyrin and biliverdin) are generally poor predictors of colour, both intra- and interspecifically. Here, we show that the constrained diversity of eggshell coloration can be accurately predicted by colour mixing models based on the relative contribution of both pigments and we demonstrate that the models' predictions can be improved by accounting for the reflectance of the eggshell's calcium carbonate matrix. The establishment of these proximate links between pigmentation and colour will enable future tests of hypotheses on the functions of perceived avian eggshell colours that depend on eggshell chemistry. More generally, colour mixing models are not limited to avian eggshell colours but apply to any natural colour. Our approach illustrates how modelling can aid the understanding of constraints on phenotypic diversity. PMID:25994009

  6. Not so colourful after all: eggshell pigments constrain avian eggshell colour space.

    PubMed

    Hanley, Daniel; Grim, Tomáš; Cassey, Phillip; Hauber, Mark E

    2015-05-01

    Birds' eggshells are renowned for their striking colours and varied patterns. Although often considered exceptionally diverse, we report that avian eggshell coloration, sampled here across the full phylogenetic diversity of birds, occupies only 0.08-0.10% of the avian perceivable colour space. The concentrations of the two known tetrapyrrole eggshell pigments (protoporphyrin and biliverdin) are generally poor predictors of colour, both intra- and interspecifically. Here, we show that the constrained diversity of eggshell coloration can be accurately predicted by colour mixing models based on the relative contribution of both pigments and we demonstrate that the models' predictions can be improved by accounting for the reflectance of the eggshell's calcium carbonate matrix. The establishment of these proximate links between pigmentation and colour will enable future tests of hypotheses on the functions of perceived avian eggshell colours that depend on eggshell chemistry. More generally, colour mixing models are not limited to avian eggshell colours but apply to any natural colour. Our approach illustrates how modelling can aid the understanding of constraints on phenotypic diversity. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

  7. Drosophila Eggshell Production: Identification of New Genes and Coordination by Pxt

    PubMed Central

    Tootle, Tina L.; Williams, Dianne; Hubb, Alexander; Frederick, Rebecca; Spradling, Allan

    2011-01-01

    Drosophila ovarian follicles complete development using a spatially and temporally controlled maturation process in which they resume meiosis and secrete a multi-layered, protective eggshell before undergoing arrest and/or ovulation. Microarray analysis revealed more than 150 genes that are expressed in a stage-specific manner during the last 24 hours of follicle development. These include all 30 previously known eggshell genes, as well as 19 new candidate chorion genes and 100 other genes likely to participate in maturation. Mutations in pxt, encoding a putative Drosophila cyclooxygenase, cause many transcripts to begin expression prematurely, and are associated with eggshell defects. Somatic activity of Pxt is required, as RNAi knockdown of pxt in the follicle cells recapitulates both the temporal expression and eggshell defects. One of the temporally regulated genes, cyp18a1, which encodes a cytochromome P450 protein mediating ecdysone turnover, is downregulated in pxt mutant follicles, and cyp18a1 mutation itself alters eggshell gene expression. These studies further define the molecular program of Drosophila follicle maturation and support the idea that it is coordinated by lipid and steroid hormonal signals. PMID:21637834

  8. Proteomics Analysis Identifies Orthologs of Human Chitinase-Like Proteins as Inducers of Tube Morphogenesis Defects in Drosophila melanogaster.

    PubMed

    Zimmerman, Sandra G; Merrihew, Gennifer E; MacCoss, Michael J; Berg, Celeste A

    2017-06-01

    Elevated levels of human chitinase-like proteins (CLPs) are associated with numerous chronic inflammatory diseases and several cancers, often correlating with poor prognosis. Nevertheless, there is scant knowledge of their function. The CLPs normally mediate immune responses and wound healing and, when upregulated, they can promote disease progression by remodeling tissue, activating signaling cascades, stimulating proliferation and migration, and by regulating adhesion. We identified Imaginal disc growth factors (Idgfs), orthologs of human CLPs CHI3L1, CHI3L2, and OVGP1, in a proteomics analysis designed to discover factors that regulate tube morphogenesis in a Drosophila melanogaster model of tube formation. We implemented a novel approach that uses magnetic beads to isolate a small population of specialized ovarian cells, cells that nonautonomously regulate morphogenesis of epithelial tubes that form and secrete eggshell structures called dorsal appendages (DAs). Differential mass spectrometry analysis of these cells detected elevated levels of four of the six Idgf family members (Idgf1, Idgf2, Idgf4, and Idgf6) in flies mutant for bullwinkle (bwk), which encodes a transcription factor and is a known regulator of DA-tube morphogenesis. We show that, during oogenesis, dysregulation of Idgfs (either gain or loss of function) disrupts the formation of the DA tubes. Previous studies demonstrate roles for Drosophila Idgfs in innate immunity, wound healing, and cell proliferation and motility in cell culture. Here, we identify a novel role for Idgfs in both normal and aberrant tubulogenesis processes. Copyright © 2017 by the Genetics Society of America.

  9. Direct visualization of identified and newly synthesized proteins in situ

    PubMed Central

    Dieck, Susanne tom; Kochen, Lisa; Hanus, Cyril; Bartnik, Ina; Nassim-Assir, Belquis; Merk, Katrin; Mosler, Thorsten; Garg, Sakshi; Bunse, Stefanie; Tirrell, David A.; Schuman, Erin M.

    2015-01-01

    Protein synthesis is a dynamic process to tune the cellular proteome to internal and external demands. Metabolic labeling approaches identify the general proteomic response but missing is a tool to visualize within cells specific newly synthesized proteins. Here we describe a technique that couples non-canonical amino acid tagging or puromycylation with the proximity-ligation assay to visualize identified newly synthesized proteins and monitor their origin, redistribution and turnover in situ. PMID:25775042

  10. Utilizing protein structure to identify non-random somatic mutations

    PubMed Central

    2013-01-01

    Background Human cancer is caused by the accumulation of somatic mutations in tumor suppressors and oncogenes within the genome. In the case of oncogenes, recent theory suggests that there are only a few key “driver” mutations responsible for tumorigenesis. As there have been significant pharmacological successes in developing drugs that treat cancers that carry these driver mutations, several methods that rely on mutational clustering have been developed to identify them. However, these methods consider proteins as a single strand without taking their spatial structures into account. We propose an extension to current methodology that incorporates protein tertiary structure in order to increase our power when identifying mutation clustering. Results We have developed iPAC (identification of Protein Amino acid Clustering), an algorithm that identifies non-random somatic mutations in proteins while taking into account the three dimensional protein structure. By using the tertiary information, we are able to detect both novel clusters in proteins that are known to exhibit mutation clustering as well as identify clusters in proteins without evidence of clustering based on existing methods. For example, by combining the data in the Protein Data Bank (PDB) and the Catalogue of Somatic Mutations in Cancer, our algorithm identifies new mutational clusters in well known cancer proteins such as KRAS and PI3KC α. Further, by utilizing the tertiary structure, our algorithm also identifies clusters in EGFR, EIF2AK2, and other proteins that are not identified by current methodology. The R package is available at: http://www.bioconductor.org/packages/2.12/bioc/html/iPAC.html. Conclusion Our algorithm extends the current methodology to identify oncogenic activating driver mutations by utilizing tertiary protein structure when identifying nonrandom somatic residue mutation clusters. PMID:23758891

  11. The location of protoporphyrin in the eggshell of brown-shelled eggs.

    PubMed

    Samiullah, S; Roberts, J R

    2013-10-01

    Protoporphyrin has been identified as the main eggshell pigment in brown-shelled eggs. However, there has been some uncertainty concerning the distribution of the pigment within the shell (and cuticle) in brown-shelled eggs. Most previous studies have suggested that the bulk of the shell pigment is deposited in the cuticle of the shell. The present study measured the levels of protoporphyrin in intact eggshells and in shells from which the cuticle had been removed, using eggs from flocks at 3 different ages. This enabled the calculation of the relative amount of protoporphyrin in the calcareous eggshell and the cuticle layer of the eggshell. The majority of the protoporphyrin pigment was located in the calcareous part of the eggshell (80-87%) with a minority contained within the cuticle (13-20%). These findings suggest that studies focused on maintenance of shell color in brown-shelled eggs need to consider the stage of egg formation at which the reduction in pigment deposition is occurring.

  12. How the oxygen isotope ratio of rain water influences the isotope ratio of chicken eggshell carbonate

    NASA Astrophysics Data System (ADS)

    Price, Gregory; Grimes, Stephen

    2015-04-01

    The stable oxygen isotope ratio of chicken eggshell carbonate was analysed from chicken eggs laid under free range, and organic farming regimes from across the UK. The eggshell carbonate oxygen isotope data shows a clear depletion in delta18O distribution from the southwest to the northeast. Although consistently offset by around 1 permil, the same isotopic distribution as that seen in eggshell carbonate is observed in the delta18O ratio of rainfall and groundwater from across the UK. This distribution is related to the Rayleigh distillation of rainfall driven by westerly winds across the UK landmass. The clear relationship observed between eggshell delta18O values and that of rainwater presumably reflects the nature of free range chickens which must be drinking locally derived rainwater and supplementing their diet and water intake with locally derived food. These results suggest that the oxygen isotope value of chicken eggshells can be used as a forensic tool to identify the locality that free range and organic eggs were laid within the UK. Furthermore, if suitable material is preserved in the archaeological and geological record then such a relationship can potentially be used to establish the oxygen isotope value of rainwater from which ancient and / or ancestral birds lived.

  13. Interaction of Proteins Identified in Human Thyroid Cells

    PubMed Central

    Pietsch, Jessica; Riwaldt, Stefan; Bauer, Johann; Sickmann, Albert; Weber, Gerhard; Grosse, Jirka; Infanger, Manfred; Eilles, Christoph; Grimm, Daniela

    2013-01-01

    Influence of gravity forces on the regulation of protein expression by healthy and malignant thyroid cells was studied with the aim to identify protein interactions. Western blot analyses of a limited number of proteins suggested a time-dependent regulation of protein expression by simulated microgravity. After applying free flow isoelectric focusing and mass spectrometry to search for differently expressed proteins by thyroid cells exposed to simulated microgravity for three days, a considerable number of candidates for gravi-sensitive proteins were detected. In order to show how proteins sensitive to microgravity could directly influence other proteins, we investigated all polypeptide chains identified with Mascot scores above 100, looking for groups of interacting proteins. Hence, UniProtKB entry numbers of all detected proteins were entered into the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and processed. The program indicated that we had detected various groups of interacting proteins in each of the three cell lines studied. The major groups of interacting proteins play a role in pathways of carbohydrate and protein metabolism, regulation of cell growth and cell membrane structuring. Analyzing these groups, networks of interaction could be established which show how a punctual influence of simulated microgravity may propagate via various members of interaction chains. PMID:23303277

  14. Shifts in bacterial communities of eggshells and antimicrobial activities in eggs during incubation in a ground-nesting passerine.

    PubMed

    Grizard, Stéphanie; Versteegh, Maaike A; Ndithia, Henry K; Salles, Joana F; Tieleman, B Irene

    2015-01-01

    Microbial invasion of egg contents is a cause of embryonic death. To counter infection risks, the embryo is protected physically by the eggshell and chemically by antimicrobial proteins. If microbial pressure drives embryo mortality, then females may have evolved, through natural selection, to adapt their immune investment into eggs. Although frequently hypothesized, this match between immune allocation and microorganisms has not been explored yet. To examine if correlations between microbes on eggs and immunity in eggs exist, we collected eggs from red-capped larks (Calandrella cinerea) and simultaneously examined their bacterial communities and antimicrobial components--pH, lysozyme and ovotransferrin--during natural incubation. Using molecular techniques, we find that bacterial communities are highly dynamic: bacterial abundance increases from the onset to late incubation, Shannon's α-diversity index increases during early incubation stages, and β-diversity analysis shows that communities from 1 day-old clutches are phylogenetically more similar to each other than the older ones. Regarding the antimicrobials, we notice a decrease of pH and lysozyme concentration, while ovotransferrin concentration increases during incubation. Interestingly, we show that two eggs of the same clutch share equivalent immune protection, independent of clutch age. Lastly, our results provide limited evidence of significant correlation between antimicrobial compounds and bacterial communities. Our study examined simultaneously, for the first time in a wild bird, the dynamics of bacterial communities present on eggshells and of albumen-associated antimicrobial components during incubation and investigated their relationship. However, the link between microorganisms and immunity of eggs remains to be elucidated further. Identifying invading microbes and their roles in embryo mortality, as well as understanding the role of the eggshell microbiome, might be key to better understand

  15. Shifts in Bacterial Communities of Eggshells and Antimicrobial Activities in Eggs during Incubation in a Ground-Nesting Passerine

    PubMed Central

    Grizard, Stéphanie; Versteegh, Maaike A.; Ndithia, Henry K.; Salles, Joana F.; Tieleman, B. Irene

    2015-01-01

    Microbial invasion of egg contents is a cause of embryonic death. To counter infection risks, the embryo is protected physically by the eggshell and chemically by antimicrobial proteins. If microbial pressure drives embryo mortality, then females may have evolved, through natural selection, to adapt their immune investment into eggs. Although frequently hypothesized, this match between immune allocation and microorganisms has not been explored yet. To examine if correlations between microbes on eggs and immunity in eggs exist, we collected eggs from red-capped larks (Calandrella cinerea) and simultaneously examined their bacterial communities and antimicrobial components—pH, lysozyme and ovotransferrin—during natural incubation. Using molecular techniques, we find that bacterial communities are highly dynamic: bacterial abundance increases from the onset to late incubation, Shannon’s α-diversity index increases during early incubation stages, and β-diversity analysis shows that communities from 1 day-old clutches are phylogenetically more similar to each other than the older ones. Regarding the antimicrobials, we notice a decrease of pH and lysozyme concentration, while ovotransferrin concentration increases during incubation. Interestingly, we show that two eggs of the same clutch share equivalent immune protection, independent of clutch age. Lastly, our results provide limited evidence of significant correlation between antimicrobial compounds and bacterial communities. Our study examined simultaneously, for the first time in a wild bird, the dynamics of bacterial communities present on eggshells and of albumen-associated antimicrobial components during incubation and investigated their relationship. However, the link between microorganisms and immunity of eggs remains to be elucidated further. Identifying invading microbes and their roles in embryo mortality, as well as understanding the role of the eggshell microbiome, might be key to better understand

  16. Nutritional supplement of hatchery eggshell membrane improves poultry performance and provides resistance against endotoxin stress

    USDA-ARS?s Scientific Manuscript database

    Eggshells are significant part of hatchery waste which consist of calcium carbonate crust, membranes, and proteins and peptides of embryonic origins along with other entrapped contaminants including microbes. We hypothesized that using this product as a nutritional additive in poultry diet may confe...

  17. Dietary supplementation with sodium bicarbonate improves calcium absorption and eggshell quality of laying hens during peak production.

    PubMed

    Jiang, M J; Zhao, J P; Jiao, H C; Wang, X J; Zhang, Q; Lin, H

    2015-01-01

    The advantage of supplemental sodium bicarbonate (NaHCO3) on eggshell quality in laying hens changes with age. Besides increasing calcium (Ca) secretion in the eggshell gland, it may improve Ca absorption in the intestine or kidney. Hy-Line Brown layers (n = 384), 25 weeks of age, were allocated to two treatment groups in two experiments, each of which included 4 replicates of 24 hens. Hens were fed a basal diet (control) or the basal diet containing 3 g NaHCO3 g/kg for 50 or 20 weeks in Experiment 1 or 2, respectively. A 24-h continuous lighting regimen was used to allow hens to consume the dietary supplements during the period of active eggshell formation. In Experiment 1, particularly from 25 to 50 weeks of age, and in Experiment 2, NaHCO3 supplementation favoured hen-d egg production at the expense of lower egg weight. The increased eggshell thickness should have nothing to do with the additional eggshell formation, because of the unchanged egg mass and daily eggshell calcification. At 35 weeks of age in both experiments, NaHCO3 supplementation increased duodenal expression of calbindin-d28k (CaBP-D28k) protein, contributing to higher Ca retention and balance. From 50 to 75 weeks of age in Experiment 1, the hens had little response to NaHCO3 supplementation and showed a negative trend on eggshell thickness and strength. It is concluded that dietary supplementation with 3 g NaHCO3 g/kg improves Ca absorption and eggshell quality of laying hens during the peak but not late production period, with the introduction of continuous lighting.

  18. Shotgun Proteomics Identifies Proteins Specific for Acute Renal Transplant Rejection

    SciTech Connect

    Sigdel, Tara K.; Kaushal, Amit; Gritsenko, Marina A.; Norbeck, Angela D.; Qian, Weijun; Xiao, Wenzhong; Camp, David G.; Smith, Richard D.; Sarwal, Minnie M.

    2010-01-04

    Acute rejection (AR) remains the primary risk factor for renal transplant outcome; development of non-invasive diagnostic biomarkers for AR is an unmet need. We used shotgun proteomics using LC-MS/MS and ELISA to analyze a set of 92 urine samples, from patients with AR, stable grafts (STA), proteinuria (NS), and healthy controls (HC). A total of 1446 urinary proteins were identified along with a number of NS specific, renal transplantation specific and AR specific proteins. Relative abundance of identified urinary proteins was measured by protein-level spectral counts adopting a weighted fold-change statistic, assigning increased weight for more frequently observed proteins. We have identified alterations in a number of specific urinary proteins in AR, primarily relating to MHC antigens, the complement cascade and extra-cellular matrix proteins. A subset of proteins (UMOD, SERPINF1 and CD44), have been further cross-validated by ELISA in an independent set of urine samples, for significant differences in the abundance of these urinary proteins in AR. This label-free, semi-quantitative approach for sampling the urinary proteome in normal and disease states provides a robust and sensitive method for detection of urinary proteins for serial, non-invasive clinical monitoring for graft rejection after

  19. Genetic parameters for eggshell traits in ostriches.

    PubMed

    Brand, Z; Cloete, S W P; Malecki, I A; Brown, C R

    2012-01-01

    1. A study was conducted on ~14000 ostrich eggs to estimate genetic parameters for eggshell traits that could benefit the hatchability of ostrich eggs. Traits measured included the number of pores on the eggshell, the average diameter of these pores, the total area of pores on the eggshell, permeability (pore area/shell thickness) and eggshell thickness. 2. Heritability estimates ranged from 0·16 for total pore area to 0·41 for the natural logarithm of pore count. The heritability estimates for water loss on 21 and 35 d (WL21 and WL35) of incubation were high at 0·23 and 0·24, respectively. 3. On a genetic level, pore count was negatively correlated with average pore diameter (-0·73) and shell thickness (-0·28), whereas it was positively correlated with total pore area (0·58), WL21 (0·24) and WL35 (0·34). The direct and maternal genetic correlations of pore count with total pore area (0·58) and permeability (0·59) were high and significant. Permeability was positively correlated to WL21 and WL35, both on the direct and maternal genetic levels. 4. The estimated genetic parameters indicate that it should be possible to select for the various eggshell traits in ostrich eggs, or for permeability and water loss. However, as a trait with an intermediate optimum, direct selection for permeability and other eggshell traits would not be straightforward, and the possible application of these results to improve hatchability of ostrich eggs in the future needs consideration.

  20. Biodegradation of thermoplastic starch/eggshell powder composites.

    PubMed

    Bootklad, Munlika; Kaewtatip, Kaewta

    2013-09-12

    Thermoplastic starch (TPS) was prepared using compression molding and chicken eggshell was used as a filler. The effect of the eggshell powder (EP) on the properties of TPS was compared with the effect of commercial calcium carbonate (CC). The organic compound on the surface of the eggshell powder acted as a coupling agent that resulted in a strong adhesion between the eggshell powder and the TPS matrix, as confirmed by SEM micrographs. The biodegradation was determined by the soil burial test. The TPS/EP composites were more rapidly degraded than the TPS/CC composites. In addition, the eggshell powder improved the water resistance and thermal stability of the TPS.

  1. The cuticle modulates ultraviolet reflectance of avian eggshells.

    PubMed

    Fecheyr-Lippens, Daphne C; Igic, Branislav; D'Alba, Liliana; Hanley, Daniel; Verdes, Aida; Holford, Mande; Waterhouse, Geoffrey I N; Grim, Tomas; Hauber, Mark E; Shawkey, Matthew D

    2015-05-11

    Avian eggshells are variedly coloured, yet only two pigments, biliverdin and protoporphyrin IX, are known to contribute to the dramatic diversity of their colours. By contrast, the contributions of structural or other chemical components of the eggshell are poorly understood. For example, unpigmented eggshells, which appear white to the human eye, vary in their ultraviolet (UV) reflectance, which may be detectable by birds. We investigated the proximate mechanisms for the variation in UV-reflectance of unpigmented bird eggshells using spectrophotometry, electron microscopy, chemical analyses, and experimental manipulations. We specifically tested how UV-reflectance is affected by the eggshell cuticle, the outermost layer of most avian eggshells. The chemical dissolution of the outer eggshell layers, including the cuticle, increased UV-reflectance for only eggshells that contained a cuticle. Our findings demonstrate that the outer eggshell layers, including the cuticle, absorb UV-light, probably because they contain higher levels of organic components and other chemicals, such as calcium phosphates, compared to the predominantly calcite-based eggshell matrix. These data highlight the need to examine factors other than the known pigments in studies of avian eggshell colour.

  2. The cuticle modulates ultraviolet reflectance of avian eggshells

    PubMed Central

    Fecheyr-Lippens, Daphne C.; Igic, Branislav; D'Alba, Liliana; Hanley, Daniel; Verdes, Aida; Holford, Mande; Waterhouse, Geoffrey I. N.; Grim, Tomas; Hauber, Mark E.; Shawkey, Matthew D.

    2015-01-01

    ABSTRACT Avian eggshells are variedly coloured, yet only two pigments, biliverdin and protoporphyrin IX, are known to contribute to the dramatic diversity of their colours. By contrast, the contributions of structural or other chemical components of the eggshell are poorly understood. For example, unpigmented eggshells, which appear white to the human eye, vary in their ultraviolet (UV) reflectance, which may be detectable by birds. We investigated the proximate mechanisms for the variation in UV-reflectance of unpigmented bird eggshells using spectrophotometry, electron microscopy, chemical analyses, and experimental manipulations. We specifically tested how UV-reflectance is affected by the eggshell cuticle, the outermost layer of most avian eggshells. The chemical dissolution of the outer eggshell layers, including the cuticle, increased UV-reflectance for only eggshells that contained a cuticle. Our findings demonstrate that the outer eggshell layers, including the cuticle, absorb UV-light, probably because they contain higher levels of organic components and other chemicals, such as calcium phosphates, compared to the predominantly calcite-based eggshell matrix. These data highlight the need to examine factors other than the known pigments in studies of avian eggshell colour. PMID:25964661

  3. Chicken eggshell as suitable calcium source at home.

    PubMed

    Brun, Lucas R; Lupo, Maela; Delorenzi, Damián A; Di Loreto, Verónica E; Rigalli, Alfredo

    2013-09-01

    Taken into consideration that the deficiency of calcium (Ca) in the diet is a common problem, the aim of this work was to study the chicken eggshell as Ca source at home. It was evaluated: (1) different mechanisms to process eggshells and find an easy way to determine the required amount of Ca at home and; (2) the flavor and the texture for eggshell fortified food. Chemical and mechanical methods of eggshell processing were evaluated. Changes in flavor and texture were evaluated in volunteers coordinated by a professional chef. A single eggshell contains 2.07 ± 0.18 g of Ca; therefore half an eggshell could provide the amount of Ca needed by adult human beings per day. The best way to use chicken eggshell as Ca dietary supplement is powdered to add to bread, pizza or spaghetti as there were small changes in texture and no changes in flavor.

  4. Localization of ligand binding site in proteins identified in silico.

    PubMed

    Brylinski, Michal; Kochanczyk, Marek; Broniatowska, Elzbieta; Roterman, Irena

    2007-07-01

    Knowledge-based models for protein folding assume that the early-stage structural form of a polypeptide is determined by the backbone conformation, followed by hydrophobic collapse. Side chain-side chain interactions, mostly of hydrophobic character, lead to the formation of the hydrophobic core, which seems to stabilize the structure of the protein in its natural environment. The fuzzy-oil-drop model is employed to represent the idealized hydrophobicity distribution in the protein molecule. Comparing it with the one empirically observed in the protein molecule reveals that they are not in agreement. It is shown in this study that the irregularity of hydrophobic distributions is aim-oriented. The character and strength of these irregularities in the organization of the hydrophobic core point to the specificity of a particular protein's structure/function. When the location of these irregularities is determined versus the idealized fuzzy-oil-drop, function-related areas in the protein molecule can be identified. The presented model can also be used to identify ways in which protein-protein complexes can possibly be created. Active sites can be predicted for any protein structure according to the presented model with the free prediction server at http://www.bioinformatics.cm-uj.krakow.pl/activesite. The implication based on the model presented in this work suggests the necessity of active presence of ligand during the protein folding process simulation.

  5. Thermal emissivity of avian eggshells.

    PubMed

    Björn, Lars Olof; Bengtson, Sven-Axel; Li, Shaoshan; Hecker, Christoph; Ullah, Saleem; Roos, Arne; Nilsson, Annica M

    2016-04-01

    The hypothesis has been tested that evolution has resulted in lower thermal emissivity of eggs of birds breeding openly in cold climates than of eggs of birds that nest under protective covering or in warmer climates. Directional thermal emissivity has been estimated from directional-hemispherical reflectance spectra. Due to several methodological difficulties the absolute emissivity is not accurately determined, but differences between species are obvious. Most notably, small waders of the genus Calidris, breeding in cold climates on the tundra, and in most cases with uniparental nest attendance, have low directional emissivity of their eggshells, about 0.92 when integration is carried out for wavelengths up to 16μm. Species belonging to Galloanserinae have the highest directional emissivity, about 0.96, of their eggs. No differences due to climate or breeding conditions were found within this group. Eggs of most other birds tested possess intermediate emissivity, but the values for Pica pica and Corvus corone cornix are as low as for Calidris. Large species-dependent differences in spectral reflectance were found at specific wavelengths. For instance, at 4.259μm the directional-hemispherical reflectance for galliforms range from 0.05 to 0.09, while for Fratercula arctica and Fulmarus glacialis it is about 0.3. The reflection peaks at 6.5 and 11.3μm due to calcite are differentially attenuated in different species. In conclusion, the hypothesis that evolution has resulted in lower thermal emissivity of bird eggs being exposed in cold climates is not supported by our results. The emissivity is not clearly related to nesting habits or climate, and it is unlikely that the small differences observed are ecologically important. The spectral differences between eggs that nevertheless exist should be taken into account when using infrared thermometers for estimating the surface temperature of avian eggs.

  6. Proteomic approaches to identifying carbonylated proteins in brain tissue.

    PubMed

    Linares, María; Marín-Garcíía, Patricia; Méndez, Darío; Puyet, Antonio; Diez, Amalia; Bautista, José M

    2011-04-01

    Oxidative stress plays a critical role in the pathogenesis of a number of diseases. The carbonyl end products of protein oxidation are among the most commonly measured markers of oxidation in biological samples. Protein carbonyl functional groups may be derivatized with 2,4-dinitrophenylhydrazine (DNPH) to render a stable 2,4-dinitrophenylhydrazone-protein (DNP-protein) and the carbonyl contents of individual proteins then determined by two-dimensional electrophoresis followed by immunoblotting using specific anti-DNP antibodies. Unfortunately, derivatization of proteins with DNPH could affect their mass spectrometry (MS) identification. This problem can be overcome using nontreated samples for protein identification. Nevertheless, derivatization could also affect their mobility, which might be solved by performing the derivatization step after the initial electrophoresis. Here, we compare two-dimensional redox proteome maps of mouse cerebellum acquired by performing the DNPH derivatization step before or after electrophoresis and detect differences in protein patterns. When the same approach is used for protein detection and identification, both methods were found to be useful to identify carbonylated proteins. However, whereas pre-DNPH derivatized proteins were successfully analyzed, high background staining complicated the analysis when the DNPH reaction was performed after transblotting. Comparative data on protein identification using both methods are provided.

  7. Leveraging protein quaternary structure to identify oncogenic driver mutations.

    PubMed

    Ryslik, Gregory A; Cheng, Yuwei; Modis, Yorgo; Zhao, Hongyu

    2016-03-22

    Identifying key "driver" mutations which are responsible for tumorigenesis is critical in the development of new oncology drugs. Due to multiple pharmacological successes in treating cancers that are caused by such driver mutations, a large body of methods have been developed to differentiate these mutations from the benign "passenger" mutations which occur in the tumor but do not further progress the disease. Under the hypothesis that driver mutations tend to cluster in key regions of the protein, the development of algorithms that identify these clusters has become a critical area of research. We have developed a novel methodology, QuartPAC (Quaternary Protein Amino acid Clustering), that identifies non-random mutational clustering while utilizing the protein quaternary structure in 3D space. By integrating the spatial information in the Protein Data Bank (PDB) and the mutational data in the Catalogue of Somatic Mutations in Cancer (COSMIC), QuartPAC is able to identify clusters which are otherwise missed in a variety of proteins. The R package is available on Bioconductor at: http://bioconductor.jp/packages/3.1/bioc/html/QuartPAC.html . QuartPAC provides a unique tool to identify mutational clustering while accounting for the complete folded protein quaternary structure.

  8. A multidimensional proteomic approach to identify hypertrophy-associated proteins.

    PubMed

    Lindsey, Merry L; Goshorn, Danielle K; Comte-Walters, Susana; Hendrick, Jennifer W; Hapke, Elizabeth; Zile, Michael R; Schey, Kevin

    2006-04-01

    Left ventricular hypertrophy (LVH) is a leading cause of congestive heart failure. The exact mechanisms that control cardiac growth and regulate the transition to failure are not fully understood, in part due to the lack of a complete inventory of proteins associated with LVH. We investigated the proteomic basis of LVH using the transverse aortic constriction model of pressure overload in mice coupled with a multidimensional approach to identify known and novel proteins that may be relevant to the development and maintenance of LVH. We identified 123 proteins that were differentially expressed during LVH, including LIM proteins, thioredoxin, myoglobin, fatty acid binding protein 3, the abnormal spindle-like microcephaly protein (ASPM), and cytoskeletal proteins such as actin and myosin. In addition, proteins with unknown functions were identified, providing new directions for future research in this area. We also discuss common pitfalls and strategies to overcome the limitations of current proteomic technologies. Together, the multidimensional approach provides insight into the proteomic changes that occur in the LV during hypertrophy.

  9. A critical evaluation of the utility of eggshells for estimating mercury concentrations in avian eggs

    USGS Publications Warehouse

    Peterson, Sarah; Ackerman, Joshua T.; Eagles-Smith, Collin A.; Hartman, C. Alex; Herzog, Mark P.

    2017-01-01

    Eggshells are a potential tool for non-lethally sampling contaminant concentrations in bird eggs, yet few studies have examined their utility to represent mercury exposure. We assessed mercury concentrations in eggshell components for 23 bird species and determined whether they correlated with total mercury (THg) in egg contents. We designed a multi-experiment analysis to examine how THg is partitioned into eggshell components, specifically hardened eggshells, material adhered to the eggshell, and inner eggshell membranes. THg concentrations in eggshells were much lower than in egg contents, and almost all of the THg within the eggshell was contained within material adhered to eggshells and inner eggshell membranes, and specifically not within calcium-rich hardened eggshells. Despite having very little mercury, THg concentrations in hardened eggshells had the strongest correlation with egg contents among all eggshell components. However, species with the same THg concentrations in eggshells had different THg concentrations in egg contents, indicating that there is no global predictive equation among species for the relationship between eggshell and egg content THg concentrations. Further, for all species, THg concentrations in eggshells decreased with relative embryo age. Although the majority of mercury in eggshells was contained within other eggshell components and not within hardened eggshells, THg in hardened eggshells can be used to estimate THg concentrations in egg contents, if embryo age and species are addressed.

  10. Effects of reducing dietary protein, methionine, choline, folic acid, and vitamin B12 during the late stages of the egg production cycle on performance and eggshell quality.

    PubMed

    Keshavarz, K

    2003-09-01

    A series of four experiments was conducted to determine whether-shell quality during the late stages of egg production can be improved by using diets that are effective in reducing egg size. The experiments involved dietary manipulation of protein, methionine, choline, folic acid, and vitamin B12. In experiment 1, reducing dietary protein in combination of reducing the dietary methionine and choline or this diet without supplemental folic acid and vitamin B12 resulted in reduced egg weight and improved shell quality. However, egg production also was drastically reduced. In experiment 2, reducing the dietary level of methionine, without adding supplemental choline, folic acid, and vitamin B12 reduced egg size and improved shell quality, but egg production was reduced as well. In this experiment reducing the dietary methionine without supplemental folic acid and vitamin B12 reduced egg size and improved shell quality with no adverse effect on egg production. In experiment 3, reducing the dietary level of methionine and choline or reducing the dietary level of choline, folic acid, and vitamin B12 reduced egg size and improved shell quality without adverse effects on egg production. On the other hand, reducing dietary methionine, folic acid, vitamin B12, and supplemental choline reduced egg weight and improved shell quality but lowered egg production. In experiment 4, reducing dietary methionine together with reducing choline and vitamin B12 reduced egg size and improved shell quality with no adverse effect on egg production. The results of this series of experiments generally indicate that certain manipulations of the combination of methionine, choline, folic acid, and vitamin B12 have the potential to reduce egg weight and improve shell quality without affecting egg production during the latter stages of the egg production cycle.

  11. Crystallization studies on avian eggshell membranes: implications for the molecular factors controlling eggshell formation.

    PubMed

    Wu, T M; Rodriguez, J P; Fink, D J; Carrino, D A; Blackwell, J; Caplan, A I; Heuer, A H

    1995-02-01

    The avian eggshell is a natural biopolymer and mineral composite. It is a very useful model for biomimetic mineralization, since it is among the fastest forming hard tissues known. Isolated eggshell membranes, which were demineralized in vitro, were used to investigate the in vitro modulation of CaCO3 crystal deposition by organic matrix materials. Crystallization on the demineralized eggshell membrane occurred almost exclusively at the peripheries of residual calcium reserve assemblies, which contain a high concentration of sulfur. Similar structures are observed for eggshell membranes after natural demineralization. The characteristic rhombohedral crystal morphologies of the calcite crystals grown in this in vitro system are much less regular when grown in the presence of organic matrix or partially purified dermatan sulfate proteoglycans obtained from the eggshell. The effect of these macromolecules on the morphology and size of CaCO3 crystals is concentration-dependent. These studies indicate the complexity of the molecular and ionic interactions involved in the initiation and formation of the eggshell, with the focus on the role of the organic matrix.

  12. What Does the Eggshell Cuticle Do? A Functional Comparison of Avian Eggshell Cuticles.

    PubMed

    D'Alba, Liliana; Torres, Roxana; Waterhouse, Geoffrey I N; Eliason, Chad; Hauber, Mark E; Shawkey, Matthew D

    The avian eggshell is a highly ordered structure with several layers (mammillae, palisades, and vertical crystal layer) composed of calcium carbonate (∼96%) and minerals within an organic matrix. The cuticle is a noncalcified layer that covers the eggshells of most bird species. Eggshells are multifunctional structures that have evolved in response to diverse embryonic requirements and challenges, including protection from microbial infection, nest flooding, and exposure to solar radiation. However, experimental evidence for these functions across diverse taxa is currently limited. Here we investigated the effects of nanosphere cuticles on (1) bacterial attachment and transshell penetration, (2) eggshell wettability, (3) water vapor conductance, and (4) regulation of ultraviolet (UV) reflectance in seven ground-nesting bird species. We found considerable interspecific variation in ultrastructure and chemical composition of cuticles. Experimental removal of the cuticle confirmed that all nanospheres were highly effective at decreasing attachment of bacteria to shell surfaces and at preventing bacterial penetration. Cuticles also greatly decreased the amount of UV reflected by eggshells. In species with particularly small nanospheres, gas exchange was reduced by the presence of cuticle. Our results support the hypothesis that microbes and solar UV radiation can cause strong selection on bird eggs but also show that we need a greater understanding about the effects of specific nesting conditions (e.g., hydric and gaseous milieu) on embryo well-being and eggshell structure variation.

  13. Fossil struthionid eggshells from Laetoli, Tanzania: Taxonomic and biostratigraphic significance

    NASA Astrophysics Data System (ADS)

    Harrison, Terry; Msuya, Charles P.

    2005-04-01

    Recent paleontological investigations at Laetoli and neighboring localities in northern Tanzania have produced a large collection of fossil ostrich eggshells from the Pliocene-aged Laetolil Beds (˜3.5-4.5 Ma) and Ndolanya Beds (˜2.6-2.7 Ma). A detailed analysis of the morphology of the eggshells and their taxonomic affinities indicates that two different species of Struthio are represented. In the Lower Laetolil Beds and in the Upper Laetolil Beds below Tuff 3 a new species is recognized— Struthio kakesiensis. This is replaced in the Upper Laetolil Beds by Struthio camelus, the modern species of ostrich. Since radiometric age determinations are available for the stratigraphic sequence at Laetoli, it is possible to precisely date the first appearance of S. camelus at ˜3.6-3.8 Ma. Comparisons of the Laetoli material with specimens from the well-dated sequences at Lothagam and Kanapoi in northern Kenya, allow the taxonomic and biochronological analysis to be extended back in time to the late Miocene. At about 6.5 Ma, Diamantornis and elephant birds were replaced in East Africa by ostriches belonging to the genus Struthio. Three time-successive species of ostriches are identified in the fossil record of East Africa, beginning with Struthio. cf. karingarabensis (˜6.5-4.2 Ma), followed by S. kakesiensis (˜4.5-3.6 Ma) and then S. camelus (˜3.8 Ma onwards). A similar sequence of taxa has previously been recorded from localities in Namibia, but at these sites there is no possibility to precisely calibrate the ages of the different species using radiometric dating. Nevertheless, the broadly similar evolutionary sequence and the close correspondence in inferred ages for the succession of species in East Africa and Namibia suggest that ostrich eggshells are a very useful tool for biochronological correlation of paleontological sites in sub-Saharan Africa.

  14. Exploiting genomic data to identify proteins involved in abalone reproduction.

    PubMed

    Mendoza-Porras, Omar; Botwright, Natasha A; McWilliam, Sean M; Cook, Mathew T; Harris, James O; Wijffels, Gene; Colgrave, Michelle L

    2014-08-28

    Aside from their critical role in reproduction, abalone gonads serve as an indicator of sexual maturity and energy balance, two key considerations for effective abalone culture. Temperate abalone farmers face issues with tank restocking with highly marketable abalone owing to inefficient spawning induction methods. The identification of key proteins in sexually mature abalone will serve as the foundation for a greater understanding of reproductive biology. Addressing this knowledge gap is the first step towards improving abalone aquaculture methods. Proteomic profiling of female and male gonads of greenlip abalone, Haliotis laevigata, was undertaken using liquid chromatography-mass spectrometry. Owing to the incomplete nature of abalone protein databases, in addition to searching against two publicly available databases, a custom database comprising genomic data was used. Overall, 162 and 110 proteins were identified in females and males respectively with 40 proteins common to both sexes. For proteins involved in sexual maturation, sperm and egg structure, motility, acrosomal reaction and fertilization, 23 were identified only in females, 18 only in males and 6 were common. Gene ontology analysis revealed clear differences between the female and male protein profiles reflecting a higher rate of protein synthesis in the ovary and higher metabolic activity in the testis. A comprehensive mass spectrometry-based analysis was performed to profile the abalone gonad proteome providing the foundation for future studies of reproduction in abalone. Key proteins involved in both reproduction and energy balance were identified. Genomic resources were utilised to build a database of molluscan proteins yielding >60% more protein identifications than in a standard workflow employing public protein databases. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. S-linked protein homocysteinylation: identifying targets based on structural, physicochemical and protein-protein interactions of homocysteinylated proteins.

    PubMed

    Silla, Yumnam; Sundaramoorthy, Elayanambi; Talwar, Puneet; Sengupta, Shantanu

    2013-05-01

    An elevated level of homocysteine, a thiol-containing amino acid is associated with a wide spectrum of disease conditions. A majority (>80 %) of the circulating homocysteine exist in protein-bound form. Homocysteine can bind to free cysteine residues in the protein or could cleave accessible cysteine disulfide bonds via thiol disulfide exchange reaction. Binding of homocysteine to proteins could potentially alter the structure and/or function of the protein. To date only 21 proteins have been experimentally shown to bind homocysteine. In this study we attempted to identify other proteins that could potentially bind to homocysteine based on the criteria that such proteins will have significant 3D structural homology with the proteins that have been experimentally validated and have solvent accessible cysteine residues either with high dihedral strain energy (for cysteine-cysteine disulfide bonds) or low pKa (for free cysteine residues). This analysis led us to the identification of 78 such proteins of which 68 proteins had 154 solvent accessible disulfide cysteine pairs with high dihedral strain energy and 10 proteins had free cysteine residues with low pKa that could potentially bind to homocysteine. Further, protein-protein interaction network was built to identify the interacting partners of these putative homocysteine binding proteins. We found that the 21 experimentally validated proteins had 174 interacting partners while the 78 proteins identified in our analysis had 445 first interacting partners. These proteins are mainly involved in biological activities such as complement and coagulation pathway, focal adhesion, ECM-receptor, ErbB signalling and cancer pathways, etc. paralleling the disease-specific attributes associated with hyperhomocysteinemia.

  16. Identifying folding nucleus based on residue contact networks of proteins.

    PubMed

    Li, Jie; Wang, Jun; Wang, Wei

    2008-06-01

    In the native structure of a protein, all the residues are tightly parked together in a specific order following its folding and every residue contacts with some spatially neighbor residues. A residue contact network can be constructed by defining the residues as nodes and the native contacts as edges. During the folding of small single-domain proteins, there is a set of contacts (or bonds), defined as the folding nucleus (FN), which is formed around the transition state, i.e., a rate-limiting barrier located at about the middle between the unfolded states and the native state on the free energy landscape. Such a FN plays an essential role in the folding dynamics and the residues, which form the related contacts called as folding nucleus residues (FNRs). In this work, the FNRs in proteins are identified by using quantities which characterize the topology of residue contact networks of proteins. By comparing the specificities of residues with the network quantities K(R), L(R), and D(R), up to 90% FNRs of six typical proteins found experimentally are identified. It is found that the FNRs behave the full-closeness centrals rather than degree or closeness centers in the residue contact network, implying that they are important to the folding cooperativity of proteins. Our study shows that the FNRs can be identified solely from the native structures of proteins based on the analysis of residue contact network without any knowledge of the transition state ensemble. (c) 2008 Wiley-Liss, Inc.

  17. Proteomic Analysis of the Soybean Symbiosome Identifies New Symbiotic Proteins*

    PubMed Central

    Clarke, Victoria C.; Loughlin, Patrick C.; Gavrin, Aleksandr; Chen, Chi; Brear, Ella M.; Day, David A.; Smith, Penelope M.C.

    2015-01-01

    Legumes form a symbiosis with rhizobia in which the plant provides an energy source to the rhizobia bacteria that it uses to fix atmospheric nitrogen. This nitrogen is provided to the legume plant, allowing it to grow without the addition of nitrogen fertilizer. As part of the symbiosis, the bacteria in the infected cells of a new root organ, the nodule, are surrounded by a plant-derived membrane, the symbiosome membrane, which becomes the interface between the symbionts. Fractions containing the symbiosome membrane (SM) and material from the lumen of the symbiosome (peribacteroid space or PBS) were isolated from soybean root nodules and analyzed using nongel proteomic techniques. Bicarbonate stripping and chloroform-methanol extraction of isolated SM were used to reduce complexity of the samples and enrich for hydrophobic integral membrane proteins. One hundred and ninety-seven proteins were identified as components of the SM, with an additional fifteen proteins identified from peripheral membrane and PBS protein fractions. Proteins involved in a range of cellular processes such as metabolism, protein folding and degradation, membrane trafficking, and solute transport were identified. These included a number of proteins previously localized to the SM, such as aquaglyceroporin nodulin 26, sulfate transporters, remorin, and Rab7 homologs. Among the proteome were a number of putative transporters for compounds such as sulfate, calcium, hydrogen ions, peptide/dicarboxylate, and nitrate, as well as transporters for which the substrate is not easy to predict. Analysis of the promoter activity for six genes encoding putative SM proteins showed nodule specific expression, with five showing expression only in infected cells. Localization of two proteins was confirmed using GFP-fusion experiments. The data have been deposited to the ProteomeXchange with identifier PXD001132. This proteome will provide a rich resource for the study of the legume-rhizobium symbiosis. PMID

  18. Automatically identifying gene/protein terms in MEDLINE abstracts.

    PubMed

    Yu, Hong; Hatzivassiloglou, Vasileios; Rzhetsky, Andrey; Wilbur, W John

    2002-01-01

    Natural language processing (NLP) techniques are used to extract information automatically from computer-readable literature. In biology, the identification of terms corresponding to biological substances (e.g., genes and proteins) is a necessary step that precedes the application of other NLP systems that extract biological information (e.g., protein-protein interactions, gene regulation events, and biochemical pathways). We have developed GPmarkup (for "gene/protein-full name mark up"), a software system that automatically identifies gene/protein terms (i.e., symbols or full names) in MEDLINE abstracts. As a part of marking up process, we also generated automatically a knowledge source of paired gene/protein symbols and full names (e.g., LARD for lymphocyte associated receptor of death) from MEDLINE. We found that many of the pairs in our knowledge source do not appear in the current GenBank database. Therefore our methods may also be used for automatic lexicon generation. GPmarkup has 73% recall and 93% precision in identifying and marking up gene/protein terms in MEDLINE abstracts. A random sample of gene/protein symbols and full names and a sample set of marked up abstracts can be viewed at http://www.cpmc.columbia.edu/homepages/yuh9001/GPmarkup/. Contact. hy52@columbia.edu. Voice: 212-939-7028; fax: 212-666-0140.

  19. 40 CFR 174.529 - Bacillus thuringiensis modified Cry1Ab protein as identified under OECD Unique Identifier SYN...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... protein as identified under OECD Unique Identifier SYN-IR67B-1 in cotton; exemption from the requirement... Tolerance Exemptions § 174.529 Bacillus thuringiensis modified Cry1Ab protein as identified under OECD... Bacillus thuringiensis modified Cry1Ab protein as identified under OECD Unique Identifier SYN-IR67B-1 are...

  20. Advanced oxidation process sanitization of eggshell surfaces.

    PubMed

    Gottselig, Steven M; Dunn-Horrocks, Sadie L; Woodring, Kristy S; Coufal, Craig D; Duong, Tri

    2016-06-01

    The microbial quality of eggs entering the hatchery represents an important critical control point for biosecurity and pathogen reduction programs in integrated poultry production. The development of safe and effective interventions to reduce microbial contamination on the surface of eggs will be important to improve the overall productivity and microbial food safety of poultry and poultry products. The hydrogen peroxide (H2O2) and ultraviolet (UV) light advanced oxidation process is a potentially important alternative to traditional sanitizers and disinfectants for egg sanitation. The H2O2/UV advanced oxidation process was demonstrated previously to be effective in reducing surface microbial contamination on eggs. In this study, we evaluated treatment conditions affecting the efficacy of H2O2/UV advanced oxidation in order to identify operational parameters for the practical application of this technology in egg sanitation. The effect of the number of application cycles, UV intensity, duration of UV exposure, and egg rotation on the recovery of total aerobic bacteria from the surface of eggs was evaluated. Of the conditions evaluated, we determined that reduction of total aerobic bacteria from naturally contaminated eggs was optimized when eggs were sanitized using 2 repeated application cycles with 5 s exposure to 14 mW cm(-2) UV light, and that rotation of the eggs between application cycles was unnecessary. Additionally, using these optimized conditions, the H2O2/UV process reduced Salmonella by greater than 5 log10 cfu egg(-1) on the surface of experimentally contaminated eggs. This study demonstrates the potential for practical application of the H2O2/UV advanced oxidation process in egg sanitation and its effectiveness in reducing Salmonella on eggshell surfaces.

  1. Diverse fragment clustering and water exclusion identify protein hot spots.

    PubMed

    Kulp, John L; Kulp, John L; Pompliano, David L; Guarnieri, Frank

    2011-07-20

    Simulated annealing of chemical potential located the highest affinity positions of eight organic probes and water on eight static structures of hen egg white lysozyme (HEWL) in various conformational states. In all HELW conformations, a diverse set of organic probes clustered in the known binding site (hot spot). Fragment clusters at other locations were excluded by tightly-bound waters so that only the hot-spot cluster remained in each case. The location of the hot spot was correctly predicted irrespective of the protein conformation and without accounting for protein flexibility during the simulations. Any one of the static structures could have been used to locate the hot spot. A site on a protein where a diversity of organic probes is calculated to cluster, but where water specifically does not bind, identifies a potential small-molecule binding site or protein-protein interaction hot spot.

  2. Proteomic Approach to Identify Nuclear Proteins in Wheat Grain.

    PubMed

    Bancel, Emmanuelle; Bonnot, Titouan; Davanture, Marlène; Branlard, Gérard; Zivy, Michel; Martre, Pierre

    2015-10-02

    The nuclear proteome of the grain of the two cultivated wheat species Triticum aestivum (hexaploid wheat; genomes A, B, and D) and T. monococcum (diploid wheat; genome A) was analyzed in two early stages of development using shotgun-based proteomics. A procedure was optimized to purify nuclei, and an improved protein sample preparation was developed to efficiently remove nonprotein substances (starch and nucleic acids). A total of 797 proteins corresponding to 528 unique proteins were identified, 36% of which were classified in functional groups related to DNA and RNA metabolism. A large number (107 proteins) of unknown functions and hypothetical proteins were also found. Some identified proteins may be multifunctional and may present multiple localizations. On the basis of the MS/MS analysis, 368 proteins were present in the two species, and in two stages of development, some qualitative differences between species and stages of development were also found. All of these data illustrate the dynamic function of the grain nucleus in the early stages of development.

  3. Proteins identified from care solution extractions of silicone hydrogels.

    PubMed

    Emch, Andrew J; Nichols, Jason J

    2009-02-01

    The purpose of this study was to investigate the quantity and identify the proteins extracted from two different types of silicone hydrogel contact lenses by several multipurpose care solutions after 1 day of wear. Ten subjects were recruited to wear galyfilcon A lenses (Acuvue Advance, Vistakon) followed by lotrafilcon B lenses (O2 Optix, CIBA Vision) each for four consecutive days. Each day, subjects inserted a new pair of lenses for 8 h of wear after which both lenses were removed using forceps (lenses were not rubbed or rinsed after removal). Lenses were pooled in one of four commercially available care solutions for a 24-h soak followed by precipitation, resuspension in water, and quantification by Bradford assay and identification by mass spectrometry. Protein recovery from care solutions was as follows (quantities are in microg/lens): AQuify (galyfilcon A: 0.56, lotrafilcon B: 1.24), Complete MoisturePlus (galyfilcon A: 1.44, lotrafilcon B: 1.47), Opti-Free Express (galyfilcon A: 2.31, lotrafilcon B: 5.67), and ReNu MoistureLoc (galyfilcon A: 1.17, lotrafilcon B: 4.38). For each care solution, greater quantities of protein were removed from lotrafilcon B (3.19 +/- 2.19 microg/lens) than from galyfilcon A (1.37 +/- 0.72 microg/lens). Lactoferrin, lysozyme, and lipocalin were the most commonly identified, whereas various keratin compounds and other unique proteins were also detected. Opti-Free Express was consistently associated with the more efficient removal of proteins from these silicone hydrogels. More total protein was removed from lotrafilcon B than from galyfilcon A (approximately 2 x more protein) for all four care solutions, and 12 total unique protein species were recovered from galyfilcon A, whereas only 10 were recovered from lotrafilcon B. The higher quantities of protein extracted from lotrafilcon B may be due to stronger protein binding with this material and/or to differences in solution efficacy.

  4. SitesIdentify: a protein functional site prediction tool

    PubMed Central

    2009-01-01

    Background The rate of protein structures being deposited in the Protein Data Bank surpasses the capacity to experimentally characterise them and therefore computational methods to analyse these structures have become increasingly important. Identifying the region of the protein most likely to be involved in function is useful in order to gain information about its potential role. There are many available approaches to predict functional site, but many are not made available via a publicly-accessible application. Results Here we present a functional site prediction tool (SitesIdentify), based on combining sequence conservation information with geometry-based cleft identification, that is freely available via a web-server. We have shown that SitesIdentify compares favourably to other functional site prediction tools in a comparison of seven methods on a non-redundant set of 237 enzymes with annotated active sites. Conclusion SitesIdentify is able to produce comparable accuracy in predicting functional sites to its closest available counterpart, but in addition achieves improved accuracy for proteins with few characterised homologues. SitesIdentify is available via a webserver at http://www.manchester.ac.uk/bioinformatics/sitesidentify/ PMID:19922660

  5. Immobilization of the urease on eggshell membrane and its application in biosensor.

    PubMed

    D'Souza, S F; Kumar, Jitendra; Jha, Sandeep Kumar; Kubal, B S

    2013-03-01

    Eggshell membrane is a natural material, essentially made up of protein fibers having flexibility in the aqueous solution and possessing gas and water permeability. It is used as a biomembrane for immobilization of urease for the development of a potentiometric urea biosensor. Eggshell membrane was treated with polyethyleneimine (PEI) to impart polycation characteristics. Urease was immobilized on the PEI treated eggshell membrane through adsorption. SEM study was carried out to observe the changes in surface morphology after immobilization. FTIR study of membrane was carried out to observe the changes in IR spectra after immobilization of enzyme. Immobilized membrane was associated with ammonium ion selective electrode. Biosensor exhibited sigmoidal responses for the urea concentration range from 0.5 to 10mM. The response time of the biosensor was 120 s. A single membrane was reused for 270 reactions without loss of activity. The urease-eggshell membranes were stable for 2 months when stored in buffer even at room temperature. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Identifying protein complexes in protein-protein interaction networks by using clique seeds and graph entropy.

    PubMed

    Chen, Bolin; Shi, Jinhong; Zhang, Shenggui; Wu, Fang-Xiang

    2013-01-01

    The identification of protein complexes plays a key role in understanding major cellular processes and biological functions. Various computational algorithms have been proposed to identify protein complexes from protein-protein interaction (PPI) networks. In this paper, we first introduce a new seed-selection strategy for seed-growth style algorithms. Cliques rather than individual vertices are employed as initial seeds. After that, a result-modification approach is proposed based on this seed-selection strategy. Predictions generated by higher order clique seeds are employed to modify results that are generated by lower order ones. The performance of this seed-selection strategy and the result-modification approach are tested by using the entropy-based algorithm, which is currently the best seed-growth style algorithm to detect protein complexes from PPI networks. In addition, we investigate four pairs of strategies for this algorithm in order to improve its accuracy. The numerical experiments are conducted on a Saccharomyces cerevisiae PPI network. The group of best predictions consists of 1711 clusters, with the average f-score at 0.68 after removing all similar and redundant clusters. We conclude that higher order clique seeds can generate predictions with higher accuracy and that our improved entropy-based algorithm outputs more reasonable predictions than the original one.

  7. Irregularly calcified eggs and eggshells of Caiman latirostris (Alligatoridae: Crocodylia)

    NASA Astrophysics Data System (ADS)

    Fernández, Mariela Soledad; Simoncini, Melina Soledad; Dyke, Gareth

    2013-05-01

    We describe irregularly calcified egg and eggshell morphologies for the first time in nests of the broad-snouted caiman, Caiman latirostris. Research is based on detailed descriptions of 270 eggs from a total sample of 46,800 collected between 2005 and 2011 in Santa Fe Province, Argentina, and encompasses animals from both natural habitats and held in captivity. We discuss possible reasons for the occurrence of eggs with different mineralisation patterns in our extensive C. latirostris field sample and its conservation significance; the chemistry of egg laying in amniotes is sensitive to environmental contamination which, in turn, has biological implications. Based on our egg sample, we identify two caiman eggshell abnormalities: (1) regularly calcified eggs with either calcitic nodules or superficial wrinkles at one egg end and (2) irregularly calcified eggs with structural gaps that weaken the shell. Some recently laid clutches we examined included eggs with most of the shell broken and detached from the flexible membrane. Most type 1 regularly calcified eggs lost their initial calcified nodules during incubation, suggesting that these deposits do not affect embryo survival rates. In contrast, irregularly calcified caiman eggs have a mean hatching success rate of 8.9 % (range 0-38 %) across our sample compared to a mean normal success of 75 %. Most irregularly calcified caiman eggs probably die because of infections caused by fungi and bacteria in the organic nest material, although another possible explanation that merits further investigation could be an increase in permeability, leading to embryo dehydration.

  8. Identifying the singleplex and multiplex proteins based on transductive learning for protein subcellular localization prediction.

    PubMed

    Cao, Junzhe; Liu, Wenqi; He, Jianjun; Gu, Hong

    2013-07-01

    A new method is proposed to identify whether a query protein is singleplex or multiplex for improving the quality of protein subcellular localization prediction. Based on the transductive learning technique, this approach utilizes the information from the both query proteins and known proteins to estimate the subcellular location number of every query protein so that the singleplex and multiplex proteins can be recognized and distinguished. Each query protein is then dealt with by a targeted single-label or multi-label predictor to achieve a high-accuracy prediction result. We assess the performance of the proposed approach by applying it to three groups of protein sequences datasets. Simulation experiments show that the proposed approach can effectively identify the singleplex and multiplex proteins. Through a comparison, the reliably of this method for enhancing the power of predicting protein subcellular localization can also be verified.

  9. Collection and separation of Aedes taeniorhynchus eggshells from mangrove soil.

    PubMed

    Ritchie, S A; Addison, D S

    1991-03-01

    Two methods to separate eggshells of Aedes taeniorhynchus from mangrove soil were compared. Selective sieving, using nested sieves with 0.185 and 0.170-mm screen openings, and water flotation both removed over 99% of the soil. However, water flotation recovered a significantly greater percentage of eggshells (62% vs. 34%). There was no significant difference in the recovery rate of viable eggs and new and old eggshells using water flotation.

  10. Identifying Antioxidant Proteins by Using Optimal Dipeptide Compositions.

    PubMed

    Feng, Pengmian; Chen, Wei; Lin, Hao

    2016-06-01

    Antioxidant proteins are a kind of molecules that can terminate cellular and DNA damages caused by free radical intermediates. The use of antioxidant proteins for prevention of diseases has been intensively studied in recent years. Thus, accurate identification of antioxidant proteins is essential for understanding their roles in pharmacology. In this study, a support vector machine-based predictor called AodPred was developed for identifying antioxidant proteins. In this predictor, the sequence was formulated by using the optimal 3-gap dipeptides obtained by using feature selection method. It was observed by jackknife cross-validation test that AodPred can achieve an overall accuracy of 74.79 % in identifying antioxidant proteins. As a user-friendly tool, AodPred is freely accessible at http://lin.uestc.edu.cn/server/AntioxiPred . To maximize the convenience of the vast majority of experimental scientists, a step-by-step guide is provided on how to use the web server to obtain the desired results.

  11. Eggshell waste as catalyst: A review.

    PubMed

    Laca, Amanda; Laca, Adriana; Díaz, Mario

    2017-07-15

    Agricultural wastes are some of the most emerging problems in food industries because of their disposal cost. However, it is also an opportunity for the bioeconomy society if new uses for these residual materials can be found. Eggshells, considered a hazardous waste by UE regulations, are discarded, amounting hundreds of thousands of tonnes worldwide. This egg processing waste is a valuable source material, which can be used in different fields such as fodder or fertilizer production. Additionally, this residue offers interesting characteristics to be used in other applications, like its employment as an environment-friendly catalyst. In the present review we provide a global view of eggshell waste uses as catalyst in different processes. According to reviewed researching works, a huge variety of added value products can be obtained by using this catalyst which emphasised the interest of further investigations in order to widen the possible uses of this cheap green catalyst. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Crystal orientation regulation in ostrich eggshells

    NASA Astrophysics Data System (ADS)

    Feng, Q. L.; Zhu, X.; Li, H. D.; Kim, T. N.

    2001-12-01

    The structure and crystallographic orientation of different layers (the cone layer, palisade layer and the crystal layer) in ostrich eggshells were studied by scanning electron microscope (SEM), X-ray diffraction (XRD) and transmission electron microscope (TEM) with selected area electron diffraction (SAED). It is found that the degree of 0 0 1 preferred orientation enhanced from inner to outer ostrich eggshells. A crystallographic orientation regulation was observed for the first time. The adjacent 1-5 calcite crystals with the same three-dimensional orientation are proposed to form a crystalline cluster with the size of several microns. The a-axes in the neighboring clusters orient from several degrees to tens of degrees.

  13. The eggshell is required for meiotic fidelity, polar-body extrusion and polarization of the C. elegans embryo

    PubMed Central

    Johnston, Wendy L; Krizus, Aldis; Dennis, James W

    2006-01-01

    Background Fertilization restores the diploid state and begins the process by which the single-cell oocyte is converted into a polarized, multicellular organism. In the nematode, Caenorhabditis elegans, two of the earliest events following fertilization are secretion of the chitinous eggshell and completion of meiosis, and in this report we demonstrate that the eggshell is essential for multiple developmental events at the one-cell stage. Results We show that the GLD (Germline differentiation abnormal)-1-regulated hexosamine pathway enzyme, glucosamine-6-phosphate N-acetyltransferase (GNA)-2, is required for synthesis of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), the substrate for eggshell chitin synthesis by chitin synthase-1 (CHS-1). Furthermore, while chs-1(RNAi) or combined RNAi with the chitin-binding proteins, CEJ-1 and B0280.5, does not interfere with normal meiotic timing, lagging chromosomes are observed at meiosis, and polar-body extrusion fails. We also demonstrate that chitin, and either CEJ-1 or B0280.5, are essential for the osmotic/permeability barrier and for movement of the sperm pronucleus/centrosome complex to the cortex, which is associated with the initiation of polarization. Conclusion Our results indicate that the eggshell is required in single-cell C. elegans development, playing an essential role in multiple actin-dependent early events. Furthermore, the earliest meiotic roles precede osmotic barrier formation, indicating that the role of the eggshell is not limited to generation of the osmotic barrier. PMID:17042944

  14. Identifying Unexpected Therapeutic Targets via Chemical-Protein Interactome

    PubMed Central

    Yang, Lun; Chen, Jian; Shi, Leming; Hudock, Michael P.; Wang, Kejian; He, Lin

    2010-01-01

    Drug medications inevitably affect not only their intended protein targets but also other proteins as well. In this study we examined the hypothesis that drugs that share the same therapeutic effect also share a common therapeutic mechanism by targeting not only known drug targets, but also by interacting unexpectedly on the same cryptic targets. By constructing and mining an Alzheimer's disease (AD) drug-oriented chemical-protein interactome (CPI) using a matrix of 10 drug molecules known to treat AD towards 401 human protein pockets, we found that such cryptic targets exist. We recovered from CPI the only validated therapeutic target of AD, acetylcholinesterase (ACHE), and highlighted several other putative targets. For example, we discovered that estrogen receptor (ER) and histone deacetylase (HDAC), which have recently been identified as two new therapeutic targets of AD, might already have been targeted by the marketed AD drugs. We further established that the CPI profile of a drug can reflect its interacting character towards multi-protein sets, and that drugs with the same therapeutic attribute will share a similar interacting profile. These findings indicate that the CPI could represent the landscape of chemical-protein interactions and uncover “behind-the-scenes” aspects of the therapeutic mechanisms of existing drugs, providing testable hypotheses of the key nodes for network pharmacology or brand new drug targets for one-target pharmacology paradigm. PMID:20221449

  15. Identifying Unstable Regions of Proteins Involved in Misfolding Diseases

    NASA Astrophysics Data System (ADS)

    Guest, Will; Cashman, Neil; Plotkin, Steven

    2009-05-01

    Protein misfolding is a necessary step in the pathogenesis of many diseases, including Creutzfeldt-Jakob disease (CJD) and familial amyotrophic lateral sclerosis (fALS). Identifying unstable structural elements in their causative proteins elucidates the early events of misfolding and presents targets for inhibition of the disease process. An algorithm was developed to calculate the Gibbs free energy of unfolding for all sequence-contiguous regions of a protein using three methods to parameterize energy changes: a modified G=o model, changes in solvent-accessible surface area, and all-atoms molecular dynamics. The entropic effects of disulfide bonds and post-translational modifications are treated analytically. It incorporates a novel method for finding local dielectric constants inside a protein to accurately handle charge effects. We have predicted the unstable parts of prion protein and superoxide dismutase 1, the proteins involved in CJD and fALS respectively, and have used these regions as epitopes to prepare antibodies that are specific to the misfolded conformation and show promise as therapeutic agents.

  16. Eggshell spottiness reflects maternally transferred antibodies in blue tits.

    PubMed

    Holveck, Marie-Jeanne; Grégoire, Arnaud; Staszewski, Vincent; Guerreiro, Romain; Perret, Philippe; Boulinier, Thierry; Doutrelant, Claire

    2012-01-01

    Blue-green and brown-spotted eggshells in birds have been proposed as sexual signals of female physiological condition and egg quality, reflecting maternal investment in the egg. Testing this hypothesis requires linking eggshell coloration to egg content, which is lacking for brown protoporphyrin-based pigmentation. As protoporphyrins can induce oxidative stress, and a large amount in eggshells should indicate either high female and egg quality if it reflects the female's high oxidative tolerance, or conversely poor quality if it reflects female physiological stress. Different studies supported either predictions but are difficult to compare given the methodological differences in eggshell-spottiness measurements. Using the blue tit Cyanistes caeruleus as a model species, we aimed at disentangling both predictions in testing if brown-spotted eggshell could reflect the quality of maternal investment in antibodies and carotenoids in the egg, and at improving between-study comparisons in correlating several common measurements of eggshell coloration (spectral and digital measures, spotted surface, pigmentation indices). We found that these color variables were weakly correlated highlighting the need for comparable quantitative measurements between studies and for multivariate regressions incorporating several eggshell-color characteristics. When evaluating the potential signaling function of brown-spotted eggshells, we thus searched for the brown eggshell-color variables that best predicted the maternal transfer of antibodies and carotenoids to egg yolks. We also tested the effects of several parental traits and breeding parameters potentially affecting this transfer. While eggshell coloration did not relate to yolk carotenoids, the eggs with larger and less evenly-distributed spots had higher antibody concentrations, suggesting that both the quantity and distribution of brown pigments reflected the transfer of maternal immune compounds in egg yolks. As yolk antibody

  17. Eggshell Spottiness Reflects Maternally Transferred Antibodies in Blue Tits

    PubMed Central

    Holveck, Marie-Jeanne; Grégoire, Arnaud; Staszewski, Vincent; Guerreiro, Romain; Perret, Philippe; Boulinier, Thierry; Doutrelant, Claire

    2012-01-01

    Blue-green and brown-spotted eggshells in birds have been proposed as sexual signals of female physiological condition and egg quality, reflecting maternal investment in the egg. Testing this hypothesis requires linking eggshell coloration to egg content, which is lacking for brown protoporphyrin-based pigmentation. As protoporphyrins can induce oxidative stress, and a large amount in eggshells should indicate either high female and egg quality if it reflects the female's high oxidative tolerance, or conversely poor quality if it reflects female physiological stress. Different studies supported either predictions but are difficult to compare given the methodological differences in eggshell-spottiness measurements. Using the blue tit Cyanistes caeruleus as a model species, we aimed at disentangling both predictions in testing if brown-spotted eggshell could reflect the quality of maternal investment in antibodies and carotenoids in the egg, and at improving between-study comparisons in correlating several common measurements of eggshell coloration (spectral and digital measures, spotted surface, pigmentation indices). We found that these color variables were weakly correlated highlighting the need for comparable quantitative measurements between studies and for multivariate regressions incorporating several eggshell-color characteristics. When evaluating the potential signaling function of brown-spotted eggshells, we thus searched for the brown eggshell-color variables that best predicted the maternal transfer of antibodies and carotenoids to egg yolks. We also tested the effects of several parental traits and breeding parameters potentially affecting this transfer. While eggshell coloration did not relate to yolk carotenoids, the eggs with larger and less evenly-distributed spots had higher antibody concentrations, suggesting that both the quantity and distribution of brown pigments reflected the transfer of maternal immune compounds in egg yolks. As yolk antibody

  18. An in vivo platform for identifying inhibitors of protein aggregation

    PubMed Central

    Mahood, Rachel A.; Jackson, Matthew P.; Revill, Charlotte H.; Foster, Richard J.; Smith, D. Alastair; Ashcroft, Alison E.; Brockwell, David J.; Radford, Sheena E.

    2015-01-01

    Protein aggregation underlies an array of human diseases, yet only one small molecule therapeutic has been successfully developed to date. Here, we introduce an in vivo system, based on a β-lactamase tripartite fusion construct, capable of identifying aggregation-prone sequences in the periplasm of Escherichia coli and inhibitors that prevent their aberrant self-assembly. We demonstrate the power of the system using a range of proteins, from small unstructured peptides (islet amyloid polypeptide and amyloid β) to larger, folded immunoglobulin domains. Configured in a 48-well format, the split β-lactamase sensor readily differentiates between aggregation-prone and soluble sequences. Performing the assay in the presence of 109 compounds enabled a rank ordering of inhibition and revealed a new inhibitor of IAPP aggregation. This platform can be applied to both amyloidogenic and other aggregation-prone systems, independent of sequence or size, and can identify small molecules or other factors able to ameliorate or inhibit protein aggregation. PMID:26656088

  19. Effects of DDT on eggshell quality and calcium adenosine triphosphatase.

    PubMed

    Kolaja, G J; Hinton, D E

    1977-11-01

    Adult mallard ducks were fed a diet containing 50 ppm DDT for 6 months. Eggs laid during this period were collected and eggshell weight, thickness, and calcium were determined. Chronic ingestion of DDT resulted in production of eggshells that were significantly thinner and lighter than those of controls. Total calcium of thinned eggshells was also reduced; however, calcium per gram of eggshell was not altered, indicating that other eggshell constituents were not incorporated as well. Calcium adenosine triphosphatase activity in the microsomal fraction of eggshell gland epithelium was assayed in control and DDT-fed ducks. Enzyme activity in DDT-fed ducks was reduced to 65% of control values. Since Ca-ATPase has been shown to be associated with calcium transport, enzyme inhibition may be responsible for decreased eggshell weight and thickness. Electron microscopic evaluation of microsomal fractions showed elements of the plasma membrane, including cilia and microvilli, as well as rough and smooth endoplasmic reticulum. Inhibition of calcium transport at the plasma membrane of mucosal epithelium is proposed as a possible mechanism of DDT-induced eggshell thinning.

  20. Chemical composition of chicken eggshell and shell membranes.

    PubMed

    Nakano, T; Ikawa, N I; Ozimek, L

    2003-03-01

    This study was undertaken to determine the occurrence of uronic acid in chicken eggshell membranes and to compare chemical compositions among the inner and outer eggshell membranes and the organic matter of eggshell. We report here for the first time the occurrence of uronic acid in chicken eggshell membranes. Uronic acid concentrations were similar (P > 0.05) between the inner shell membrane and outer shell membrane but approximately fivefold higher (P < 0.05) in the organic matter of eggshell. Sialic acid concentrations were the highest (P < 0.05) in the organic matter of eggshell and higher (P < 0.05) in the inner than in the outer shell membrane. Nitrogen concentrations were the lowest (P < 0.05) in the organic matter of eggshell but relatively constant between the two shell membranes. Amino acid analysis showed that the contents of glycine and alanine were higher (P < 0.05) and those of proline and hydroxyproline were lower (P < 0.05) in the organic matter of eggshell compared to shell membranes.

  1. New clues to identify proteins correlated with Attractin.

    PubMed

    Li, J; Yang, J; Cheng, D; Shen, S-L; Xiong, C-L

    2014-09-01

    In our previous study, the age-dependent testis vacuolisation and sperm dysfunction were found in Attractin (Atrn)-deficient mice, Atrn(mg-3J) , which is a null or nearly null allele. To explore the potential mechanism involved in these pathological changes, Attractin knock-down in mouse Sertoli cells TM4 (psiAtrn-TM4) was successfully established by stable RNA interference. The TM4 transfected by psiRNA-hH1 (psiRNA-TM4) was the control group, in which the expression of Atrn was not affected. The proteomic changes among the psiAtrn-TM4, primary cultures of Sertoli cells of Atrn(mg-3J) (mu-Sc) and control cells (psiRNA-TM4) were compared by two-dimensional gel electrophoresis. Fifteen differentially expressed protein spots of those cells were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry and the NCBI proteins database. Except the decreased expression of superoxide dismutase (SOD), there were several novel proteins associated with Atrn function, including downregulated ATP synthase, peroxiredoxin 2 and upregulated caspase 6, ketohexokinase, etc., in psiAtrn-TM4 and mu-Sc. These data suggest that these differentially expressed proteins may be associated with the function of Atrn in Sertoli cells, thus providing a new clue to interpret the mechanism of testis degeneration in Atrn mutants. © 2013 Blackwell Verlag GmbH.

  2. Identifying subcellular protein localization with fluorescent protein fusions after transient expression in onion epidermal cells.

    PubMed

    Nebenführ, Andreas

    2014-01-01

    Most biochemical functions of plant cells are carried out by proteins which act at very specific places within these cells, for example, within different organelles. Identifying the subcellular localization of proteins is therefore a useful tool to narrow down the possible functions that a novel or unknown protein may carry out. The discovery of genetically encoded fluorescent markers has made it possible to tag specific proteins and visualize them in vivo under a variety of conditions. This chapter describes a simple method to use transient expression of such fluorescently tagged proteins in onion epidermal cells to determine their subcellular localization relative to known markers.

  3. ’In Vitro’ Inhibition of Microsomal Calcium Atpase from Eggshell Gland of Mallard Duck.

    DTIC Science & Technology

    1976-12-01

    shell gland homogenate. Previous studies have shown DDT-induced eggshell thinning to be due to decreased calcium content of eggshells . Since calcium...ATPase has been shown to be associated with calcium transport in eggshell gland, the inhibition of this enzyme in vitro offers a possible explanation for DDT-induced eggshell thinning.

  4. Characteristics of glycosaminoglycans in chicken eggshells and the influence of disaccharide composition on eggshell properties.

    PubMed

    Liu, Z; Sun, X; Cai, C; He, W; Zhang, F; Linhardt, R J

    2016-12-01

    Glycosaminoglycans (GAG) are linear, highly negatively charged polysaccharides that may perform an important role in biomineralization. GAG were isolated from chicken eggshell membranes and calcified shells. Disaccharide compositional analysis was performed using liquid chromatography-mass spectrometry. All 4 groups of GAG - hyaluronan (HA), keratan sulfate (KS), chondroitin sulfate (CS), and heparan sulfate (HS) - were detected in shell membranes and in calcified shells. HA was the most plentiful GAG in shell membranes, and CS was the most abundant in calcified shells. The CS present, in both membranes and calcified shells, consisted primarily of 6SCS-C, 4SCS-A, and 0SCS-0 disaccharides. Neither 4S6SCS-E nor 2SCS was detectable in shell components. Small amounts of 2S4SCS-B were detected in membranes and TriSCS, and 2S4SCS-B and 2S6SCS-D were detected in calcified shells. HS in calcified shells contained all disaccharides except for 2S6S. In shell membranes, HS contained primarily NS and 0S as well as small amounts of TriS, NS2S, NS6SHS, and 6S, but neither 2S6S nor 2S was detectable. The disaccharide composition of membrane CS, as well as membrane and calcified shell HS, were very similar in all eggshells. In contrast, the composition of calcified shell CS disaccharides was highly variable. In membranes, both HA and KS content showed a correlation with egg shape index. The 4SCS-A content correlated with eggshell strength, and 0SCS-0 correlated with eggshell strength and calcified shell thickness. HS content and its disaccharide composition showed no apparent correlation to properties of calcified shells. In calcified shells, only HS 6S correlated with egg shape index. This study suggests that GAG content and disaccharide composition of shell membranes might impact the quality of chicken eggshells. © 2016 Poultry Science Association Inc.

  5. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells

    PubMed Central

    Kim, Dae In; Raida, Manfred; Burke, Brian

    2012-01-01

    We have developed a new technique for proximity-dependent labeling of proteins in eukaryotic cells. Named BioID for proximity-dependent biotin identification, this approach is based on fusion of a promiscuous Escherichia coli biotin protein ligase to a targeting protein. BioID features proximity-dependent biotinylation of proteins that are near-neighbors of the fusion protein. Biotinylated proteins may be isolated by affinity capture and identified by mass spectrometry. We apply BioID to lamin-A (LaA), a well-characterized intermediate filament protein that is a constituent of the nuclear lamina, an important structural element of the nuclear envelope (NE). We identify multiple proteins that associate with and/or are proximate to LaA in vivo. The most abundant of these include known interactors of LaA that are localized to the NE, as well as a new NE-associated protein named SLAP75. Our results suggest BioID is a useful and generally applicable method to screen for both interacting and neighboring proteins in their native cellular environment. PMID:22412018

  6. Ovarian dual oxidase (Duox) activity is essential for insect eggshell hardening and waterproofing.

    PubMed

    Dias, Felipe A; Gandara, Ana Caroline P; Queiroz-Barros, Fernanda G; Oliveira, Raquel L L; Sorgine, Marcos H F; Braz, Glória R C; Oliveira, Pedro L

    2013-12-06

    In insects, eggshell hardening involves cross-linking of chorion proteins via their tyrosine residues. This process is catalyzed by peroxidases at the expense of H2O2 and confers physical and biological protection to the developing embryo. Here, working with Rhodnius prolixus, the insect vector of Chagas disease, we show that an ovary dual oxidase (Duox), a NADPH oxidase, is the source of the H2O2 that supports dityrosine-mediated protein cross-linking and eggshell hardening. RNAi silencing of Duox activity decreased H2O2 generation followed by a failure in embryo development caused by a reduced resistance to water loss, which, in turn, caused embryos to dry out following oviposition. Phenotypes of Duox-silenced eggs were reversed by incubation in a water-saturated atmosphere, simultaneous silencing of the Duox and catalase genes, or H2O2 injection into the female hemocoel. Taken together, our results show that Duox-generated H2O2 fuels egg chorion hardening and that this process plays an essential role during eggshell waterproofing.

  7. A new scheme to characterize and identify protein ubiquitination sites.

    PubMed

    Nguyen, Van-Nui; Huang, Kai-Yao; Huang, Chien-Hsun; Lai, K Robert; Lee, Tzong-Yi

    2016-02-08

    Protein ubiquitination, involving the conjugation of ubiquitin on lysine residue, serves as an important modulator of many cellular functions in eukaryotes. Recent advancements in proteomic technology have stimulated increasing interest in identifying ubiquitination sites. However, most computational tools for predicting ubiquitination sites are focused on small-scale data. With an increasing number of experimentally verified ubiquitination sites, we were motivated to design a predictive model for identifying lysine ubiquitination sites for large-scale proteome dataset. This work assessed not only single features, such as amino acid composition (AAC), amino acid pair composition (AAPC) and evolutionary information, but also the effectiveness of incorporating two or more features into a hybrid approach to model construction. The support vector machine (SVM) was applied to generate the prediction models for ubiquitination site identification. Evaluation by five-fold cross-validation showed that the SVM models learned from the combination of hybrid features delivered a better prediction performance. Additionally, a motif discovery tool, MDDLogo, was adopted to characterize the potential substrate motifs of ubiquitination sites. The SVM models integrating the MDDLogo-identified substrate motifs could yield an average accuracy of 68.70%. Furthermore, the independent testing result showed that the MDDLogo-clustered SVM models could provide a promising accuracy (78.50%) and perform better than other prediction tools. Two cases have demonstrated the effective prediction of ubiquitination sites with corresponding substrate motifs.

  8. Mutational analysis of the major coat protein of M13 identifies residues that control protein display.

    PubMed Central

    Weiss, G. A.; Wells, J. A.; Sidhu, S. S.

    2000-01-01

    We have reported variants of the M13 bacteriophage major coat protein (P8) that enable high copy display of monomeric and oligomeric proteins, such as human growth hormone and steptavidin, on the surface of phage particles (Sidhu SS, Weiss GA, Wells JA. 2000. High copy display of large proteins on phage for functional selections. J Mol Biol 296:487-495). Here, we explore how an optimized P8 variant (opti-P8) could evolve the ability to efficiently display a protein fused to its N-terminus. Reversion of individual opti-P8 residues back to the wild-type P8 residue identifies a limited set of hydrophobic residues responsible for the high copy protein display. These hydrophobic amino acids bracket a conserved hydrophobic face on the P8 alpha helix thought to be in contact with the phage coat. Mutations additively combine to promote high copy protein display, which was further enhanced by optimization of the linker between the phage coat and the fusion protein. These data are consistent with a model in which protein display-enhancing mutations allow for better packing of the fusion protein into the phage coat. The high tolerance for phage coat protein mutations observed here suggests that filamentous phage coat proteins could readily evolve new capabilities. PMID:10794407

  9. Dataset of integrin-linked kinase protein: Protein interactions in cardiomyocytes identified by mass spectrometry.

    PubMed

    Traister, Alexandra; Lu, Mingliang; Coles, John G; Maynes, Jason T

    2016-06-01

    Using hearts from mice overexpressing integrin linked kinase (ILK) behind the cardiac specific promoter αMHC, we have performed immunoprecipitation and mass spectrometry to identify novel ILK protein:protein interactions that regulate cardiomyocyte activity and calcium flux. Integrin linked kinase complexes were captured from mouse heart lysates using a commercial antibody, with subsequent liquid chromatography tandem mass spectral analysis. Interacting partners were identified using the MASCOT server, and important interactions verified using reverse immunoprecipitation and mass spectrometry. All ILK interacting proteins were identified in a non-biased manner, and are stored in the ProteomeXchange Consortium via the PRIDE partner repository (reference ID PRIDE: PXD001053). The functional role of identified ILK interactions in cardiomyocyte function and arrhythmia were subsequently confirmed in human iPSC-cardiomyocytes.

  10. FunMod: a Cytoscape plugin for identifying functional modules in undirected protein-protein networks.

    PubMed

    Natale, Massimo; Benso, Alfredo; Di Carlo, Stefano; Ficarra, Elisa

    2014-08-01

    The characterization of the interacting behaviors of complex biological systems is a primary objective in protein-protein network analysis and computational biology. In this paper we present FunMod, an innovative Cytoscape version 2.8 plugin that is able to mine undirected protein-protein networks and to infer sub-networks of interacting proteins intimately correlated with relevant biological pathways. This plugin may enable the discovery of new pathways involved in diseases. In order to describe the role of each protein within the relevant biological pathways, FunMod computes and scores three topological features of the identified sub-networks. By integrating the results from biological pathway clustering and topological network analysis, FunMod proved to be useful for the data interpretation and the generation of new hypotheses in two case studies.

  11. Antimicrobial Characteristics of Heated Eggshell Powder.

    PubMed

    Ohshima, Yuki; Takada, Daisuke; Namai, Satoe; Sawai, Jun; Kikuchi, Mikio; Hotta, Mikinori

    2015-01-01

    Eggshells have high bioavailability and can be used as a source of calcium. The main component is CaCO3, which, when heated, is converted to CaO. Seashells are also mainly composed of CaCO3 and were previously found to exhibit antimicrobial activity after being heated. In this study, heated eggshell powder (HESP) was found to have antimicrobial activity against bacterial vegetative cells, fungi and bacterial spores. Parameters, such as the minimum inhibitory concentration, were determined with kinetic analysis using an indirect conductimetric assay. Moreover, HESP was able to kill the Bacillus subtilis spores. There were no significant differences in the activity between HESP, heated scallop-shell powder and pure CaO. The MIC values for HESP against bacteria and fungi were 0.29-0.43 and 1.3-1.5 mg/mL, respectively. Against B. subtilis spores, a reduction of two orders of magnitude of viability was confirmed following 20 min of treatment at 10 mg/mL at 60 ℃. The active oxygen generated from the HESP slurry was examined with chemiluminescence. The intensity of this increased with increasing concentrations of the HESP slurry. This suggests that HESP could be used as a natural antimicrobial agent. Although a high pH is the main contributor to this antimicrobial activity, active oxygen species generated from HESP are likely to be the main antimicrobial agents..

  12. Molecular preservation in Late Cretaceous sauropod dinosaur eggshells

    PubMed Central

    Schweitzer, M.H; Chiappe, L; Garrido, A.C; Lowenstein, J.M; Pincus, S.H

    2005-01-01

    Exceptionally preserved sauropod eggshells discovered in Upper Cretaceous (Campanian) deposits in Patagonia, Argentina, contain skeletal remains and soft tissues of embryonic Titanosaurid dinosaurs. To preserve these labile embryonic remains, the rate of mineral precipitation must have superseded post-mortem degradative processes, resulting in virtually instantaneous mineralization of soft tissues. If so, mineralization may also have been rapid enough to retain fragments of original biomolecules in these specimens. To investigate preservation of biomolecular compounds in these well-preserved sauropod dinosaur eggshells, we applied multiple analytical techniques. Results demonstrate organic compounds and antigenic structures similar to those found in extant eggshells. PMID:15888409

  13. Microstructure and chemical composition of giant avian eggshells.

    PubMed

    Dauphin, Yannicke; Cuif, Jean-Pierre; Salomé, Murielle; Susini, Jean; Williams, C Terry

    2006-11-01

    The microstructure and composition of the layers of two giant avian eggshells were investigated using a combination of scanning electron microscopy, electron probe microanalyses, and X-ray absorption near-edge structure spectroscopy (XANES). The two species have some similarities and differences in their microstructure and composition; the composition is not homogeneous throughout the eggshell thickness. XANES studies show that sulfur is associated with amino acids in the inner organic membranes, whereas in the mineralised layers the sulfur is mainly associated with sulfated polysaccharides. These results are similar to those obtained on chicken eggshells, and confirm the active role of sulfated acidic polysaccharides in biomineralisation processes of carbonate skeletons.

  14. Quantitative Tagless Copurification: A Method to Validate and Identify Protein-Protein Interactions

    DOE PAGES

    Shatsky, Maxim; Dong, Ming; Liu, Haichuan; ...

    2016-04-20

    Identifying protein-protein interactions (PPIs) at an acceptable false discovery rate (FDR) is challenging. Previously we identified several hundred PPIs from affinity purification - mass spectrometry (AP-MS) data for the bacteria Escherichia coli and Desulfovibrio vulgaris. These two interactomes have lower FDRs than any of the nine interactomes proposed previously for bacteria and are more enriched in PPIs validated by other data than the nine earlier interactomes. To more thoroughly determine the accuracy of ours or other interactomes and to discover further PPIs de novo, here we present a quantitative tagless method that employs iTRAQ MS to measure the copurification ofmore » endogenous proteins through orthogonal chromatography steps. 5273 fractions from a four-step fractionation of a D. vulgaris protein extract were assayed, resulting in the detection of 1242 proteins. Protein partners from our D. vulgaris and E. coli AP-MS interactomes copurify as frequently as pairs belonging to three benchmark data sets of well-characterized PPIs. In contrast, the protein pairs from the nine other bacterial interactomes copurify two- to 20-fold less often. We also identify 200 high confidence D. vulgaris PPIs based on tagless copurification and colocalization in the genome. These PPIs are as strongly validated by other data as our AP-MS interactomes and overlap with our AP-MS interactome for D.vulgaris within 3% of expectation, once FDRs and false negative rates are taken into account. Finally, we reanalyzed data from two quantitative tagless screens of human cell extracts. We estimate that the novel PPIs reported in these studies have an FDR of at least 85% and find that less than 7% of the novel PPIs identified in each screen overlap. Our results establish that a quantitative tagless method can be used to validate and identify PPIs, but that such data must be analyzed carefully to minimize the FDR.« less

  15. Quantitative Tagless Copurification: A Method to Validate and Identify Protein-Protein Interactions*

    PubMed Central

    Shatsky, Maxim; Dong, Ming; Liu, Haichuan; Yang, Lee Lisheng; Choi, Megan; Singer, Mary E.; Geller, Jil T.; Fisher, Susan J.; Hall, Steven C.; Hazen, Terry C.; Brenner, Steven E.; Butland, Gareth; Jin, Jian; Witkowska, H. Ewa; Chandonia, John-Marc; Biggin, Mark D.

    2016-01-01

    Identifying protein-protein interactions (PPIs) at an acceptable false discovery rate (FDR) is challenging. Previously we identified several hundred PPIs from affinity purification - mass spectrometry (AP-MS) data for the bacteria Escherichia coli and Desulfovibrio vulgaris. These two interactomes have lower FDRs than any of the nine interactomes proposed previously for bacteria and are more enriched in PPIs validated by other data than the nine earlier interactomes. To more thoroughly determine the accuracy of ours or other interactomes and to discover further PPIs de novo, here we present a quantitative tagless method that employs iTRAQ MS to measure the copurification of endogenous proteins through orthogonal chromatography steps. 5273 fractions from a four-step fractionation of a D. vulgaris protein extract were assayed, resulting in the detection of 1242 proteins. Protein partners from our D. vulgaris and E. coli AP-MS interactomes copurify as frequently as pairs belonging to three benchmark data sets of well-characterized PPIs. In contrast, the protein pairs from the nine other bacterial interactomes copurify two- to 20-fold less often. We also identify 200 high confidence D. vulgaris PPIs based on tagless copurification and colocalization in the genome. These PPIs are as strongly validated by other data as our AP-MS interactomes and overlap with our AP-MS interactome for D.vulgaris within 3% of expectation, once FDRs and false negative rates are taken into account. Finally, we reanalyzed data from two quantitative tagless screens of human cell extracts. We estimate that the novel PPIs reported in these studies have an FDR of at least 85% and find that less than 7% of the novel PPIs identified in each screen overlap. Our results establish that a quantitative tagless method can be used to validate and identify PPIs, but that such data must be analyzed carefully to minimize the FDR. PMID:27099342

  16. Quantitative Tagless Copurification: A Method to Validate and Identify Protein-Protein Interactions

    SciTech Connect

    Shatsky, Maxim; Dong, Ming; Liu, Haichuan; Yang, Lee Lisheng; Choi, Megan; Singer, Mary; Geller, Jil; Fisher, Susan; Hall, Steven; Hazen, Terry C.; Brenner, Steven; Butland, Gareth; Jin, Jian; Witkowska, H. Ewa; Chandonia, John-Marc; Biggin, Mark D.

    2016-04-20

    Identifying protein-protein interactions (PPIs) at an acceptable false discovery rate (FDR) is challenging. Previously we identified several hundred PPIs from affinity purification - mass spectrometry (AP-MS) data for the bacteria Escherichia coli and Desulfovibrio vulgaris. These two interactomes have lower FDRs than any of the nine interactomes proposed previously for bacteria and are more enriched in PPIs validated by other data than the nine earlier interactomes. To more thoroughly determine the accuracy of ours or other interactomes and to discover further PPIs de novo, here we present a quantitative tagless method that employs iTRAQ MS to measure the copurification of endogenous proteins through orthogonal chromatography steps. 5273 fractions from a four-step fractionation of a D. vulgaris protein extract were assayed, resulting in the detection of 1242 proteins. Protein partners from our D. vulgaris and E. coli AP-MS interactomes copurify as frequently as pairs belonging to three benchmark data sets of well-characterized PPIs. In contrast, the protein pairs from the nine other bacterial interactomes copurify two- to 20-fold less often. We also identify 200 high confidence D. vulgaris PPIs based on tagless copurification and colocalization in the genome. These PPIs are as strongly validated by other data as our AP-MS interactomes and overlap with our AP-MS interactome for D.vulgaris within 3% of expectation, once FDRs and false negative rates are taken into account. Finally, we reanalyzed data from two quantitative tagless screens of human cell extracts. We estimate that the novel PPIs reported in these studies have an FDR of at least 85% and find that less than 7% of the novel PPIs identified in each screen overlap. Our results establish that a quantitative tagless method can be used to validate and identify PPIs, but that such data must be analyzed carefully to minimize the FDR.

  17. Ultrastructure of eggs of Ascaris lumbricoides Linnaeus, 1758. I. Egg-shells.

    PubMed

    Lýsek, H; Malínský, J; Janisch, R

    1985-01-01

    Under the light microscope the chitin-protein layer of egg-shells in ascarids appears to be a regular, hyaline and nonstructural layer of 1.5 to 2.00 microns in thickness. The outer uterine layer is usually removed during the preparation. The lipid (ascaroside) layer covers the inner surface of the chitinous layer and seems to be irregularly undulated and regularly thick over the whole surface, with the thickness up to 1 micron. In electron micrographs the fibrous structure of the lipid layer is not evident as a rule. This is probably due to washing the lipids away from this layer during the dehydration of deeper layers of egg-shells that are imperfectly fixed with glutaraldehyde. A very low permeability of the egg-shells is typical of geohelminth eggs. The layer lipid shows a distinct lamellate structure only after a prolonged fixation with osmium at higher temperature. This is supported by the studies using the method of freeze-fracturing.

  18. Identifying true protein complex constituents in interaction proteomics: the example of the DMXL2 protein complex.

    PubMed

    Li, Ka Wan; Chen, Ning; Klemmer, Patricia; Koopmans, Frank; Karupothula, Ramesh; Smit, August B

    2012-08-01

    A typical high-sensitivity antibody affinity purification-mass spectrometry experiment easily identifies hundreds of protein interactors. However, most of these are non-valid resulting from multiple causes other than interaction with the bait protein. To discriminate true interactors from off-target recognition, we propose to differentially include an (peptide) antigen during the antibody incubation in the immuno-precipitation experiment. This contrasts the specific antibody-bait protein interactions, versus all other off-target protein interactions. To exemplify the power of the approach, we studied the DMXL2 interactome. From the initial six immuno-precipitations, we identified about 600 proteins. When filtering for interactors present in all anti-DMXL2 antibody immuno-precipitation experiments, absent in the bead controls, and competed off by the peptide antigen, this hit list is reduced to ten proteins, including known and novel interactors of DMXL2. Together, our approach enables the use of a wide range of available antibodies in large-scale protein interaction proteomics, while gaining specificity of the interactions. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Identifying relevant positions in proteins by Critical Variable Selection.

    PubMed

    Grigolon, Silvia; Franz, Silvio; Marsili, Matteo

    2016-06-21

    Evolution in its course has found a variety of solutions to the same optimisation problem. The advent of high-throughput genomic sequencing has made available extensive data from which, in principle, one can infer the underlying structure on which biological functions rely. In this paper, we present a new method aimed at the extraction of sites encoding structural and functional properties from a set of protein primary sequences, namely a multiple sequence alignment. The method, called critical variable selection, is based on the idea that subsets of relevant sites correspond to subsequences that occur with a particularly broad frequency distribution in the dataset. By applying this algorithm to in silico sequences, to the response regulator receiver and to the voltage sensor domain of ion channels, we show that this procedure recovers not only the information encoded in single site statistics and pairwise correlations but also captures dependencies going beyond pairwise correlations. The method proposed here is complementary to statistical coupling analysis, in that the most relevant sites predicted by the two methods differ markedly. We find robust and consistent results for datasets as small as few hundred sequences that reveal a hidden hierarchy of sites that are consistent with the present knowledge on biologically relevant sites and evolutionary dynamics. This suggests that critical variable selection is capable of identifying a core of sites encoding functional and structural information in a multiple sequence alignment.

  20. Dynamics of bacterial and fungal communities associated with eggshells during incubation

    PubMed Central

    Grizard, Stéphanie; Dini-Andreote, Francisco; Tieleman, B Irene; Salles, Joana F

    2014-01-01

    Microorganisms are closely associated with eggs and may play a determinant role in embryo survival. Yet, the majority of studies focusing on this association relied on culture-based methodology, eventually leading to a skewed assessment of microbial communities. By targeting the 16S rRNA gene and internal transcribed spacer (ITS) region, we, respectively, described bacterial and fungal communities on eggshells of the homing pigeon Columba livia. We explored their structure, abundance, and composition. Firstly, we showed that sampling technique affected the outcome of the results. While broadly used, the egg swabbing procedure led to a lower DNA extraction efficiency and provided different profiles of bacterial communities than those based on crushed eggshell pieces. Secondly, we observed shifts in bacterial and fungal communities during incubation. At late incubation, bacterial communities showed a reduction in diversity, while their abundance increased, possibly due to the competitive advantage of some species. When compared to their bacterial counterparts, fungal communities also decreased in diversity at late incubation. In that case, however, the decline was associated with a diminution of their overall abundance. Conclusively, our results showed that although incubation might inhibit microbial growth when compared to unincubated eggs, we observed the selective growth of specific bacterial species during incubation. Moreover, we showed that fungi are a substantial component of the microbial communities associated with eggshells and require further investigations in avian ecology. Identifying the functional roles of these microorganisms is likely to provide news insights into the evolutionary strategies that control embryo survival. We aimed to describe the dynamics of bacterial and fungal communities on homing pigeon eggshell surfaces. We investigated these communities at early and late incubation stages. PMID:24772289

  1. 15. April 1963 SPIRAL STAIRS AND EGGSHELL DORMER Shaker ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    15. April 1963 SPIRAL STAIRS AND EGG-SHELL DORMER - Shaker Centre Family Trustees' Office, South side of Village Road, North of U.S. Route 68 & State Route 33 intersection, Shakertown, Mercer County, KY

  2. Antimicrobial activity of lipophilic avian eggshell surface extracts.

    PubMed

    Wellman-Labadie, Olivier; Lemaire, Simon; Mann, Karlheinz; Picman, Jaroslav; Hincke, Maxwell T

    2010-09-22

    The avian eggshell cuticle is the waxy outermost layer of the mineralized eggshell in direct contact with the environment. In this study, lipophilic eggshell surface extracts from three domestic species were evaluated for their antimicrobial activity. Chicken and goose extracts demonstrated potent bactericidal activity against both Gram-positive and Gram-negative bacteria, while activity could not be detected for duck eggshell surface extracts. Using the chicken as a model species, evaluation of albumen, fecal material, and uropygial gland extracts eliminated these as a potential source of the observed activity. Results suggest that lipophilic components are incorporated into the egg during its formation and play a role in antimicrobial defense. This study represents the first successful extraction and evaluation of lipophilic antimicrobial components from the avian egg.

  3. Nanosized hydroxyapatite powder synthesized from eggshell and phosphoric acid.

    PubMed

    Lee, Sang-Jin; Yoon, Young-Soo; Lee, Myung-Hyun; Oh, Nam-Sik

    2007-11-01

    The present research describes synthesis of highly sinterable, nano-sized hydroxyapatite (HAp) powders using a wet chemical route with recycled eggshell and phosphoric acid as calcium and phosphorous sources. The raw eggshell was easily turned to CaO by the calcining process, and phosphoric acid was mixed with the calcined eggshell by the wet, ball-milling method. The crystalline development and microstructures of the synthesized powders and sintered samples were examined by X-ray diffractometry and scanning electron microscopy, respectively. The observed phases on the powder synthesis process were dependent on the mixing ratio (wt%) of the calcined eggshell to phosphoric acid and the heating temperature. The ball-milled, nano-sized HAp powder, which has an average particle size of 70 nm, was fully densified at 1300 degrees C for 1h. The Ca/P ratio for stoichiometric composition of HAp was controlled by adjustment of the mixing ratio.

  4. Automated Analysis of Fluorescence Microscopy Images to Identify Protein-Protein Interactions

    PubMed Central

    Doktycz, M. J.; Qi, H.; Morrell-Falvey, J. L.

    2006-01-01

    The identification of protein interactions is important for elucidating biological networks. One obstacle in comprehensive interaction studies is the analyses of large datasets, particularly those containing images. Development of an automated system to analyze an image-based protein interaction dataset is needed. Such an analysis system is described here, to automatically extract features from fluorescence microscopy images obtained from a bacterial protein interaction assay. These features are used to relay quantitative values that aid in the automated scoring of positive interactions. Experimental observations indicate that identifying at least 50% positive cells in an image is sufficient to detect a protein interaction. Based on this criterion, the automated system presents 100% accuracy in detecting positive interactions for a dataset of 16 images. Algorithms were implemented using MATLAB and the software developed is available on request from the authors. PMID:23165043

  5. Automated Analysis of Fluorescence Microscopy Images to Identify Protein-Protein Interactions

    DOE PAGES

    Venkatraman, S.; Doktycz, M. J.; Qi, H.; ...

    2006-01-01

    The identification of protein interactions is important for elucidating biological networks. One obstacle in comprehensive interaction studies is the analyses of large datasets, particularly those containing images. Development of an automated system to analyze an image-based protein interaction dataset is needed. Such an analysis system is described here, to automatically extract features from fluorescence microscopy images obtained from a bacterial protein interaction assay. These features are used to relay quantitative values that aid in the automated scoring of positive interactions. Experimental observations indicate that identifying at least 50% positive cells in an image is sufficient to detect a protein interaction.more » Based on this criterion, the automated system presents 100% accuracy in detecting positive interactions for a dataset of 16 images. Algorithms were implemented using MATLAB and the software developed is available on request from the authors.« less

  6. Eggshell defects detection based on color processing

    NASA Astrophysics Data System (ADS)

    Garcia-Alegre, Maria C.; Ribeiro, Angela; Guinea, Domingo; Cristobal, Gabriel

    2000-03-01

    The automatic classification of defective eggs constitutes a fundamental issue at the poultry industry for both economical and sanitary reasons. The early separation of eggs with spots and cracks is a relevant task as the stains can leak while progressing on the conveyor-belts, degrading all the mechanical parts. Present work is focused on the implementation of an artificial vision system for detecting in real time defective eggs at the poultry farm. First step of the algorithmic process is devoted to the detection of the egg shape to fix the region of interest. A color processing is then performed only on the eggshell to obtain an image segmentation that allows the discrimination of defective eggs from clean ones in critic time. The results are presented to demonstrate the validity of the proposed visual process on a wide sample of both defective and non-defective eggs.

  7. 40 CFR 174.529 - Bacillus thuringiensis modified Cry1Ab protein as identified under OECD Unique Identifier SYN...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... protein as identified under OECD Unique Identifier SYN-IR67B-1 in cotton; exemption from the requirement... Unique Identifier SYN-IR67B-1 in cotton; exemption from the requirement of a tolerance. Residues of... exempt from the requirement of a tolerance when used as a plant-incorporated protectant in cotton;...

  8. 40 CFR 174.529 - Bacillus thuringiensis modified Cry1Ab protein as identified under OECD Unique Identifier SYN...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... protein as identified under OECD Unique Identifier SYN-IR67B-1 in cotton; exemption from the requirement... Unique Identifier SYN-IR67B-1 in cotton; exemption from the requirement of a tolerance. Residues of... exempt from the requirement of a tolerance when used as a plant-incorporated protectant in cotton;...

  9. 40 CFR 174.529 - Bacillus thuringiensis modified Cry1Ab protein as identified under OECD Unique Identifier SYN...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... protein as identified under OECD Unique Identifier SYN-IR67B-1 in cotton; exemption from the requirement... Unique Identifier SYN-IR67B-1 in cotton; exemption from the requirement of a tolerance. Residues of... exempt from the requirement of a tolerance when used as a plant-incorporated protectant in cotton;...

  10. 40 CFR 174.529 - Bacillus thuringiensis modified Cry1Ab protein as identified under OECD Unique Identifier SYN...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... protein as identified under OECD Unique Identifier SYN-IR67B-1 in cotton; exemption from the requirement... Unique Identifier SYN-IR67B-1 in cotton; exemption from the requirement of a tolerance. Residues of... exempt from the requirement of a tolerance when used as a plant-incorporated protectant in cotton;...

  11. Application of waste eggshell as low-cost solid catalyst for biodiesel production.

    PubMed

    Wei, Ziku; Xu, Chunli; Li, Baoxin

    2009-06-01

    Waste eggshell was investigated in triglyceride transesterification with a view to determine its viability as a solid catalyst for use in biodiesel synthesis. Effect of calcination temperature on structure and activity of eggshell catalysts was investigated. Reusability of eggshell catalysts was also examined. It was found that high active, reusable solid catalyst was obtained by just calcining eggshell. Utilization of eggshell as a catalyst for biodiesel production not only provides a cost-effective and environmental friendly way of recycling this solid eggshell waste, significantly reducing its environmental effects, but also reduces the price of biodiesel to make biodiesel competitive with petroleum diesel.

  12. Eggshell thickness and DDE residue levels in vlulture eggs

    USGS Publications Warehouse

    Kiff, L.F.; Peakall, David B.; Morrison, M.L.; Wilbur, S.R.; Wilbur, Sanford R.; Jackson, Jerome A.

    1983-01-01

    Post-DDT (post-1947) eggshell thickness was examined in samples of Turkey Vulture, Black Vulture, and Crested Caracara eggs from several parts of the United States. Highly significant post-DDT decreases in eggshell thickness indices of at least 10 percent were found in Turkey Vulture eggs from California, Florida, and Texas and in Black Vulture eggs from Texas and Florida. Over one-third of the Black VUlture eggs and about 30 percent of the Turkey Vulture eggs from Texas showed thinning exceeding 20 percent, a level associated with reproductive failure and population decline in other species. A strong negative correlation was found between eggshell thickness indices and DDE residues extracted from eggshell membranes in California and Texas samples of Turkey Vulture eggs and in Texas Black Vulture eggs. Crested Caracara eggs from Texas and Florida showed mean changes in eggshell thickness indices of only -5.6 and -8.2 percent, respectively, although thinning in a few eggs from both states exceeded 20 percent. Most of the post-DDT Old World vulture eggs examined appeared to be of normal thickness, with low DDE residue levels in eggshell membranes; but single eggs of Egyptian Vulture from India, Cinereous Vulture from Spain, and White-headed Vulture from Zambia showed apparent thinning. Further monitoring of vulture populations in tropical regions, where DDT use is still increasing, is recommended.

  13. A new scoring function for protein-protein docking that identifies native structures with unprecedented accuracy.

    PubMed

    Moreira, Irina S; Martins, João M; Coimbra, João T S; Ramos, Maria J; Fernandes, Pedro A

    2015-01-28

    Protein-protein (P-P) 3D structures are fundamental to structural biology and drug discovery. However, most of them have never been determined. Many docking algorithms were developed for that purpose, but they have a very limited accuracy in generating native-like structures and identifying the most correct one, in particular when a single answer is asked for. With such a low success rate it is difficult to point out one docked structure as being native-like. Here we present a new, high accuracy, scoring method to identify the 3D structure of P-P complexes among a set of trial poses. It incorporates alanine scanning mutagenesis experimental data that need to be obtained a priori. The scoring scheme works by matching the computational and the experimental alanine scanning mutagenesis results. The size of the trial P-P interface area is also taken into account. We show that the method ranks the trial structures and identifies the native-like structures with unprecedented accuracy (∼94%), providing the correct P-P 3D structures that biochemists and molecular biologists need to pursue their studies. With such a success rate, the bottleneck of protein-protein docking moves from the scoring to searching algorithms.

  14. A coevolution analysis for identifying protein-protein interactions by Fourier transform

    PubMed Central

    Yin, Changchuan; Yau, Stephen S. -T.

    2017-01-01

    Protein-protein interactions (PPIs) play key roles in life processes, such as signal transduction, transcription regulations, and immune response, etc. Identification of PPIs enables better understanding of the functional networks within a cell. Common experimental methods for identifying PPIs are time consuming and expensive. However, recent developments in computational approaches for inferring PPIs from protein sequences based on coevolution theory avoid these problems. In the coevolution theory model, interacted proteins may show coevolutionary mutations and have similar phylogenetic trees. The existing coevolution methods depend on multiple sequence alignments (MSA); however, the MSA-based coevolution methods often produce high false positive interactions. In this paper, we present a computational method using an alignment-free approach to accurately detect PPIs and reduce false positives. In the method, protein sequences are numerically represented by biochemical properties of amino acids, which reflect the structural and functional differences of proteins. Fourier transform is applied to the numerical representation of protein sequences to capture the dissimilarities of protein sequences in biophysical context. The method is assessed for predicting PPIs in Ebola virus. The results indicate strong coevolution between the protein pairs (NP-VP24, NP-VP30, NP-VP40, VP24-VP30, VP24-VP40, and VP30-VP40). The method is also validated for PPIs in influenza and E.coli genomes. Since our method can reduce false positive and increase the specificity of PPI prediction, it offers an effective tool to understand mechanisms of disease pathogens and find potential targets for drug design. The Python programs in this study are available to public at URL (https://github.com/cyinbox/PPI). PMID:28430779

  15. A coevolution analysis for identifying protein-protein interactions by Fourier transform.

    PubMed

    Yin, Changchuan; Yau, Stephen S-T

    2017-01-01

    Protein-protein interactions (PPIs) play key roles in life processes, such as signal transduction, transcription regulations, and immune response, etc. Identification of PPIs enables better understanding of the functional networks within a cell. Common experimental methods for identifying PPIs are time consuming and expensive. However, recent developments in computational approaches for inferring PPIs from protein sequences based on coevolution theory avoid these problems. In the coevolution theory model, interacted proteins may show coevolutionary mutations and have similar phylogenetic trees. The existing coevolution methods depend on multiple sequence alignments (MSA); however, the MSA-based coevolution methods often produce high false positive interactions. In this paper, we present a computational method using an alignment-free approach to accurately detect PPIs and reduce false positives. In the method, protein sequences are numerically represented by biochemical properties of amino acids, which reflect the structural and functional differences of proteins. Fourier transform is applied to the numerical representation of protein sequences to capture the dissimilarities of protein sequences in biophysical context. The method is assessed for predicting PPIs in Ebola virus. The results indicate strong coevolution between the protein pairs (NP-VP24, NP-VP30, NP-VP40, VP24-VP30, VP24-VP40, and VP30-VP40). The method is also validated for PPIs in influenza and E.coli genomes. Since our method can reduce false positive and increase the specificity of PPI prediction, it offers an effective tool to understand mechanisms of disease pathogens and find potential targets for drug design. The Python programs in this study are available to public at URL (https://github.com/cyinbox/PPI).

  16. Development of a Capillary Electrophoresis Platform for Identifying Inhibitors of Protein-Protein Interactions

    PubMed Central

    Rauch, Jennifer N.; Nie, Jing; Buchholz, Tonia J.; Gestwicki, Jason E.; Kennedy, Robert T.

    2013-01-01

    Methods for identifying chemical inhibitors of protein-protein interactions (PPIs) are often prone to discovery of false positives, particularly those caused by molecules that induce protein aggregation. Thus, there is interest in developing new platforms that might allow earlier identification of these problematic compounds. Capillary electrophoresis (CE) has been evaluated as a method to screen for PPI inhibitors using the challenging system of Hsp70 interacting with its co-chaperone Bag3. In the method, Hsp70 is labeled with a fluorophore, mixed with Bag3, and the resulting bound and free Hsp70 separated and detected by CE with laser-induced fluorescence detection. The method used a chemically modified CE capillary to prevent protein adsorption. Inhibitors of the Hsp70-Bag3 interaction were detected by observing a reduction in the bound to free ratio. The method was used to screen a library of 3,443 compounds and results compared to those from a flow cytometry protein interaction assay. CE was found to produce a lower hit rate with more compounds that reconfirmed in subsequent testing suggesting greater specificity. This finding was attributed to use of electropherograms to detect artifacts such as aggregators and to differences in protein modifications required to perform the different assays. Increases in throughput are required to make the CE method suitable for primary screens but at the current stage of development it is attractive as a secondary screen to test hits found by higher throughput methods. PMID:24060167

  17. Development of a capillary electrophoresis platform for identifying inhibitors of protein-protein interactions.

    PubMed

    Rauch, Jennifer N; Nie, Jing; Buchholz, Tonia J; Gestwicki, Jason E; Kennedy, Robert T

    2013-10-15

    Methods for identifying chemical inhibitors of protein-protein interactions (PPIs) are often prone to discovery of false positives, particularly those caused by molecules that induce protein aggregation. Thus, there is interest in developing new platforms that might allow earlier identification of these problematic compounds. Capillary electrophoresis (CE) has been evaluated as a method to screen for PPI inhibitors using the challenging system of Hsp70 interacting with its co-chaperone Bag3. In the method, Hsp70 is labeled with a fluorophore, mixed with Bag3, and the resulting bound and free Hsp70 are separated and detected by CE with laser-induced fluorescence detection. The method used a chemically modified CE capillary to prevent protein adsorption. Inhibitors of the Hsp70-Bag3 interaction were detected by observing a reduction in the bound-to-free ratio. The method was used to screen a library of 3443 compounds, and the results were compared to those from a flow cytometry protein interaction assay. CE was found to produce a lower hit rate with more compounds that were reconfirmed in subsequent testing, suggesting greater specificity. This finding was attributed to the use of electropherograms to detect artifacts such as aggregators and to differences in protein modifications required to perform the different assays. Increases in throughput are required to make the CE method suitable for primary screens, but at the current stage of development it is attractive as a secondary screen to test hits found by higher-throughput methods.

  18. Proteomic approaches to identify cold-regulated plasma membrane proteins.

    PubMed

    Takahashi, Daisuke; Nakayama, Takato; Miki, Yushi; Kawamura, Yukio; Uemura, Matsuo

    2014-01-01

    Plasma membrane is the primary determinant of freezing tolerance in plants because of its central role in freeze-thaw cycle. Changes in the plasma membrane proteins have been one of the major research areas in plant cold acclimation. To obtain comprehensive profiles of the plasma membrane proteomes and their changes during the cold acclimation process, a plasma membrane purification method using a dextran-polyethylene glycol two polymer system and a mass spectrometry-based shotgun proteomics method using nano-LC-MS/MS for the plasma membrane proteins are described. The proteomic results obtained are further applied to label-free protein semiquantification.

  19. DDE-induced eggshell thinning in birds: effects of p,p'-DDE on the calcium and prostaglandin metabolism of the eggshell gland.

    PubMed

    Lundholm, C D

    1997-10-01

    1. The focus of this review is the effects and mechanism of action of p,p'-DDE on eggshell formation in birds. Inhibition of prostaglandin synthesis in the eggshell gland mucosa is a probable mechanism for p,p'-DDE-induced eggshell thinning. 2. The duck is sensitive to p,p'-DDE-induced eggshell thinning but the domestic fowl is not, and studies comparing the two species in regard to the calcium and prostaglandin metabolism of the eggshell gland have shown that eggshell thinning induced by p,p'-DDE in ducks is accompanied by reduced activity of prostaglandin synthetase, reduced levels of prostaglandin E2, and reduced uptake of 45Ca by the eggshell gland mucosa. The content of calcium, bicarbonate, chloride, sodium, and potassium are also reduced in the eggshell gland lumen in ducks exhibiting eggshell thinning. None of these effects are seen in the domestic fowl. 3. Inhibition of prostaglandin synthesis is a specific effect of p,p'-DDE. The detrimental effects of p,p'-DDE on the eggshell gland seem to be unique when comparing the compound with structurally related substances, i.e., similar treatment regimens with o,p'-DDE, p,p'-DDT, o,p'-DDT, and p,p'-DDD do not cause eggshell thinning in ducks. Neither do they inhibit prostaglandin synthesis in the eggshell gland mucosa. 4. Administration of other compounds that do inhibit prostaglandin synthesis, e.g., indomethacin, does cause the same effects as those seen with p,p'-DDE, i.e., eggshell thinning and the described effects on the calcium and prostaglandin metabolism of the eggshell gland.

  20. Informatics View on the Challenges of Identifying Missing Proteins from Shotgun Proteomics.

    PubMed

    Choong, Wai-Kok; Chang, Hui-Yin; Chen, Ching-Tai; Tsai, Chia-Feng; Hsu, Wen-Lian; Chen, Yu-Ju; Sung, Ting-Yi

    2015-12-04

    Protein experiment evidence at protein level from mass spectrometry and antibody experiments are essential to characterize the human proteome. neXtProt (2014-09 release) reported 20 055 human proteins, including 16 491 proteins identified at protein level and 3564 proteins unidentified. Excluding 616 proteins at uncertain level, 2948 proteins were regarded as missing proteins. Missing proteins were unidentified partially due to MS limitations and intrinsic properties of proteins, for example, only appearing in specific diseases or tissues. Despite such reasons, it is desirable to explore issues affecting validation of missing proteins from an "ideal" shotgun analysis of human proteome. We thus performed in silico digestions on the human proteins to generate all in silico fully digested peptides. With these presumed peptides, we investigated the identification of proteins without any unique peptide, the effect of sequence variants on protein identification, difficulties in identifying olfactory receptors, and highly similar proteins. Among all proteins with evidence at transcript level, G protein-coupled receptors and olfactory receptors, based on InterPro classification, were the largest families of proteins and exhibited more frequent variants. To identify missing proteins, the above analyses suggested including sequence variants in protein FASTA for database searching. Furthermore, evidence of unique peptides identified from MS experiments would be crucial for experimentally validating missing proteins.

  1. Study of the deposition process of eggshell pigments using an improved dissolution method.

    PubMed

    Wang, X-T; Deng, X-M; Zhao, C-J; Li, J-Y; Xu, G-Y; Lian, L-S; Wu, C-X

    2007-10-01

    An improved dissolution method called layer-by-layer dissolution was adopted to study the process of eggshell deposition, which is opposite to the process of eggshell dissolution. In the present study, blue and brown eggshells from 2 Chinese indigenous chicken breeds, Dongxing and Shouguang, respectively, were analyzed with layer-by-layer dissolution. The results showed that the deposition velocity of the eggshell pigments in the top (first) eggshell layer was the highest compared with other layers, which were biliverdin and protoporphyrin in blue eggshell or primarily protoporphyrin in brown eggshell. It was also revealed that the deposition processes of biliverdin and protoporphyrin were synchronous in the blue eggshell of the Dongxiang chicken in the present study.

  2. Factors influencing bacterial eggshell contamination in conventional cages, furnished cages and free-range systems for laying hens under commercial conditions.

    PubMed

    Huneau-Salaün, A; Michel, V; Huonnic, D; Balaine, L; Le Bouquin, S

    2010-04-01

    1. The aim was to assess eggshell contamination in various laying hen-housing systems and to identify factors influencing this contamination. 2. Fifty-eight laying hen farms in France were studied, including 21 flocks housed in conventional cages, 7 in furnished cages and 30 kept on-floor. 3. Sixty eggs per flock were analysed to obtain counts of the total mesophilic flora. Data on equipment and hen management were collected. 4. Mean bacterial count on eggshells tended to be higher in on-floor systems (4.82 +/- 0.51 log CFU/eggshell) than in cage systems (4.57 +/- 0.58 log CFU/eggshell, P = 0.09). 5. Contamination increased with age of the hens, airborne dust concentration, manual packing of the eggs, and packing in plastic rather than in recycled-pulp egg-flats. 6. The effect of the housing system on eggshell contamination, previously described in experimental assays, was confirmed under production conditions.

  3. Identifying Protein Stabilizing Ligands Using GroEL

    PubMed Central

    Naik, Subhashchandra; Haque, Inamul; Degner, Nick; Kornilayev, Boris; Bomhoff, Gregory; Hodges, Jacob; Khorassani, Ara-Azad; Katayama, Hiroo; Morris, Jill; Kelly, Jeffery; Seed, John; Fisher, Mark T.

    2010-01-01

    Over the past five years, it has become increasingly apparent to researchers that the initial promise and excitement of using gene replacement therapies to ameliorate folding diseases are still far from being broadly or easily applicable. Because a large number of human diseases are protein folding diseases (~30 to 50%), many researchers now realize that more directed approaches to target and reverse the fundamental misfolding reactions preceding disease are highly feasible and offer the potential of developing more targeted drug therapies. This is also true with a large number of so called “orphan protein folding diseases”. The development of a broad-based general screening array method using the chaperonin as a detection platform will enable us to screen large chemical combinatorial libraries for specific ligands against the elusive transient, primary reactions that often lead to protein misfolding. This development will provide a highly desirable tool for the pharmaceutical, academic and medical professions. PMID:19802819

  4. U-Th Burial Dates on Ostrich Eggshell

    NASA Astrophysics Data System (ADS)

    Sharp, W. D.; Fylstra, N. D.; Tryon, C. A.; Faith, J. T.; Peppe, D. J.

    2015-12-01

    Obtaining precise and accurate dates at archaeological sites beyond the range of radiocarbon dating is challenging but essential for understanding human origins. Eggshells of ratites (large flightless birds including ostrich, emu and others) are common in many archaeological sequences in Africa, Australia and elsewhere. Ancient eggshells are geochemically suitable for the U-Th technique (1), which has about ten times the range of radiocarbon dating (>500 rather than 50 ka), making eggshells attractive dating targets. Moreover, C and N isotopic studies of eggshell provide insights into paleovegetation and paleoprecipitation central to assessing past human-environment interactions (2,3). But until now, U-Th dates on ratite eggshell have not accounted for the secondary origin of essentially all of their U. We report a novel approach to U-Th dating of eggshell that explicitly accounts for secondary U uptake that begins with burial. Using ostrich eggshell (OES) from Pleistocene-Holocene east African sites, we have measured U and 232Th concentration profiles across OES by laser ablation ICP-MS. U commonly peaks at 10s to 100s of ppb and varies 10-fold or more across the ~2 mm thickness of OES, with gradients modulated by the layered structure of the eggshell. Common Th is high near the shell surfaces, but low in the middle "pallisade" layer of OES, making it optimal for U-Th dating. We determine U-Th ages along the U concentration gradient by solution ICP-MS analyses of two or more fractions of the pallisade layer. We then estimate OES burial dates using a simple model for diffusive uptake of uranium. Comparing such "U-Th burial dates" with radiocarbon dates for OES calcite from the same shells, we find good agreement in 7 out of 9 cases, consistent with rapid burial and confirming the accuracy of the approach. The remaining 2 eggshells have anomalous patterns of apparent ages that reveal they are unsuitable for U-Th dating, thereby providing reliability criteria innate

  5. Eggshell color in brown-egg laying hens - a review.

    PubMed

    Samiullah, S; Roberts, J R; Chousalkar, K

    2015-10-01

    The major pigment in eggshells of brown-egg laying hens is protoporphyrin IX, but traces of biliverdin and its zinc chelates are also present. The pigment appears to be synthesized in the shell gland. The protoporphyrin IX synthetic pathway is well defined, but precisely where and how it is synthesized in the shell gland of the brown-egg laying hen is still ambiguous. The pigment is deposited onto all shell layers including the shell membranes, but most of it is concentrated in the outermost layer of the calcareous shell and in the cuticle. Recently, the genes that are involved in pigment synthesis have been identified, but the genetic control of synthesis and deposition of brown pigment in the commercial laying hen is not fully understood. The brown coloration of the shell is an important shell quality parameter and has a positive influence on consumer preference. The extent of pigment deposition is influenced by the housing system, hen age, hen strain, diet, stressors, and certain diseases such as infectious bronchitis. In this article, the physiological and biochemical characteristics of the brown pigment in commercial brown-egg layers are reviewed in relation to its various functions in the poultry industry. © 2015 Poultry Science Association Inc.

  6. An unbiased expression screen for synaptogenic proteins identifies the LRRTM protein family as synaptic organizers.

    PubMed

    Linhoff, Michael W; Laurén, Juha; Cassidy, Robert M; Dobie, Frederick A; Takahashi, Hideto; Nygaard, Haakon B; Airaksinen, Matti S; Strittmatter, Stephen M; Craig, Ann Marie

    2009-03-12

    Delineating the molecular basis of synapse development is crucial for understanding brain function. Cocultures of neurons with transfected fibroblasts have demonstrated the synapse-promoting activity of candidate molecules. Here, we performed an unbiased expression screen for synaptogenic proteins in the coculture assay using custom-made cDNA libraries. Reisolation of NGL-3/LRRC4B and neuroligin-2 accounts for a minority of positive clones, indicating that current understanding of mammalian synaptogenic proteins is incomplete. We identify LRRTM1 as a transmembrane protein that induces presynaptic differentiation in contacting axons. All four LRRTM family members exhibit synaptogenic activity, LRRTMs localize to excitatory synapses, and artificially induced clustering of LRRTMs mediates postsynaptic differentiation. We generate LRRTM1(-/-) mice and reveal altered distribution of the vesicular glutamate transporter VGLUT1, confirming an in vivo synaptic function. These results suggest a prevalence of LRR domain proteins in trans-synaptic signaling and provide a cellular basis for the reported linkage of LRRTM1 to handedness and schizophrenia.

  7. Changes in brown eggshell color as the hen ages.

    PubMed

    Odabaşi, A Z; Miles, R D; Balaban, M O; Portier, K M

    2007-02-01

    The color of eggshells from eggs laid by commercial-type Hy-Line brown hens 25 wk of age was studied over a period of 10 mo. Color measurements were made by a color machine vision system and were analyzed using a mixed model to calculate between and within hen variances and to investigate the effect of time on shell color. Hens laid eggs with lighter colored shells as the flock aged, as evidenced by the lightness (L*) values increasing in time. A decrease in pigmentation was associated with a decrease in the amount of redness (a*) in the eggshell. When L* and a* values were corrected for egg weight, the rate of change in the L* and a* values decreased, indicating that size of the egg was a major factor affecting the color of the eggshell. These findings quantified the observations that older hens lay lighter colored eggs due to an increase in egg size associated with no proportionate change in the quantity of pigment deposited over the shell surface. Using a 2-stage sampling analysis and the variances between and within hens, sample sizes required to estimate the color of eggshells within 5% of the true mean were calculated. Accordingly, 11 eggs would need to be collected from each of the 51 hens housed for a study of brown eggshell color using the L*, a*, and b* (yellowness) coordinates.

  8. Atmospheric pressure plasma jet treatment of Salmonella Enteritidis inoculated eggshells.

    PubMed

    Moritz, Maike; Wiacek, Claudia; Koethe, Martin; Braun, Peggy G

    2017-03-20

    Contamination of eggshells with Salmonella Enteritidis remains a food safety concern. In many cases human salmonellosis within the EU can be traced back to raw or undercooked eggs and egg products. Atmospheric pressure plasma is a novel decontamination method that can reduce a wide range of pathogens. The aim of this work was to evaluate the possibility of using an effective short time cold plasma treatment to inactivate Salmonella Enteritidis on the eggshell. Therefore, artificially contaminated eggshells were treated with an atmospheric pressure plasma jet under different experimental settings with various exposure times (15-300s), distances from the plasma jet nozzle to the eggshell surface (5, 8 or 12mm), feed gas compositions (Ar, Ar with 0.2, 0.5 or 1.0% O2), gas flow rates (5 and 7slm) and different inoculations of Salmonella Enteritidis (10(1)-10(6)CFU/cm(2)). Atmospheric pressure plasma could reduce Salmonella Enteritidis on eggshells significantly. Reduction factors ranged between 0.22 and 2.27 log CFU (colony-forming units). Exposure time and, particularly at 10(4)CFU/cm(2) inoculation, feed gas had a major impact on Salmonella reduction. Precisely, longer exposure times led to higher reductions and Ar as feed gas was more effective than ArO2 mixtures.

  9. Identifying Novel Candidate Genes Related to Apoptosis from a Protein-Protein Interaction Network

    PubMed Central

    Wang, Baoman; Yuan, Fei; Kong, Xiangyin; Hu, Lan-Dian; Cai, Yu-Dong

    2015-01-01

    Apoptosis is the process of programmed cell death (PCD) that occurs in multicellular organisms. This process of normal cell death is required to maintain the balance of homeostasis. In addition, some diseases, such as obesity, cancer, and neurodegenerative diseases, can be cured through apoptosis, which produces few side effects. An effective comprehension of the mechanisms underlying apoptosis will be helpful to prevent and treat some diseases. The identification of genes related to apoptosis is essential to uncover its underlying mechanisms. In this study, a computational method was proposed to identify novel candidate genes related to apoptosis. First, protein-protein interaction information was used to construct a weighted graph. Second, a shortest path algorithm was applied to the graph to search for new candidate genes. Finally, the obtained genes were filtered by a permutation test. As a result, 26 genes were obtained, and we discuss their likelihood of being novel apoptosis-related genes by collecting evidence from published literature. PMID:26543496

  10. Paramagnetic Ligand Tagging To Identify Protein Binding Sites

    PubMed Central

    2015-01-01

    Transient biomolecular interactions are the cornerstones of the cellular machinery. The identification of the binding sites for low affinity molecular encounters is essential for the development of high affinity pharmaceuticals from weakly binding leads but is hindered by the lack of robust methodologies for characterization of weakly binding complexes. We introduce a paramagnetic ligand tagging approach that enables localization of low affinity protein–ligand binding clefts by detection and analysis of intermolecular protein NMR pseudocontact shifts, which are invoked by the covalent attachment of a paramagnetic lanthanoid chelating tag to the ligand of interest. The methodology is corroborated by identification of the low millimolar volatile anesthetic interaction site of the calcium sensor protein calmodulin. It presents an efficient route to binding site localization for low affinity complexes and is applicable to rapid screening of protein–ligand systems with varying binding affinity. PMID:26289584

  11. Protein networks identify novel symbiogenetic genes resulting from plastid endosymbiosis

    PubMed Central

    Méheust, Raphaël; Zelzion, Ehud; Bhattacharya, Debashish; Lopez, Philippe; Bapteste, Eric

    2016-01-01

    The integration of foreign genetic information is central to the evolution of eukaryotes, as has been demonstrated for the origin of the Calvin cycle and of the heme and carotenoid biosynthesis pathways in algae and plants. For photosynthetic lineages, this coordination involved three genomes of divergent phylogenetic origins (the nucleus, plastid, and mitochondrion). Major hurdles overcome by the ancestor of these lineages were harnessing the oxygen-evolving organelle, optimizing the use of light, and stabilizing the partnership between the plastid endosymbiont and host through retargeting of proteins to the nascent organelle. Here we used protein similarity networks that can disentangle reticulate gene histories to explore how these significant challenges were met. We discovered a previously hidden component of algal and plant nuclear genomes that originated from the plastid endosymbiont: symbiogenetic genes (S genes). These composite proteins, exclusive to photosynthetic eukaryotes, encode a cyanobacterium-derived domain fused to one of cyanobacterial or another prokaryotic origin and have emerged multiple, independent times during evolution. Transcriptome data demonstrate the existence and expression of S genes across a wide swath of algae and plants, and functional data indicate their involvement in tolerance to oxidative stress, phototropism, and adaptation to nitrogen limitation. Our research demonstrates the “recycling” of genetic information by photosynthetic eukaryotes to generate novel composite genes, many of which function in plastid maintenance. PMID:26976593

  12. Protein networks identify novel symbiogenetic genes resulting from plastid endosymbiosis.

    PubMed

    Méheust, Raphaël; Zelzion, Ehud; Bhattacharya, Debashish; Lopez, Philippe; Bapteste, Eric

    2016-03-29

    The integration of foreign genetic information is central to the evolution of eukaryotes, as has been demonstrated for the origin of the Calvin cycle and of the heme and carotenoid biosynthesis pathways in algae and plants. For photosynthetic lineages, this coordination involved three genomes of divergent phylogenetic origins (the nucleus, plastid, and mitochondrion). Major hurdles overcome by the ancestor of these lineages were harnessing the oxygen-evolving organelle, optimizing the use of light, and stabilizing the partnership between the plastid endosymbiont and host through retargeting of proteins to the nascent organelle. Here we used protein similarity networks that can disentangle reticulate gene histories to explore how these significant challenges were met. We discovered a previously hidden component of algal and plant nuclear genomes that originated from the plastid endosymbiont: symbiogenetic genes (S genes). These composite proteins, exclusive to photosynthetic eukaryotes, encode a cyanobacterium-derived domain fused to one of cyanobacterial or another prokaryotic origin and have emerged multiple, independent times during evolution. Transcriptome data demonstrate the existence and expression of S genes across a wide swath of algae and plants, and functional data indicate their involvement in tolerance to oxidative stress, phototropism, and adaptation to nitrogen limitation. Our research demonstrates the "recycling" of genetic information by photosynthetic eukaryotes to generate novel composite genes, many of which function in plastid maintenance.

  13. Linear Discriminant Analysis Identifies Mitochondrially Localized Proteins in Neurospora crassa.

    PubMed

    Wirsing, Lisette; Klawonn, Frank; Sassen, Wiebke Anna; Lünsdorf, Heinrich; Probst, Corinna; Hust, Michael; Mendel, Ralf R; Kruse, Tobias; Jänsch, Lothar

    2015-09-04

    Besides their role as powerhouses, mitochondria play a pivotal role in the spatial organization of numerous enzymatic functions. They are connected to the ER, and many pathways are organized through the mitochondrial membranes. Thus, the precise definition of mitochondrial proteomes remains a challenging task. Here, we have established a proteomic strategy to accurately determine the mitochondrial localization of proteins from the fungal model organism Neurospora crassa. This strategy relies on both highly pure mitochondria as well as the quantitative monitoring of mitochondrial components along their consecutive enrichment. Pure intact mitochondria were obtained by a multistep approach combining differential and density Percoll (ultra) centrifugations. When compared with three other intermediate enrichment stages, peptide sequencing and quantitative profiling of pure mitochondrial fractions revealed prototypic regulatory profiles of per se mitochondrial components. These regulatory profiles constitute a distinct cluster defining the mitochondrial compartment and support linear discriminant analyses, which rationalized the annotation process. In total, this approach experimentally validated the mitochondrial localization of 512 proteins including 57 proteins that had not been reported for N. crassa before.

  14. Nature's technical ceramic: the avian eggshell.

    PubMed

    Hahn, Eric N; Sherman, Vincent R; Pissarenko, Andrei; Rohrbach, Samuel D; Fernandes, Daniel J; Meyers, Marc A

    2017-01-01

    Avian eggshells may break easily when impacted at a localized point; however, they exhibit impressive resistance when subjected to a well-distributed compressive load. For example, a common demonstration of material strength is firmly squeezing a chicken egg along its major axis between one's hands without breaking it. This research provides insight into the underlying mechanics by evaluating both macroscopic and microstructural features. Eggs of different size, varying from quail (30 mm) to ostrich (150 mm), are investigated. Compression experiments were conducted along the major axis of the egg using force-distributing rubber cushions between steel plates and the egg. The force at failure increases with egg size, reaching loads upwards of 5000 N for ostrich eggs. The corresponding strength, however, decreases with increasing shell thickness (intimately related to egg size); this is rationalized by a micro-defects model. Failure occurs by axial splitting parallel to the loading direction-the result of hoop tensile stresses due to the applied compressive load. Finite-element analysis is successfully employed to correlate the applied compressive force to tensile breaking strength for the eggs, and the influence of geometric ratio and microstructural heterogeneities on the shell's strength and fracture toughness is established.

  15. Biological activities of peptide concentrates obtained from hydrolysed eggshell membrane byproduct by optimisation with response surface methodology.

    PubMed

    Santana, Ana; Melo, Armindo; Tavares, Tânia; Ferreira, Isabel M P L V O

    2016-11-09

    The increase of hen egg consumption demands profitable applications for eggshells, including their membranes, in order to minimize environmental and public health problems that could result from their accumulation. This work presents an innovative application for eggshell membranes to obtain an added-value food ingredient that combines maximized ACE-inhibitory and antioxidant activities. Firstly, the use of acetic acid 5% (v/v); and 3-mercaptopropionic acid 1.25 M enabled 63% recovery of eggshell membrane proteins. Secondly, the extracted proteins were hydrolysed by alcalase from Bacillus licheniformis, viscozyme L and protease from Bacillus amyloliquefaciens. Hydrolysis conditions were optimized using response surface methodology experimental design. The ACE-inhibitory activity (IC50) was 34.5 ± 2.1 μg mL(-1), 63.0 ± 4.2 μg mL(-1) and 43.0 ± 8.5 μg mL(-1) for each enzyme, respectively, and the antioxidant activity was ca. 4.0 μmoltrolox equivalent mg(-1)hydrolysed protein. The combination of both bioactive properties is of potential interest to control cardiovascular diseases.

  16. Traits of eggshells and shell membranes of translucent eggs.

    PubMed

    Wang, De-He; Li, Ya-Jie; Liu, Long; Liu, Jing-Shou; Bao, Man; Yang, Ning; Zhuo-Cheng, Hou; Ning, Zhong-Hua

    2017-02-01

    Translucent eggshells negatively affect the appearance of eggs and decrease their economic value. Translocation and accumulation of water from the contents to the shells of eggs are frequent occurrences. Causes of translucent eggshell formation have been investigated, but the primary reason is uncertain. In previous studies, scientists have found that the thickness of the eggshell membrane was significantly different between translucent and opaque eggs. However, there are some conflicts among studies. We performed 2 experiments with 3 breeding flocks of chickens to target the reasons for egg translucence. In experiment 1, eggs of 1,024 Brown-Egg Dwarf Layers (DWL) were used. Approximately 1,600 eggs were collected over 2 consecutive days. They were stored for 3 days, and then 120 translucent and 120 opaque eggs were selected for measurement of egg quality traits and weight loss over several weeks. In experiment 2, we used DWL and White Leghorn pure line (WLL) for assessment of eggshell ultrastructure and membrane traits. We chose 120 translucent and 120 opaque eggs from 3,500 DWL eggs and 125 translucent and 125 opaque eggs from 5,028 WLL eggs. The results are as follows: (1) translucent eggs had greater eggshell strength and lower ultimate failure stress of shell membrane than opaque eggs in both DWL and WLL groups, (2) translucent eggs had thicker shells and thinner shell membranes than opaque eggs in DWL, (3) no significant differences were found in either gas pore or bubble pore traits between translucent and opaque eggs in either line, and (4) no significant differences were detected in internal egg quality or weight loss between translucent and opaque eggs in either line. In summary, the present study suggests that variations in both eggshells and shell membrane structures are implicated in the formation of translucent eggs.

  17. Identifying and tracking proteins through the marine water column: insights into the inputs and preservation mechanisms of protein in sediments

    PubMed Central

    Moore, Eli K.; Nunn, Brook L.; Goodlett, David R.; Harvey, H. Rodger

    2012-01-01

    Proteins generated during primary production represent an important fraction of marine organic nitrogen and carbon, and have the potential to provide organism-specific information in the environment. The Bering Sea is a highly productive system dominated by seasonal blooms and was used as a model system for algal proteins to be tracked through the water column and incorporated into detrital sedimentary material. Samples of suspended and sinking particles were collected at multiple depths along with surface sediments on the continental shelf and deeper basin of the Bering Sea. Modified standard proteomic preparations were used in conjunction with high pressure liquid chromatography-tandem mass spectrometry to identify the suite of proteins present and monitor changes in their distribution. In surface waters 207 proteins were identified, decreasing through the water column to 52 proteins identified in post-bloom shelf surface sediments and 24 proteins in deeper (3490 m) basin sediments. The vast majority of identified proteins in all samples were diatom in origin, reflecting their dominant contribution of biomass during the spring bloom. Identified proteins were predominantly from metabolic, binding/structural, and transport-related protein groups. Significant linear correlations were observed between the number of proteins identified and the concentration of total hydrolysable amino acids normalized to carbon and nitrogen. Organelle-bound, transmembrane, photosynthetic, and other proteins involved in light harvesting were preferentially retained during recycling. These findings suggest that organelle and membrane protection represent important mechanisms that enhance the preservation of protein during transport and incorporation into sediments. PMID:22711915

  18. Hierarchical structure and mechanical properties of snake (Naja atra) and turtle (Ocadia sinensis) eggshells.

    PubMed

    Chang, Yin; Chen, Po-Yu

    2016-02-01

    After hundreds of million years of evolution, natural armors have evolved in various organisms, and has manifested in diverse forms such as eggshells, abalone shells, alligator osteoderms, turtle shells, and fish scales. Eggshells serve as multifunctional shields for successful embryogenesis, such as protection, moisture control and thermal regulation. Unlike calcareous avian eggshells which are brittle and hard, reptilians have leathery eggshells that are tough and flexible. Reptilian eggshells can withstand collision damages when laid in holes and dropped onto each other, and reduce abrasion caused by buried sand. In this study, we investigate structure and mechanical properties of eggshells of Taiwan cobra snake (Naja atra) and Chinese striped-neck turtle (Ocadia sinensis). From Acid Fuchsin Orange G (AFOG) staining and ATR-FTIR examination, we found that both eggshells are mainly composed of keratin. The mechanical properties of demineralized snake and turtle eggshells were evaluated by tensile and fracture tests and show distinctly difference. Turtle eggshells are relatively stiff and rigid, while snake eggshells behave as elastomers, which are highly extensible and reversible. The exceptional deformability (110-230% tensile strain) and toughness of snake eggshells are contributed by the wavy and random arrangement of keratin fibers as well as collagen layers. Multi-scale toughening mechanisms of snake eggshells were observed and elucidated, including crack deflection and twisting, fibers reorientation, sliding and bridging, inter-laminar shear effect, as well as the α-β phase transition of keratin. Inspirations from the structural and mechanical designs of reptilian eggshells may lead to the synthesis of tough, extensible, lightweight composites which could be further applied in the flexible devices, packaging and bio-medical fields. Amniotic eggshells serve as multifunctional shields for successful embryogenesis. The avian eggshells have been extensively

  19. Bioinformatic approaches to identifying and classifying Rab proteins.

    PubMed

    Diekmann, Yoan; Pereira-Leal, José B

    2015-01-01

    The bioinformatic annotation of Rab GTPases is important, for example, to understand the evolution of the endomembrane system. However, Rabs are particularly challenging for standard annotation pipelines because they are similar to other small GTPases and form a large family with many paralogous subfamilies. Here, we describe a bioinformatic annotation pipeline specifically tailored to Rab GTPases. It proceeds in two steps: first, Rabs are distinguished from other proteins based on GTPase-specific motifs, overall sequence similarity to other Rabs, and the occurrence of Rab-specific motifs. Second, Rabs are classified taking either a more accurate but slower phylogenetic approach or a slightly less accurate but much faster bioinformatic approach. All necessary steps can either be performed locally or using the referenced online tools. An implementation of a slightly more involved version of the pipeline presented here is available at RabDB.org.

  20. Dating lacustrine episodes in the eastern Sahara by the epimerization of isoleucine in ostrich eggshells

    USGS Publications Warehouse

    Miller, G.H.; Wendorf, F.; Ernst, R.; Schild, R.; Close, A.E.; Friedman, I.; Schwarcz, H.P.

    1991-01-01

    The eggshell of the African ostrich, Struthio camelus, closely approximates a closed system for the retention of indigenous proteinaceous residues. Epimerization of the protein amino acid isoleucine follows linear first-order kinetics in laboratory simulations nearly to racemic equilibrium, and the variation in D/L ratio within a single fragment, or between fragments of the same age, is significantly less than in other carbonate systems. These observations suggest that the extent of isoleucine epimerization (aIle/Ile ratio) in ostrich eggshell offers the potential for high-resolution geochronology of Quaternary deposits. From the simulation experiments, and dated early Holocene samples for which we have in situ mean annual sediment temperature measurements, Arrhenius parameters have been calculated; the activation energy is 30.33 kcal mol-1, similar to that of other carbonate systems. We have measured the aIle/Ile ratio in ostrich eggshell associated with lacustrine episodes at Bir Tarfawi and Bir Sahara East, two depressions in what is currently the hyperarid eastern Sahara. The ratios can be used directly to indicate qualitatively the time represented by each series of lake sediment, and to correlate disjunct lacustrine deposits within and between the basins. Uranium-series disequilibrium dating of algal mats contained within some of the lake beds indicate that a major wet interval occurred about 130 ka ago. Using the U-series date for calibration, the amino acid ratios are used to date the most recent lacustrine interval to about 100 ka B.P., and two older intervals, one about 200 ?? 25 ka B.P., and an older interval that occurred prior to 250 ka ago. ?? 1991.

  1. Identifying Interactions that Determine Fragment Binding at Protein Hotspots.

    PubMed

    Radoux, Chris J; Olsson, Tjelvar S G; Pitt, Will R; Groom, Colin R; Blundell, Tom L

    2016-05-12

    Locating a ligand-binding site is an important first step in structure-guided drug discovery, but current methods do little to suggest which interactions within a pocket are the most important for binding. Here we illustrate a method that samples atomic hotspots with simple molecular probes to produce fragment hotspot maps. These maps specifically highlight fragment-binding sites and their corresponding pharmacophores. For ligand-bound structures, they provide an intuitive visual guide within the binding site, directing medicinal chemists where to grow the molecule and alerting them to suboptimal interactions within the original hit. The fragment hotspot map calculation is validated using experimental binding positions of 21 fragments and subsequent lead molecules. The ligands are found in high scoring areas of the fragment hotspot maps, with fragment atoms having a median percentage rank of 97%. Protein kinase B and pantothenate synthetase are examined in detail. In each case, the fragment hotspot maps are able to rationalize a Free-Wilson analysis of SAR data from a fragment-based drug design project.

  2. Evaluation of Muscodor cinnamomi as an egg biofumigant for the reduction of microorganisms on eggshell surfaces and its effect on egg quality.

    PubMed

    Suwannarach, Nakarin; Kaewyana, Chariya; Yodmeeklin, Arpaporn; Kumla, Jaturong; Matsui, Kenji; Lumyong, Saisamorn

    2017-03-06

    The presence of microorganisms on the eggshell surface is a factor of consideration in determining egg quality. These microorganisms can contribute to egg spoilage and can infect the egg. In this study, 18 morphotypes of microorganisms were isolated from eggshells. Morphological, biochemical, physiological and molecular analyses were used to identify these morphotypes into 7 species; Bacillus drentensis, Staphylococcus arlettae, Stap. cohnii, Stap. kloosii, Stap. saprophyticus, Stap. sciuri and Stap. xylosus. The potential of Muscodor cinnamomi to reduce the presence of microorganisms on eggshells by biological fumigation was investigated. The result showed that 16 strains of the tested microorganisms were inactivated after the exposure of the fungal volatile organic compounds. The most abundant compound was 2-methylpropanoic acid, followed by 3-methylbutan-1-ol. Our results indicated that a 24-h period of fumigation of 100g rye grain culture of M. cinnamomi was the minimum dose that could significantly reduce the number of microorganisms on the eggshell surface. Fumigated eggs from both box and cabinet fumigation trials showed significantly lower microbial numbers on the eggshell than non-fumigated eggs during the storage period of 14days. It was found that the values of the yolk index, albumen index and the Haugh unit of the eggs decreased during this storage time. However, those values of the fumigated eggs from both fumigation trials were found to be significantly higher than the non-fumigated eggs after the 24-h fumigation period and following storage for 5, 7 and 14days. However, the values of the albumen index were not found to have significantly increased over 5days of the box trial. This study is the first to report on mycofumigation activity for the purposes of reducing the presence of microorganisms on the surface of eggshells. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Which Came First, the Eggshell or the Egg? Answering Biomineralization Riddles (442nd Brookhaven Lecture)

    SciTech Connect

    DiMasi, Elaine

    2008-11-12

    Some of the hardest and sturdiest materials are not made in the factory; they are made inside the bodies of animals through a process called biomineralization. Look no further than your refrigerator for one of the simplest products of this natural construction company: a chicken's eggshell. Made out of just about a half-millimeter of layered calcium carbonate and protein, eggshells might be thought of as fragile, but they also provide vital protection for the chick forming inside. Biomineralization, the process by which organisms form materials such as bones, mollusk shells, and other structures, has captured the attention of scientists for years. The cells in an animal's body have special ways of controlling the sizes and shapes of these mineral compounds and incorporating organic materials into the mix, making many materials that are stronger, harder, and more wear-resistant than rocks. Finding a way to mimic the properties of these sturdy and naturally made materials could lead to the medical engineering of replacement bone, teeth, and cartilage, as well as the development of new electronic and industrial materials. With collaborators at Stony Brook University, physicist Elaine DiMasi develops different biomineralization models, including a protein network that resembles real tissue. Then, the researchers use x-rays at the NSLS and a technique called shear modulation force microscopy to determine what biominerals look like and how they grow. In particular, DiMasi is interested in studying some of the earliest stages of biomineralization to find out what sets the process in motion.

  4. Protein polymorphism of human IL-18 identified by monoclonal antibodies.

    PubMed

    Seya, T; Matsumoto, M; Shiratori, I; Fukumori, Y; Toyoshima, K

    2001-11-01

    Six mAbs were raised against human "functionally inactive" recombinant IL-18, ELISA for determination of "functionally inactive" forms of IL-18 were established using two of these mAbs (#21 and #132), and inactive species of IL-18 protein were examined with human blood plasma and macrophages (Mp). In 6-day GM-CSF-treated monocytes, namely Mp, the mAb #21 recognized the IL-18 proform (24 kDa) and a 48 kDa dimer by immunoblotting. In contrast, only the 24 kDa species was detected as a relatively faint band with a commercial mAb against "active" IL-18. No IL-18 species was detected in premature monocytes. Thus, the dimeric IL-18 was produced in Mp and detectable with the mAb we established. In blood plasma of normal subjects and patients, the #21-recognizable IL-18 was also detected by ELISA, the levels of which were not consistent with those obtained with the commercially available kit for determination of "functionally active" IL-18. We designated the former as type 2 and the latter as type 1. Strikingly, IL-18 type 1 was detected in all volunteers while type 2 was detected in approximately 30% of healthy subjects, and the levels of type 2 were high (10-100 ng/ml) compared to those of type 1 (0.02-0.55 ng/ml) in their blood plasma. In patients with atopic dermatitis, the mean value of type 1 was high (200 ng/ml) compared to those of normal subjects (0.122 ng/ml) and patients with lung cancer (0.113 ng/ml). Production of high type 1 may be associated with an immunomodulatory state in atopic dermatitis. The levels and frequencies of IL-18 type 2 were not significantly changed among these populations. Hence, large amounts of type 2 species are produced in monocyte-Mp differentiation, and their levels and frequencies are unchanged in blood plasma irrespective of the levels of type 1.

  5. Contemporary techniques for detecting and identifying proteins susceptible to reversible thiol oxidation.

    PubMed

    Burgoyne, Joseph R; Eaton, Philip

    2011-10-01

    Elevated protein oxidation is a widely reported hallmark of most major diseases. Historically, this 'oxidative stress' has been considered causatively detrimental, as the protein oxidation events were interpreted simply as damage. However, recent advances have changed this antiquated view; sensitive methodology for detecting and identifying proteins susceptible to oxidation has revealed a fundamental role for this modification in physiological cell signalling during health. Reversible protein oxidation that is dynamically coupled with cellular reducing systems allows oxidative protein modifications to regulate protein function, analogous to phosphoregulation. However, the relatively labile nature of many reversible protein oxidation states hampers the reliable detection and identification of modified proteins. Consequently, specialized methods to stabilize protein oxidation in combination with techniques to detect specific types of modification have been developed. Here, these techniques are discussed, and their sensitivity, selectivity and ability to reliably identify reversibly oxidized proteins are critically assessed.

  6. Identification of discriminant proteins through antibody profiling, methods and apparatus for identifying an individual

    DOEpatents

    Thompson, Vicki S; Lacey, Jeffrey A; Gentillon, Cynthia A; Apel, William A

    2015-03-03

    A method for determining a plurality of proteins for discriminating and positively identifying an individual based from a biological sample. The method may include profiling a biological sample from a plurality of individuals against a protein array including a plurality of proteins. The protein array may include proteins attached to a support in a preselected pattern such that locations of the proteins are known. The biological sample may be contacted with the protein array such that a portion of antibodies in the biological sample reacts with and binds to the proteins forming immune complexes. A statistical analysis method, such as discriminant analysis, may be performed to determine discriminating proteins for distinguishing individuals. Proteins of interest may be used to form a protein array. Such a protein array may be used, for example, to compare a forensic sample from an unknown source with a sample from a known source.

  7. Identification of discriminant proteins through antibody profiling, methods and apparatus for identifying an individual

    DOEpatents

    Apel, William A.; Thompson, Vicki S; Lacey, Jeffrey A.; Gentillon, Cynthia A.

    2016-08-09

    A method for determining a plurality of proteins for discriminating and positively identifying an individual based from a biological sample. The method may include profiling a biological sample from a plurality of individuals against a protein array including a plurality of proteins. The protein array may include proteins attached to a support in a preselected pattern such that locations of the proteins are known. The biological sample may be contacted with the protein array such that a portion of antibodies in the biological sample reacts with and binds to the proteins forming immune complexes. A statistical analysis method, such as discriminant analysis, may be performed to determine discriminating proteins for distinguishing individuals. Proteins of interest may be used to form a protein array. Such a protein array may be used, for example, to compare a forensic sample from an unknown source with a sample from a known source.

  8. Ras1 interacts with multiple new signaling and cytoskeletal loci in Drosophila eggshell patterning and morphogenesis.

    PubMed Central

    Schnorr, J D; Holdcraft, R; Chevalier, B; Berg, C A

    2001-01-01

    Little is known about the genes that interact with Ras signaling pathways to regulate morphogenesis. The synthesis of dorsal eggshell structures in Drosophila melanogaster requires multiple rounds of Ras signaling followed by dramatic epithelial sheet movements. We took advantage of this process to identify genes that link patterning and morphogenesis; we screened lethal mutations on the second chromosome for those that could enhance a weak Ras1 eggshell phenotype. Of 1618 lethal P-element mutations tested, 13 showed significant enhancement, resulting in forked and fused dorsal appendages. Our genetic and molecular analyses together with information from the Berkeley Drosophila Genome Project reveal that 11 of these lines carry mutations in previously characterized genes. Three mutations disrupt the known Ras1 cell signaling components Star, Egfr, and Blistered, while one mutation disrupts Sec61beta, implicated in ligand secretion. Seven lines represent cell signaling and cytoskeletal components that are new to the Ras1 pathway; these are Chickadee (Profilin), Tec29, Dreadlocks, POSH, Peanut, Smt3, and MESK2, a suppressor of dominant-negative Ksr. A twelfth insertion disrupts two genes, Nrk, a "neurospecific" receptor tyrosine kinase, and Tpp, which encodes a neuropeptidase. These results suggest that Ras1 signaling during oogenesis involves novel components that may be intimately associated with additional signaling processes and with the reorganization of the cytoskeleton. To determine whether these Ras1 Enhancers function upstream or downstream of the Egf receptor, four mutations were tested for their ability to suppress an activated Egfr construct (lambdatop) expressed in oogenesis exclusively in the follicle cells. Mutations in Star and l(2)43Bb had no significant effect upon the lambdatop eggshell defect whereas smt3 and dock alleles significantly suppressed the lambdatop phenotype. PMID:11606538

  9. Ras1 interacts with multiple new signaling and cytoskeletal loci in Drosophila eggshell patterning and morphogenesis.

    PubMed

    Schnorr, J D; Holdcraft, R; Chevalier, B; Berg, C A

    2001-10-01

    Little is known about the genes that interact with Ras signaling pathways to regulate morphogenesis. The synthesis of dorsal eggshell structures in Drosophila melanogaster requires multiple rounds of Ras signaling followed by dramatic epithelial sheet movements. We took advantage of this process to identify genes that link patterning and morphogenesis; we screened lethal mutations on the second chromosome for those that could enhance a weak Ras1 eggshell phenotype. Of 1618 lethal P-element mutations tested, 13 showed significant enhancement, resulting in forked and fused dorsal appendages. Our genetic and molecular analyses together with information from the Berkeley Drosophila Genome Project reveal that 11 of these lines carry mutations in previously characterized genes. Three mutations disrupt the known Ras1 cell signaling components Star, Egfr, and Blistered, while one mutation disrupts Sec61beta, implicated in ligand secretion. Seven lines represent cell signaling and cytoskeletal components that are new to the Ras1 pathway; these are Chickadee (Profilin), Tec29, Dreadlocks, POSH, Peanut, Smt3, and MESK2, a suppressor of dominant-negative Ksr. A twelfth insertion disrupts two genes, Nrk, a "neurospecific" receptor tyrosine kinase, and Tpp, which encodes a neuropeptidase. These results suggest that Ras1 signaling during oogenesis involves novel components that may be intimately associated with additional signaling processes and with the reorganization of the cytoskeleton. To determine whether these Ras1 Enhancers function upstream or downstream of the Egf receptor, four mutations were tested for their ability to suppress an activated Egfr construct (lambdatop) expressed in oogenesis exclusively in the follicle cells. Mutations in Star and l(2)43Bb had no significant effect upon the lambdatop eggshell defect whereas smt3 and dock alleles significantly suppressed the lambdatop phenotype.

  10. Proteome Analysis. Novel Proteins Identified at the Peribacteroid Membrane from Lotus japonicus Root Nodules1

    PubMed Central

    Wienkoop, Stefanie; Saalbach, Gerhard

    2003-01-01

    The peribacteroid membrane (PBM) forms the structural and functional interface between the legume plant and the rhizobia. The model legume Lotus japonicus was chosen to study the proteins present at the PBM by proteome analysis. PBM was purified from root nodules by an aqueous polymer two-phase system. Extracted proteins were subjected to a global trypsin digest. The peptides were separated by nanoscale liquid chromatography and analyzed by tandem mass spectrometry. Searching the nonredundant protein database and the green plant expressed sequence tag database using the tandem mass spectrometry data identified approximately 94 proteins, a number far exceeding the number of proteins reported for the PBM hitherto. In particular, a number of membrane proteins like transporters for sugars and sulfate; endomembrane-associated proteins such as GTP-binding proteins and vesicle receptors; and proteins involved in signaling, for example, receptor kinases, calmodulin, 14-3-3 proteins, and pathogen response-related proteins, including a so-called HIR protein, were detected. Several ATPases and aquaporins were present, indicating a more complex situation than previously thought. In addition, the unexpected presence of a number of proteins known to be located in other compartments was observed. Two characteristic protein complexes obtained from native gel electrophoresis of total PBM proteins were also analyzed. Together, the results identified specific proteins at the PBM involved in important physiological processes and localized proteins known from nodule-specific expressed sequence tag databases to the PBM. PMID:12644660

  11. On the heritability of blue-green eggshell coloration.

    PubMed

    Morales, J; Kim, S-Y; Lobato, E; Merino, S; Tomás, G; Martínez-de la Puente, J; Moreno, J

    2010-08-01

    Avian blue-green eggshell coloration has been proposed as a female signal of genetic or phenotypic quality to males. However, little is known about the relative importance of additive genetic and environmental effects as sources of eggshell colour variation in natural populations. Using 5 years of data and animal models, we explored these effects in a free-living population of pied flycatchers. Permanent environmental and year effects were negligible, although year environmental variance (V(Year)) was significant for all but one of the traits. However, we found high-moderate narrow-sense heritabilities for some colour parameters. Within-clutch colour variability showed the highest coefficient of additive genetic variation (i.e. evolvability). Previous evidence suggests that eggshell colour is sexually selected in this species, males enhancing parental effort in clutches with higher colour variability and peak values. Eggshell colour could be driven by good-genes selection in pied flycatchers although further genetic studies should confirm this possibility.

  12. Organochlorine residues and eggshell thinning in wood storks and anhingas

    USGS Publications Warehouse

    Ohlendorf, H.M.; Klaas, E.E.; Kaiser, T.E.

    1978-01-01

    All 10 Wood Stork eggs collected at Merritt Island National Wildlife Refuge in 1973 contained residues of DDE (geometric mean 4.0 ppm wet weight) and PCBs (1.2 ppm). Nine other organochlorines were found at lower frequencies in the eggs. Eggshells from the recent period were 8.9% thinner (P < 0.001) than pre-1947 samples; decrease in eggshell thickness was more closely correlated with DDE than other organochlorines and correlation of DDE and eggshell thickness approached significance (P = 0.115).....Anhinga eggs were collected at 7 localities; 45 of the 46 eggs analyzed contained DDE residues and 24 contained PCBs. Residues of other organochlorines were found less frequently. Shell thickness of recent eggs from Louisiana and Mississippi was significantly less (-7.5%; P < 0.05) than the mean for pre-1947 eggs, but there was no significant change in shell thickness of eggs from Florida. The change in clutch mean eggshell thickness was significantly negatively correlated (P < 0.05) with the concentration of DDE in the eggs.

  13. Identifying solubility-promoting buffers for intrinsically disordered proteins prior to purification.

    PubMed

    Churion, Kelly A; Bondos, Sarah E

    2012-01-01

    Intrinsically disordered proteins are anticipated to be more prone to aggregation than folded, stable proteins. Chemical additives included in the buffer can help maintain proteins in a soluble, monomeric state. However, the array of chemicals that impact protein solubility is staggering, precluding iterative testing of chemical conditions during purification. Herein, we describe a filter-based aggregation assay to rapidly identify chemical additives that maintain solubility for a protein of interest. A hierarchical approach to buffer selection is provided, in which the type of chemical which best improves solubility is first determined, followed by identifying the optimal chemical and its most effective concentration. Finally, combinations of chemical additives can be assessed if necessary. Although this assay can be applied to purified protein, partially purified protein, or aggregated protein, this protocol specifically details the use of this assay for crude cell lysate. This approach allows identification of solubility-promoting buffers prior to the initial protein purification.

  14. A Simple Approach for an Eggshell-Based 3D-Printed Osteoinductive Multiphasic Calcium Phosphate Scaffold.

    PubMed

    Dadhich, Prabhash; Das, Bodhisatwa; Pal, Pallabi; Srivas, Pavan K; Dutta, Joy; Ray, Sabyasachi; Dhara, Santanu

    2016-05-18

    Natural origin bioceramics are widely used for bone grafts. In the present study, an eggshell-derived bioceramic scaffold is fabricated by 3D printing as a potential bone-graft analogue. The eggshell, a biological waste material, was mixed with a specific ratio of phosphoric acid and chitosan to form a precursor toward the fabrication of an osteoinductive multiphasic calcium phosphate scaffold via a coagulation-assisted extrusion and sintering for a multiscalar hierarchical porous structure with improved mechanical properties. Physicochemical characterization of the formed scaffolds was carried out for phase analysis, surface morphology, and mechanical properties. A similar scaffold was prepared using a chemically synthesized calcium phosphate powder that was compared with the natural origin one. The higher surface area associated with the interconnected porosity along with multiple phases of the natural origin scaffold facilitated higher cell adhesion and proliferation compared to the chemically synthesized one. Further, the natural origin scaffold displayed relatively higher cell differentiation activity, as is evident by protein and gene expression studies. On subcutaneous implantation for 30 days, promising vascular tissue in-growth was observed, circumventing a major foreign body response. Collagen-rich vascular extracellular matrix deposition and osteocalcin secretion indicated bonelike tissue formation. Finally, the eggshell-derived multiphasic calcium phosphate scaffold displayed improvement in the mechanical properties with higher porosity and osteoinductivity compared to the chemically derived apatite and unveiled a new paradigm for utilization of biological wastes in bone-graft application.

  15. Pathway Analysis Incorporating Protein-Protein Interaction Networks Identified Candidate Pathways for the Seven Common Diseases

    PubMed Central

    Lin, Peng-Lin; Yu, Ya-Wen

    2016-01-01

    Pathway analysis has become popular as a secondary analysis strategy for genome-wide association studies (GWAS). Most of the current pathway analysis methods aggregate signals from the main effects of single nucleotide polymorphisms (SNPs) in genes within a pathway without considering the effects of gene-gene interactions. However, gene-gene interactions can also have critical effects on complex diseases. Protein-protein interaction (PPI) networks have been used to define gene pairs for the gene-gene interaction tests. Incorporating the PPI information to define gene pairs for interaction tests within pathways can increase the power for pathway-based association tests. We propose a pathway association test, which aggregates the interaction signals in PPI networks within a pathway, for GWAS with case-control samples. Gene size is properly considered in the test so that genes do not contribute more to the test statistic simply due to their size. Simulation studies were performed to verify that the method is a valid test and can have more power than other pathway association tests in the presence of gene-gene interactions within a pathway under different scenarios. We applied the test to the Wellcome Trust Case Control Consortium GWAS datasets for seven common diseases. The most significant pathway is the chaperones modulate interferon signaling pathway for Crohn’s disease (p-value = 0.0003). The pathway modulates interferon gamma, which induces the JAK/STAT pathway that is involved in Crohn’s disease. Several other pathways that have functional implications for the seven diseases were also identified. The proposed test based on gene-gene interaction signals in PPI networks can be used as a complementary tool to the current existing pathway analysis methods focusing on main effects of genes. An efficient software implementing the method is freely available at http://puppi.sourceforge.net. PMID:27622767

  16. Ciliate pellicular proteome identifies novel protein families with characteristic repeat motifs that are common to alveolates.

    PubMed

    Gould, Sven B; Kraft, Lesleigh G K; van Dooren, Giel G; Goodman, Christopher D; Ford, Kristina L; Cassin, Andrew M; Bacic, Antony; McFadden, Geoffrey I; Waller, Ross F

    2011-03-01

    The pellicles of alveolates (ciliates, apicomplexans, and dinoflagellates) share a common organization, yet perform very divergent functions, including motility, host cell invasion, and armor. The alveolate pellicle consists of a system of flattened membrane sacs (alveoli, which are the defining feature of the group) below the plasma membrane that is supported by a membrane skeleton as well as a network of microtubules and other filamentous elements. We recently showed that a family of proteins, alveolins, are common and unique to this pellicular structure in alveolates. To identify additional proteins that contribute to this structure, a pellicle proteome study was conducted for the ciliate Tetrahymena thermophila. We found 1,173 proteins associated with this structure, 45% (529 proteins) of which represented novel proteins without matches to other functionally characterized proteins. Expression of four newly identified T. thermophila pellicular proteins as green fluorescent protein-fusion constructs confirmed pellicular location, and one new protein located in the oral apparatus. Bioinformatic analysis revealed that 21% of the putative pellicular proteins, predominantly the novel proteins, contained highly repetitive regions with strong amino acid biases for particular residues (K, E, Q, L, I, and V). When the T. thermophila novel proteins were compared with apicomplexan genomic data, 278 proteins with high sequence similarity were identified, suggesting that many of these putative pellicular components are shared between the alveolates. Of these shared proteins, 126 contained the distinctive repeat regions. Localization of two such proteins in Toxoplasma gondii confirmed their role in the pellicle and in doing so identified two new proteins of the apicomplexan invasive structure--the apical complex. Screening broadly for these repetitive domains in genomic data revealed large and actively evolving families of such proteins in alveolates, suggesting that these

  17. Sulfur-based autotrophic denitrification with eggshell for nitrate-contaminated synthetic groundwater treatment.

    PubMed

    Xu, Yaxian; Chen, Nan; Feng, Chuanping; Hao, Chunbo; Peng, Tong

    2016-12-01

    Eggshell is considered to be a waste and a significant quantity of eggshell waste is generated from food processing, baking and hatching industries. In this study, the effect of different sulfur/eggshell (w/w) ratios and temperatures was investigated to evaluate the feasibility of the sulfur-based autotrophic denitrification with eggshell (SADE) process for nitrate removal. The results showed eggshell can maintain a neutral condition in a range of pH 7.05-7.74 in the SADE process, and remove 97% of nitrate in synthetic groundwater. Compared with oyster shell and limestone, eggshell was found to be a desirable alkaline material for sulfur-based autotrophic denitrification (SAD) with no nitrite accumulation and insignificant sulfate production. Denitrification reaction was found to follow the first-order kinetic models (R(2) > .9) having nitrate removal rate constants of 0.85 and 0.93 d(-1) for raw eggshell and boiled eggshell, respectively. Sulfur/eggshell ratio of 2:3 provided the best efficiency on nitrate removal. Nitrate was removed completely by the SADE process at a low temperature of 15°C. Eggshell could be used for the SAD process due to its good effect for nitrate removal from groundwater.

  18. Identifying the hierarchy of dynamic domains in proteins using the data of molecular dynamics simulations.

    PubMed

    Yesylevskyy, Semen O

    2010-04-01

    The Hierarchical Domain-Wise Alignment (HDWA) technique of domain identification in proteins is presented. HDWA is designed to identify hierarchically organized dynamic domains in proteins using the MD trajectories by eliminating systematic motions from MD trajectories recursively in a model-free manner. The method is tested on the proteins from different structural classes.

  19. Identifying proteins that bind a known RNA sequence using the yeast three-hybrid system.

    PubMed

    Koh, Yvonne Y; Wickens, Marvin

    2014-01-01

    The yeast three-hybrid system can be used to identify a protein partner of a known RNA sequence by screening a cDNA library fused to a transcription activation domain, with a hybrid RNA as 'bait.' Most commonly, such screens are performed to identify proteins that interact with a given RNA in vivo. © 2014 Elsevier Inc. All rights reserved.

  20. Change in the chicken eggshell cuticle with hen age and egg freshness.

    PubMed

    Rodríguez-Navarro, Alejandro B; Domínguez-Gasca, Nazaret; Muñoz, Arantxa; Ortega-Huertas, Miguel

    2013-11-01

    For a fuller understanding of the functionality of the eggshell cuticle, we conducted a detailed study using a wide array of analytical techniques (scanning and transmission microscopy), energy dispersive x-rays, and attenuated total reflection-Fourier transform infrared spectroscopy to analyze the structure, morphology, and chemical composition of this organic coating. This study shows that the cuticle has a compositional gradation with an outer part richer in proteins and an inner part richer in sulfated polysaccharides and phosphates. It also shown that the cuticle composition, thickness, and degree of coverage are highly dependent on hen age and egg freshness. During the course of the first laying year, the thickness and degree of glycosylation of the cuticle decreases with hen age, and at the end of the laying cycle, the cuticle is significantly depleted in lipids. There are also well-defined compositional changes in the cuticle of freshly laid eggs as time passes and there is a notable increase in the permeability of the eggshell after 24 h due to cuticle drying. We discuss how these changes in the cuticle can affect the food safety of eggs in relation to the risk of trans-shell contamination by bacteria (i.e., Salmonellosis).

  1. A Topology Potential-Based Method for Identifying Essential Proteins from PPI Networks.

    PubMed

    Li, Min; Lu, Yu; Wang, Jianxin; Wu, Fang-Xiang; Pan, Yi

    2015-01-01

    Essential proteins are indispensable for cellular life. It is of great significance to identify essential proteins that can help us understand the minimal requirements for cellular life and is also very important for drug design. However, identification of essential proteins based on experimental approaches are typically time-consuming and expensive. With the development of high-throughput technology in the post-genomic era, more and more protein-protein interaction data can be obtained, which make it possible to study essential proteins from the network level. There have been a series of computational approaches proposed for predicting essential proteins based on network topologies. Most of these topology based essential protein discovery methods were to use network centralities. In this paper, we investigate the essential proteins' topological characters from a completely new perspective. To our knowledge it is the first time that topology potential is used to identify essential proteins from a protein-protein interaction (PPI) network. The basic idea is that each protein in the network can be viewed as a material particle which creates a potential field around itself and the interaction of all proteins forms a topological field over the network. By defining and computing the value of each protein's topology potential, we can obtain a more precise ranking which reflects the importance of proteins from the PPI network. The experimental results show that topology potential-based methods TP and TP-NC outperform traditional topology measures: degree centrality (DC), betweenness centrality (BC), closeness centrality (CC), subgraph centrality (SC), eigenvector centrality (EC), information centrality (IC), and network centrality (NC) for predicting essential proteins. In addition, these centrality measures are improved on their performance for identifying essential proteins in biological network when controlled by topology potential.

  2. Effect of Egg Washing and Correlation between Eggshell Characteristics and Egg Penetration by Various Salmonella Typhimurium Strains

    PubMed Central

    Gole, Vaibhav C.; Chousalkar, Kapil K.; Roberts, Juliet R.; Sexton, Margaret; May, Damian; Tan, Jessica; Kiermeier, Andreas

    2014-01-01

    Salmonella is an important foodborne pathogen, causing an estimated 11,992 cases of infection in Australia per year. Egg or egg product related salmonellosis is a major concern for the egg industry. Worldwide, S. Typhimurium is one of the most common serovars identified in Salmonella food poisoning cases. The current study investigated the ability of five S. Typhimurium strains to penetrate washed and unwashed eggs using whole egg and agar egg penetration methods. All S. Typhimurium strains were able to penetrate eggshells and survive in egg albumen (at 20°C) according to whole egg penetration results. Polymerase Chain Reaction results demonstrated that S. Typhimurium strain 2 (103 and 105 CFU/mL), and strain 5 (103 and 105 CFU/mL) egg penetration was significantly higher (p<0.05) in washed eggs when compared to unwashed eggs. Statistical analysis of the agar penetration experiment indicated that S. Typhimurium was able to penetrate washed eggs at a significantly higher rate when compared to unwashed eggs (p<0.05). When compared to unwashed eggs, washed eggs also had significantly damaged cuticles. Statistical analysis also indicated that eggshell penetration by S. Typhimurium was related to various eggshell ultrastructural features such as cap quality, alignment, erosion, confluence, Type B bodies and cuticle cover. PMID:24621821

  3. Effect of egg washing and correlation between eggshell characteristics and egg penetration by various Salmonella Typhimurium strains.

    PubMed

    Gole, Vaibhav C; Chousalkar, Kapil K; Roberts, Juliet R; Sexton, Margaret; May, Damian; Tan, Jessica; Kiermeier, Andreas

    2014-01-01

    Salmonella is an important foodborne pathogen, causing an estimated 11,992 cases of infection in Australia per year. Egg or egg product related salmonellosis is a major concern for the egg industry. Worldwide, S. Typhimurium is one of the most common serovars identified in Salmonella food poisoning cases. The current study investigated the ability of five S. Typhimurium strains to penetrate washed and unwashed eggs using whole egg and agar egg penetration methods. All S. Typhimurium strains were able to penetrate eggshells and survive in egg albumen (at 20°C) according to whole egg penetration results. Polymerase Chain Reaction results demonstrated that S. Typhimurium strain 2 (10(3) and 10(5) CFU/mL), and strain 5 (10(3) and 10(5) CFU/mL) egg penetration was significantly higher (p<0.05) in washed eggs when compared to unwashed eggs. Statistical analysis of the agar penetration experiment indicated that S. Typhimurium was able to penetrate washed eggs at a significantly higher rate when compared to unwashed eggs (p<0.05). When compared to unwashed eggs, washed eggs also had significantly damaged cuticles. Statistical analysis also indicated that eggshell penetration by S. Typhimurium was related to various eggshell ultrastructural features such as cap quality, alignment, erosion, confluence, Type B bodies and cuticle cover.

  4. A new computational strategy for identifying essential proteins based on network topological properties and biological information.

    PubMed

    Qin, Chao; Sun, Yongqi; Dong, Yadong

    2017-01-01

    Essential proteins are the proteins that are indispensable to the survival and development of an organism. Deleting a single essential protein will cause lethality or infertility. Identifying and analysing essential proteins are key to understanding the molecular mechanisms of living cells. There are two types of methods for predicting essential proteins: experimental methods, which require considerable time and resources, and computational methods, which overcome the shortcomings of experimental methods. However, the prediction accuracy of computational methods for essential proteins requires further improvement. In this paper, we propose a new computational strategy named CoTB for identifying essential proteins based on a combination of topological properties, subcellular localization information and orthologous protein information. First, we introduce several topological properties of the protein-protein interaction (PPI) network. Second, we propose new methods for measuring orthologous information and subcellular localization and a new computational strategy that uses a random forest prediction model to obtain a probability score for the proteins being essential. Finally, we conduct experiments on four different Saccharomyces cerevisiae datasets. The experimental results demonstrate that our strategy for identifying essential proteins outperforms traditional computational methods and the most recently developed method, SON. In particular, our strategy improves the prediction accuracy to 89, 78, 79, and 85 percent on the YDIP, YMIPS, YMBD and YHQ datasets at the top 100 level, respectively.

  5. Does contrast between eggshell ground and spot coloration affect egg rejection?

    NASA Astrophysics Data System (ADS)

    Dainson, Miri; Hauber, Mark E.; López, Analía V.; Grim, Tomáš; Hanley, Daniel

    2017-08-01

    Obligate avian brood parasitic species impose the costs of incubating foreign eggs and raising young upon their unrelated hosts. The most common host defence is the rejection of parasitic eggs from the nest. Both egg colours and spot patterns influence egg rejection decisions in many host species, yet no studies have explicitly examined the role of variation in spot coloration. We studied the American robin Turdus migratorius, a blue-green unspotted egg-laying host of the brown-headed cowbird Molothrus ater, a brood parasite that lays non-mimetic spotted eggs. We examined host responses to model eggs with variable spot coloration against a constant robin-mimetic ground colour to identify patterns of rejection associated with perceived contrast between spot and ground colours. By using avian visual modelling, we found that robins were more likely to reject eggs whose spots had greater chromatic (hue) but not achromatic (brightness) contrast. Therefore, egg rejection decision rules in the American robin may depend on the colour contrast between parasite eggshell spot and host ground coloration. Our study also suggests that egg recognition in relation to spot coloration, like ground colour recognition, is tuned to the natural variation of avian eggshell spot colours but not to unnatural spot colours.

  6. Comparative influenza protein interactomes identify the role of plakophilin 2 in virus restriction

    PubMed Central

    Wang, Lingyan; Fu, Bishi; Li, Wenjun; Patil, Girish; Liu, Lin; Dorf, Martin E.; Li, Shitao

    2017-01-01

    Cellular protein interaction networks are integral to host defence and immune signalling pathways, which are often hijacked by viruses via protein interactions. However, the comparative virus–host protein interaction networks and how these networks control host immunity and viral infection remain to be elucidated. Here, we mapped protein interactomes between human host and several influenza A viruses (IAV). Comparative analyses of the interactomes identified common and unique interaction patterns regulating innate immunity and viral infection. Functional screening of the ‘core‘ interactome consisting of common interactions identified five novel host factors regulating viral infection. Plakophilin 2 (PKP2), an influenza PB1-interacting protein, restricts IAV replication and competes with PB2 for PB1 binding. The binding competition leads to perturbation of the IAV polymerase complex, thereby limiting polymerase activity and subsequent viral replication. Taken together, comparative analyses of the influenza–host protein interactomes identified PKP2 as a natural inhibitor of IAV polymerase complex. PMID:28169297

  7. Development of 'Redox Arrays' for identifying novel glutathionylated proteins in the secretome.

    PubMed

    Mullen, Lisa; Seavill, Miles; Hammouz, Raneem; Bottazzi, Barbara; Chan, Philippe; Vaudry, David; Ghezzi, Pietro

    2015-09-29

    Proteomics techniques for analysing the redox status of individual proteins in complex mixtures tend to identify the same proteins due to their high abundance. We describe here an array-based technique to identify proteins undergoing glutathionylation and apply it to the secretome and the proteome of human monocytic cells. The method is based on incorporation of biotinylated glutathione (GSH) into proteins, which can then be identified following binding to a 1000-protein antibody array. We thus identify 38 secreted and 55 intracellular glutathionylated proteins, most of which are novel candidates for glutathionylation. Two of the proteins identified in these experiments, IL-1 sRII and Lyn, were then confirmed to be susceptible to glutathionylation. Comparison of the redox array with conventional proteomic methods confirmed that the redox array is much more sensitive, and can be performed using more than 100-fold less protein than is required for methods based on mass spectrometry. The identification of novel targets of glutathionylation, particularly in the secretome where the protein concentration is much lower, shows that redox arrays can overcome some of the limitations of established redox proteomics techniques.

  8. Development of ‘Redox Arrays’ for identifying novel glutathionylated proteins in the secretome

    PubMed Central

    Mullen, Lisa; Seavill, Miles; Hammouz, Raneem; Bottazzi, Barbara; Chan, Philippe; Vaudry, David; Ghezzi, Pietro

    2015-01-01

    Proteomics techniques for analysing the redox status of individual proteins in complex mixtures tend to identify the same proteins due to their high abundance. We describe here an array-based technique to identify proteins undergoing glutathionylation and apply it to the secretome and the proteome of human monocytic cells. The method is based on incorporation of biotinylated glutathione (GSH) into proteins, which can then be identified following binding to a 1000-protein antibody array. We thus identify 38 secreted and 55 intracellular glutathionylated proteins, most of which are novel candidates for glutathionylation. Two of the proteins identified in these experiments, IL-1 sRII and Lyn, were then confirmed to be susceptible to glutathionylation. Comparison of the redox array with conventional proteomic methods confirmed that the redox array is much more sensitive, and can be performed using more than 100-fold less protein than is required for methods based on mass spectrometry. The identification of novel targets of glutathionylation, particularly in the secretome where the protein concentration is much lower, shows that redox arrays can overcome some of the limitations of established redox proteomics techniques. PMID:26416726

  9. Quality assessment of chicken eggshell cuticle by infrared spectroscopy and staining techniques: a comparative study.

    PubMed

    Dominguez-Gasca, N; Muñoz, A; Rodriguez-Navarro, A B

    2017-07-20

    1. The cuticle is a very thin organic layer that coats the eggshell surface and plugs the eggshell pores preventing bacterial penetration. It also reduces eggshell permeability which is important to maintain internal quality of the egg. Thus, the eggshell cuticle quality is crucial to ensure the food safety and quality of eggs. 2. A new methodology to assess eggshell cuticle quality, based on attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), is compared with a more traditional method based on cuticle staining techniques. 3. Both techniques are useful to determine the amount of cuticle; however, the ATR-FTIR technique is independent of egg colour, more sensitive and provides complete information about the cuticle and its composition. Additionally, it provides information about eggshell permeability. 4. The methodology for cuticle quality assessment described in this work can be very useful for genetic selection programmes aimed to improve the safety and quality of eggs.

  10. Novel royal jelly proteins identified by gel-based and gel-free proteomics.

    PubMed

    Han, Bin; Li, Chenxi; Zhang, Lan; Fang, Yu; Feng, Mao; Li, Jianke

    2011-09-28

    Royal jelly (RJ) plays an important role in caste determination of the honeybee; the genetically same female egg develops into either a queen or worker bee depending on the time and amount of RJ fed to the larvae. RJ also has numerous health-promoting properties for humans. Gel-based and gel-free proteomics approaches and high-performance liquid chromatography-chip quadruple time-of-flight tandem mass spectrometry were applied to comprehensively investigate the protein components of RJ. Overall, 37 and 22 nonredundant proteins were identified by one-dimensional gel electrophoresis and gel-free analysis, respectively, and 19 new proteins were found by these two proteomics approaches. Major royal jelly proteins (MRJPs) were identified as the principal protein components of RJ, and proteins related to carbohydrate metabolism such as glucose oxidase, α-glucosidase precursor, and glucose dehydrogenase were also successfully identified. Importantly, the 19 newly identified proteins were mainly classified into three functional categories: oxidation-reduction (ergic53 CG6822-PA isoform A isoform 1, Sec61 CG9539-PA, and ADP/ATP translocase), protein binding (regucalcin and translationally controlled tumor protein CG4800-PA isoform 1), and lipid transport (apolipophorin-III-like protein). These new findings not only significantly increase the RJ proteome coverage but also help to provide new knowledge of RJ for honeybee biology and potential use for human health promotion.

  11. Protein-protein interaction networks identify targets which rescue the MPP+ cellular model of Parkinson’s disease

    NASA Astrophysics Data System (ADS)

    Keane, Harriet; Ryan, Brent J.; Jackson, Brendan; Whitmore, Alan; Wade-Martins, Richard

    2015-11-01

    Neurodegenerative diseases are complex multifactorial disorders characterised by the interplay of many dysregulated physiological processes. As an exemplar, Parkinson’s disease (PD) involves multiple perturbed cellular functions, including mitochondrial dysfunction and autophagic dysregulation in preferentially-sensitive dopamine neurons, a selective pathophysiology recapitulated in vitro using the neurotoxin MPP+. Here we explore a network science approach for the selection of therapeutic protein targets in the cellular MPP+ model. We hypothesised that analysis of protein-protein interaction networks modelling MPP+ toxicity could identify proteins critical for mediating MPP+ toxicity. Analysis of protein-protein interaction networks constructed to model the interplay of mitochondrial dysfunction and autophagic dysregulation (key aspects of MPP+ toxicity) enabled us to identify four proteins predicted to be key for MPP+ toxicity (P62, GABARAP, GBRL1 and GBRL2). Combined, but not individual, knockdown of these proteins increased cellular susceptibility to MPP+ toxicity. Conversely, combined, but not individual, over-expression of the network targets provided rescue of MPP+ toxicity associated with the formation of autophagosome-like structures. We also found that modulation of two distinct proteins in the protein-protein interaction network was necessary and sufficient to mitigate neurotoxicity. Together, these findings validate our network science approach to multi-target identification in complex neurological diseases.

  12. Protein-protein interaction networks identify targets which rescue the MPP+ cellular model of Parkinson’s disease

    PubMed Central

    Keane, Harriet; Ryan, Brent J.; Jackson, Brendan; Whitmore, Alan; Wade-Martins, Richard

    2015-01-01

    Neurodegenerative diseases are complex multifactorial disorders characterised by the interplay of many dysregulated physiological processes. As an exemplar, Parkinson’s disease (PD) involves multiple perturbed cellular functions, including mitochondrial dysfunction and autophagic dysregulation in preferentially-sensitive dopamine neurons, a selective pathophysiology recapitulated in vitro using the neurotoxin MPP+. Here we explore a network science approach for the selection of therapeutic protein targets in the cellular MPP+ model. We hypothesised that analysis of protein-protein interaction networks modelling MPP+ toxicity could identify proteins critical for mediating MPP+ toxicity. Analysis of protein-protein interaction networks constructed to model the interplay of mitochondrial dysfunction and autophagic dysregulation (key aspects of MPP+ toxicity) enabled us to identify four proteins predicted to be key for MPP+ toxicity (P62, GABARAP, GBRL1 and GBRL2). Combined, but not individual, knockdown of these proteins increased cellular susceptibility to MPP+ toxicity. Conversely, combined, but not individual, over-expression of the network targets provided rescue of MPP+ toxicity associated with the formation of autophagosome-like structures. We also found that modulation of two distinct proteins in the protein-protein interaction network was necessary and sufficient to mitigate neurotoxicity. Together, these findings validate our network science approach to multi-target identification in complex neurological diseases. PMID:26608097

  13. New proteins identified in epididymal fluid from the platypus (Ornithorhynchus anatinus).

    PubMed

    Dacheux, Jean-Louis; Dacheux, Francoise; Labas, Valerie; Ecroyd, Heath; Nixon, Brett; Jones, Russell C

    2009-01-01

    The platypus epididymal proteome is being studied because epididymal proteins are essential for male fertility in mammals and it is considered that knowledge of the epididymal proteome in an early mammal would be informative in assessing the convergence and divergence of proteins that are important in the function of the mammalian epididymis. Few of the epididymal proteins that have been identified in eutherian mammals were found in platypus caudal epididymal fluid, and the major epididymal proteins in the platypus (PXN-FBPL, SPARC and E-OR20) have never been identified in the epididymis of any other mammal.

  14. MAS C-Terminal Tail Interacting Proteins Identified by Mass Spectrometry- Based Proteomic Approach

    PubMed Central

    Tirupula, Kalyan C.; Zhang, Dongmei; Osbourne, Appledene; Chatterjee, Arunachal; Desnoyer, Russ; Willard, Belinda; Karnik, Sadashiva S.

    2015-01-01

    Propagation of signals from G protein-coupled receptors (GPCRs) in cells is primarily mediated by protein-protein interactions. MAS is a GPCR that was initially discovered as an oncogene and is now known to play an important role in cardiovascular physiology. Current literature suggests that MAS interacts with common heterotrimeric G-proteins, but MAS interaction with proteins which might mediate G protein-independent or atypical signaling is unknown. In this study we hypothesized that MAS C-terminal tail (Ct) is a major determinant of receptor-scaffold protein interactions mediating MAS signaling. Mass-spectrometry based proteomic analysis was used to comprehensively identify the proteins that interact with MAS Ct comprising the PDZ-binding motif (PDZ-BM). We identified both PDZ and non-PDZ proteins from human embryonic kidney cell line, mouse atrial cardiomyocyte cell line and human heart tissue to interact specifically with MAS Ct. For the first time our study provides a panel of PDZ and other proteins that potentially interact with MAS with high significance. A ‘cardiac-specific finger print’ of MAS interacting PDZ proteins was identified which includes DLG1, MAGI1 and SNTA. Cell based experiments with wild-type and mutant MAS lacking the PDZ-BM validated MAS interaction with PDZ proteins DLG1 and TJP2. Bioinformatics analysis suggested well-known multi-protein scaffold complexes involved in nitric oxide signaling (NOS), cell-cell signaling of neuromuscular junctions, synapses and epithelial cells. Majority of these protein hits were predicted to be part of disease categories comprising cancers and malignant tumors. We propose a ‘MAS-signalosome’ model to stimulate further research in understanding the molecular mechanism of MAS function. Identifying hierarchy of interactions of ‘signalosome’ components with MAS will be a necessary step in future to fully understand the physiological and pathological functions of this enigmatic receptor. PMID

  15. MAS C-Terminal Tail Interacting Proteins Identified by Mass Spectrometry- Based Proteomic Approach.

    PubMed

    Tirupula, Kalyan C; Zhang, Dongmei; Osbourne, Appledene; Chatterjee, Arunachal; Desnoyer, Russ; Willard, Belinda; Karnik, Sadashiva S

    2015-01-01

    Propagation of signals from G protein-coupled receptors (GPCRs) in cells is primarily mediated by protein-protein interactions. MAS is a GPCR that was initially discovered as an oncogene and is now known to play an important role in cardiovascular physiology. Current literature suggests that MAS interacts with common heterotrimeric G-proteins, but MAS interaction with proteins which might mediate G protein-independent or atypical signaling is unknown. In this study we hypothesized that MAS C-terminal tail (Ct) is a major determinant of receptor-scaffold protein interactions mediating MAS signaling. Mass-spectrometry based proteomic analysis was used to comprehensively identify the proteins that interact with MAS Ct comprising the PDZ-binding motif (PDZ-BM). We identified both PDZ and non-PDZ proteins from human embryonic kidney cell line, mouse atrial cardiomyocyte cell line and human heart tissue to interact specifically with MAS Ct. For the first time our study provides a panel of PDZ and other proteins that potentially interact with MAS with high significance. A 'cardiac-specific finger print' of MAS interacting PDZ proteins was identified which includes DLG1, MAGI1 and SNTA. Cell based experiments with wild-type and mutant MAS lacking the PDZ-BM validated MAS interaction with PDZ proteins DLG1 and TJP2. Bioinformatics analysis suggested well-known multi-protein scaffold complexes involved in nitric oxide signaling (NOS), cell-cell signaling of neuromuscular junctions, synapses and epithelial cells. Majority of these protein hits were predicted to be part of disease categories comprising cancers and malignant tumors. We propose a 'MAS-signalosome' model to stimulate further research in understanding the molecular mechanism of MAS function. Identifying hierarchy of interactions of 'signalosome' components with MAS will be a necessary step in future to fully understand the physiological and pathological functions of this enigmatic receptor.

  16. Heavy metals and metalloids in egg contents and eggshells of passerine birds from Arizona

    USGS Publications Warehouse

    Mora, Miguel A.

    2003-01-01

    Concentrations of inorganic elements were determined in eggs of passerine birds including the endangered southwestern willow flycatcher (Empidonax traillii extimus) from four regions in Arizona. The main aim of the study was to determine the distribution of metals in egg contents and eggshells, with emphasis on the deposition of Sr in eggshells. Seventy eggs of 11 passerine species were collected at four nesting locations during 2000. Aluminum, Ba, Cr, Cu, Mn, Se, Sr, and Zn, were detected primarily in egg contents of all bird species. Arsenic, Ni, Pb, and V were detected primarily in eggshells. A proportion of most inorganic elements accumulated in the eggshell. Concentrations of Ba, Cu, Mn, Se, Sr, and Zn in egg contents and As, Ba, Cu, and V in eggshells of yellow-breasted chats (Icteria virens) were similar among locations. However, concentrations of Mn, Ni, Sr, and Zn in eggshells were significant different among locations. Except for Cu, Mn, Se, and Zn, concentrations of inorganic elements were 2–35 times greater in eggshells than in eggs. Most concentrations of metals and metalloids in eggs and eggshells of all the bird species were below levels known to affect reproduction or that have other deleterious effects. However, I found somewhat elevated concentrations of Sr in eggshells (highest mean=1505 μg/g dw, n=3) of yellow-breasted chats and willow flycatchers, and in egg contents of yellow warblers (Dendroica petechia) and song sparrows (Melospiza melodia). Whether current observed concentrations of Sr in eggshells are affecting nesting birds in Arizona remains to be determined. Strontium and other metals could be associated with lower hatching success in some areas. This study shows that a proportion of many inorganic elements accumulates in the eggshell and that the potential effects on the proper structure and functioning of the eggshell should not be ignored.

  17. Utilizing Yeast Surface Human Proteome Display Libraries to Identify Small Molecule-Protein Interactions.

    PubMed

    Bidlingmaier, Scott; Liu, Bin

    2015-01-01

    The identification of proteins that interact with small bioactive molecules is a critical but often difficult and time-consuming step in understanding cellular signaling pathways or molecular mechanisms of drug action. Numerous methods for identifying small molecule-interacting proteins have been developed and utilized, including affinity-based purification followed by mass spectrometry analysis, protein microarrays, phage display, and three-hybrid approaches. Although all these methods have been used successfully, there remains a need for additional techniques for analyzing small molecule-protein interactions. A promising method for identifying small molecule-protein interactions is affinity-based selection of yeast surface-displayed human proteome libraries. Large and diverse libraries displaying human protein fragments on the surface of yeast cells have been constructed and subjected to FACS-based enrichment followed by comprehensive exon microarray-based output analysis to identify protein fragments with affinity for small molecule ligands. In a recent example, a proteome-wide search has been successfully carried out to identify cellular proteins binding to the signaling lipids PtdIns(4,5)P2 and PtdIns(3,4,5)P3. Known phosphatidylinositide-binding proteins such as pleckstrin homology domains were identified, as well as many novel interactions. Intriguingly, many novel nuclear phosphatidylinositide-binding proteins were discovered. Although the existence of an independent pool of nuclear phosphatidylinositides has been known about for some time, their functions and mechanism of action remain obscure. Thus, the identification and subsequent study of nuclear phosphatidylinositide-binding proteins is expected to bring new insights to this important biological question. Based on the success with phosphatidylinositides, it is expected that the screening of yeast surface-displayed human proteome libraries will be of general use for the discovery of novel small

  18. Identifying different transcribed proteins in the newly described Theraphosidae Pamphobeteus verdolaga.

    PubMed

    Estrada-Gómez, Sebastian; Vargas-Muñoz, Leidy Johana; Saldarriaga-Córdoba, Mónica; Cifuentes, Yeimy; Perafan, Carlos

    2017-04-01

    Theraphosidae spider venoms are well known for possess a complex mixture of protein and non-protein compounds in their venom. The objective of this study was to report and identify different proteins translated from the venom gland DNA information of the recently described Theraphosidae spider Pamphobeteus verdolaga. Using a venom gland transcriptomic analysis, we reported a set of the first complete sequences of seven different proteins of the recenlty described Theraphosidae spider P. verdolaga. Protein analysis indicates the presence of different proteins on the venom composition of this new spider, some of them uncommon in the Theraphosidae family. MS/MS analysis of P. verdolaga showed different fragments matching sphingomyelinases (sicaritoxin), barytoxins, hexatoxins, latroinsectotoxins, and linear (zadotoxins) peptides. Only four of the MS/MS fragments showed 100% sequence similarity with one of the transcribed proteins. Transcriptomic analysis showed the presence of different groups of proteins like phospholipases, hyaluronidases, inhibitory cysteine knots (ICK) peptides among others. The three database of protein domains used in this study (Pfam, SMART and CDD) showed congruency in the search of unique conserved protein domain for only four of the translated proteins. Those proteins matched with EF-hand proteins, cysteine rich secretory proteins, jingzhaotoxins, theraphotoxins and hexatoxins, from different Mygalomorphae spiders belonging to the families Theraphosidae, Barychelidae and Hexathelidae. None of the analyzed sequences showed a complete 100% similarity.

  19. Identifying subcellular localizations of mammalian protein complexes based on graph theory with a random forest algorithm.

    PubMed

    Li, Zhan-Chao; Lai, Yan-Hua; Chen, Li-Li; Chen, Chao; Xie, Yun; Dai, Zong; Zou, Xiao-Yong

    2013-04-05

    In the post-genome era, one of the most important and challenging tasks is to identify the subcellular localizations of protein complexes, and further elucidate their functions in human health with applications to understand disease mechanisms, diagnosis and therapy. Although various experimental approaches have been developed and employed to identify the subcellular localizations of protein complexes, the laboratory technologies fall far behind the rapid accumulation of protein complexes. Therefore, it is highly desirable to develop a computational method to rapidly and reliably identify the subcellular localizations of protein complexes. In this study, a novel method is proposed for predicting subcellular localizations of mammalian protein complexes based on graph theory with a random forest algorithm. Protein complexes are modeled as weighted graphs containing nodes and edges, where nodes represent proteins, edges represent protein-protein interactions and weights are descriptors of protein primary structures. Some topological structure features are proposed and adopted to characterize protein complexes based on graph theory. Random forest is employed to construct a model and predict subcellular localizations of protein complexes. Accuracies on a training set by a 10-fold cross-validation test for predicting plasma membrane/membrane attached, cytoplasm and nucleus are 84.78%, 71.30%, and 82.00%, respectively. And accuracies for the independent test set are 81.31%, 69.95% and 81.00%, respectively. These high prediction accuracies exhibit the state-of-the-art performance of the current method. It is anticipated that the proposed method may become a useful high-throughput tool and plays a complementary role to the existing experimental techniques in identifying subcellular localizations of mammalian protein complexes. The source code of Matlab and the dataset can be obtained freely on request from the authors.

  20. Learning to Translate Sequence and Structure to Function: Identifying DNA Binding and Membrane Binding Proteins

    PubMed Central

    Langlois, Robert E; Carson, Matthew B; Bhardwaj, Nitin; Lu, Hui

    2009-01-01

    A protein's function depends in a large part on interactions with other molecules. With an increasing number of protein structures becoming available every year, a corresponding structural annotation approach identifying such interactions grows more expedient. At the same time, machine learning has gained popularity in bioinformatics because it provides robust annotation of genes and proteins without depending solely on sequence similarity. Here we developed a machine learning protocol to identify DNA-binding proteins and membrane-binding proteins. In general, there is no theory or even rule of thumb to pick the best machine learning algorithm. Thus, a systematic comparison of several classification algorithms known to perform well was investigated. Indeed, the boosted tree classifier was found to give the best performance, achieving 93% and 88% accuracy to discriminate non-homologous DNA-binding proteins and membrane-binding proteins respectively from non-binding proteins, significantly outperforming all previously published works. We also explored the importance of a protein's attributes in function prediction and the relationships between relevant attributes. A graphical model based on boosted trees was applied to study the important features in discriminating DNA-binding proteins. In summary, the current protocol identified physical features important in DNA- and membrane-binding, rather than annotating function through sequence similarity. PMID:17436108

  1. Proteomic profiling of human plasma exosomes identifies PPARgamma as an exosome-associated protein.

    PubMed

    Looze, Christopher; Yui, David; Leung, Lester; Ingham, Matthew; Kaler, Maryann; Yao, Xianglan; Wu, Wells W; Shen, Rong-Fong; Daniels, Mathew P; Levine, Stewart J

    2009-01-16

    Exosomes are nanovesicles that are released from cells as a mechanism of cell-free intercellular communication. Only a limited number of proteins have been identified from the plasma exosome proteome. Here, we developed a multi-step fractionation scheme incorporating gel exclusion chromatography, rate zonal centrifugation through continuous sucrose gradients, and high-speed centrifugation to purify exosomes from human plasma. Exosome-associated proteins were separated by SDS-PAGE and 66 proteins were identified by LC-MS/MS, which included both cellular and extracellular proteins. Furthermore, we identified and characterized peroxisome proliferator-activated receptor-gamma (PPARgamma), a nuclear receptor that regulates adipocyte differentiation and proliferation, as well as immune and inflammatory cell functions, as a novel component of plasma-derived exosomes. Given the important role of exosomes as intercellular messengers, the discovery of PPARgamma as a component of human plasma exosomes identifies a potential new pathway for the paracrine transfer of nuclear receptors.

  2. Guaifenesin stone matrix proteomics: a protocol for identifying proteins critical to stone formation.

    PubMed

    Kolbach-Mandel, A M; Mandel, N S; Cohen, S R; Kleinman, J G; Ahmed, F; Mandel, I C; Wesson, J A

    2017-04-01

    Drug-related kidney stones are a diagnostic problem, since they contain a large matrix (protein) fraction and are frequently incorrectly identified as matrix stones. A urine proteomics study patient produced a guaifenesin stone during her participation, allowing us to both correctly diagnose her disease and identify proteins critical to this drug stone-forming process. The patient provided three random midday urine samples for proteomics studies; one of which contained stone-like sediment with two distinct fractions. These solids were characterized with optical microscopy and Fourier transform infrared spectroscopy. Immunoblotting and quantitative mass spectrometry were used to quantitatively identify the proteins in urine and stone matrix. Infrared spectroscopy showed that the sediment was 60 % protein and 40 % guaifenesin and its metabolite guaiacol. Of the 156 distinct proteins identified in the proteomic studies, 49 were identified in the two stone-components with approximately 50 % of those proteins also found in this patient's urine. Many proteins observed in this drug-related stone have also been reported in proteomic matrix studies of uric acid and calcium containing stones. More importantly, nine proteins were highly enriched and highly abundant in the stone matrix and 8 were reciprocally depleted in urine, suggesting a critical role for these proteins in guaifenesin stone formation. Accurate stone analysis is critical to proper diagnosis and treatment of kidney stones. Many matrix proteins were common to all stone types, but likely not related to disease mechanism. This protocol defined a small set of proteins that were likely critical to guaifenesin stone formation based on their high enrichment and high abundance in stone matrix, and it should be applied to all stone types.

  3. Heart in An Eggshell Calcification: Idiopathic Calcific Constrictive Pericarditis

    PubMed Central

    Song, Bong Gun; Kang, Gu Hyun; Park, Yong Hwan; Chun, Woo Jung; Oh, Ju Hyeon

    2011-01-01

    Constrictive pericarditis is caused by fibrosis and calcification of the pericardium, which inhibits diastolic filling of the heart. Chest roentgenogram can show the calcification as a mass or sheet over the heart and computed tomography scan allows anatomic delineation of the pericardium and determines the extent of calcification. We reported a case of eggshell calcification of idiopathic chronic constrictive pericarditis diagnosed by echocardiography and multi-detector computed tomography.

  4. An Experimentally Based Computer Search Identifies Unstructured Membrane-binding Sites in Proteins

    PubMed Central

    Brzeska, Hanna; Guag, Jake; Remmert, Kirsten; Chacko, Susan; Korn, Edward D.

    2010-01-01

    Programs exist for searching protein sequences for potential membrane-penetrating segments (hydrophobic regions) and for lipid-binding sites with highly defined tertiary structures, such as PH, FERM, C2, ENTH, and other domains. However, a rapidly growing number of membrane-associated proteins (including cytoskeletal proteins, kinases, GTP-binding proteins, and their effectors) bind lipids through less structured regions. Here, we describe the development and testing of a simple computer search program that identifies unstructured potential membrane-binding sites. Initially, we found that both basic and hydrophobic amino acids, irrespective of sequence, contribute to the binding to acidic phospholipid vesicles of synthetic peptides that correspond to the putative membrane-binding domains of Acanthamoeba class I myosins. Based on these results, we modified a hydrophobicity scale giving Arg- and Lys-positive, rather than negative, values. Using this basic and hydrophobic scale with a standard search algorithm, we successfully identified previously determined unstructured membrane-binding sites in all 16 proteins tested. Importantly, basic and hydrophobic searches identified previously unknown potential membrane-binding sites in class I myosins, PAKs and CARMIL (capping protein, Arp2/3, myosin I linker; a membrane-associated cytoskeletal scaffold protein), and synthetic peptides and protein domains containing these newly identified sites bound to acidic phospholipids in vitro. PMID:20018884

  5. Using the Ras Recruitment System to identify PP2A-B55-interacting proteins.

    PubMed

    Barr, Haim M; Sharf, Rakefet; Kleinberger, Tamar

    2003-01-01

    The RRS system facilitated the discovery of hitherto unknown interactions with the PP2A-B55 subunit. The advantages of the system lie in its ability to identify interactions that may not be detected by traditional yeast two-hybrid systems. The RRS can thus provide a complementary genetic approach to the identification of protein-protein interactions.

  6. Preparation and properties of calcium oxide from eggshells via calcination

    NASA Astrophysics Data System (ADS)

    Tangboriboon, N.; Kunanuruksapong, R.; Sirivat, A.

    2012-12-01

    Duck eggs are one of the most versatile cooking ingredients in which residue eggshells are discarded. Raw duck eggshells were calcined at temperatures between 300 to 900 °C, for 1, 3, and 5 h. Both the raw and calcined duck eggshells were characterized by FTIR, STA, XRD, XRF, TEM, BET, a particle size analyzer, and an impedance analyzer. The proper calcination conditions are: 900 °C and 1 h, yielding calcium oxide with a purity of 99.06 % w/w. The calcium carbonate of the rhombohedral form (CaCO3) transforms completely into the calcium oxide or lime of the face centered cubic form (CaO) at 900 °C, as shown by XRD diffraction patterns. The transmission electron microscopy (TEM) images of the calcium oxide reveal a moderately good dispersion of nearly uniform particles. The calcium oxide has a white color, a spherical shape, high porosity, and narrow particles size distribution. The percentage of ceramic yield of the calcium oxide is 53.53, as measured by STA (TG-DTA-DTG). The calcium oxide has a N2 adsorption-desorption isotherm indicating the meso-porosity range. The dielectric constant and the electrical conductivity of the calcined calcium oxide are 35 and 1:0×10-6(Ω·m)-1, respectively, at the frequency of 500 Hz.

  7. Ultrastructure of avian eggshell during resorption following egg fertilization.

    PubMed

    Chien, Y-C; Hincke, M T; McKee, M D

    2009-12-01

    For skeletal mineralization, the avian embryo mobilizes calcium from its calcitic eggshell. This occurs through dissolution of specific interior regions of the shell in a process that also weakens the shell to allow hatching. Here, we have examined eggshell ultrastructure during dissolution occurring between laying of a fertilized egg (with incubation) and hatching of the chick (Gallus gallus). We have focused on changes in shell mammillae where the majority of dissolution takes place. Using scanning electron microscopy, we describe differences in matrix-mineral structure and relationships not observed in unfertilized eggs (unresorbed eggshell). We document changes in the calcium reserve body - an essential sub-compartment of mammillae - consistent with it being an early, primary source of calcium essential for embryonic skeletal growth. Dissolution events occurring in the calcium reserve sac and in the base plate of the calcium reserve body, and similar changes in surrounding bulk mammillae structure, all correlate with advancing skeletal embryonic calcification. The changes in mammillae sub-structures can generally be characterized as mineral dissolutions revealing fine surface topographies on remaining mineral surfaces and the exposure of an extensive, intracrystalline (occluded) organic matrix network. We propose that this mineral-occluded network regulates how shell mineral is dissolved by providing dissolution channels facilitating calcium release for the embryonic skeleton.

  8. Identifying the molecular functions of electron transport proteins using radial basis function networks and biochemical properties.

    PubMed

    Le, Nguyen-Quoc-Khanh; Nguyen, Trinh-Trung-Duong; Ou, Yu-Yen

    2017-05-01

    The electron transport proteins have an important role in storing and transferring electrons in cellular respiration, which is the most proficient process through which cells gather energy from consumed food. According to the molecular functions, the electron transport chain components could be formed with five complexes with several different electron carriers and functions. Therefore, identifying the molecular functions in the electron transport chain is vital for helping biologists understand the electron transport chain process and energy production in cells. This work includes two phases for discriminating electron transport proteins from transport proteins and classifying categories of five complexes in electron transport proteins. In the first phase, the performances from PSSM with AAIndex feature set were successful in identifying electron transport proteins in transport proteins with achieved sensitivity of 73.2%, specificity of 94.1%, and accuracy of 91.3%, with MCC of 0.64 for independent data set. With the second phase, our method can approach a precise model for identifying of five complexes with different molecular functions in electron transport proteins. The PSSM with AAIndex properties in five complexes achieved MCC of 0.51, 0.47, 0.42, 0.74, and 1.00 for independent data set, respectively. We suggest that our study could be a power model for determining new proteins that belongs into which molecular function of electron transport proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Changes in the levels of different ions in the eggshell gland lumen following p,p'-DDE-induced eggshell thinning in ducks.

    PubMed

    Lundholm, C E

    1994-09-01

    Eggshell thinning in ducks was induced by administration of p,p'-DDE in the diet (40 mg/kg food) for 45 days. This treatment resulted in a 19% reduction of the Eggshell Index (EI). Shells from calcifying eggs obtained at the time of slaughter showed a 36% reduction of EI. Prostaglandin synthesis by a homogenate of eggshell gland mucosa from p,p'-DDE-treated ducks was reduced by 24%. HCO3(-)-stimulated ATPase activity by a homogenate of eggshell gland mucosa from p,p'-DDE-treated ducks was not significantly changed. The calcium content of eggshell gland mucosa was increased to 127% in p,p'-DDE-treated ducks. p,p'-DDE-treated ducks showed profound changes in the shell gland luminal content of several ions. Calcium (-43%), sodium (-15%), potassium (-15%), bicarbonate (-33%) and chloride (-29%) were all significantly reduced in p,p'-DDE-treated ducks. The content of phosphate was unchanged. These findings are discussed in relation to a proposed mechanism for p,p'-DDE-induced eggshell thinning that involves inhibition of prostaglandin synthesis in eggshell gland mucosa.

  10. A New Method for Identifying Essential Proteins Based on Network Topology Properties and Protein Complexes

    PubMed Central

    Qin, Chao; Sun, Yongqi; Dong, Yadong

    2016-01-01

    Essential proteins are indispensable to the viability and reproduction of an organism. The identification of essential proteins is necessary not only for understanding the molecular mechanisms of cellular life but also for disease diagnosis, medical treatments and drug design. Many computational methods have been proposed for discovering essential proteins, but the precision of the prediction of essential proteins remains to be improved. In this paper, we propose a new method, LBCC, which is based on the combination of local density, betweenness centrality (BC) and in-degree centrality of complex (IDC). First, we introduce the common centrality measures; second, we propose the densities Den1(v) and Den2(v) of a node v to describe its local properties in the network; and finally, the combined strategy of Den1, Den2, BC and IDC is developed to improve the prediction precision. The experimental results demonstrate that LBCC outperforms traditional topological measures for predicting essential proteins, including degree centrality (DC), BC, subgraph centrality (SC), eigenvector centrality (EC), network centrality (NC), and the local average connectivity-based method (LAC). LBCC also improves the prediction precision by approximately 10 percent on the YMIPS and YMBD datasets compared to the most recently developed method, LIDC. PMID:27529423

  11. Organic solvent extraction as a versatile procedure to identify hydrophobic chloroplast membrane proteins.

    PubMed

    Ferro, M; Seigneurin-Berny, D; Rolland, N; Chapel, A; Salvi, D; Garin, J; Joyard, J

    2000-10-01

    As a complementary approach to genome projects, proteomic analyses have been set up to identify new gene products. One of the major challenges in proteomics concerns membrane proteins, especially the minor ones. A procedure based on the differential extraction of membrane proteins in chloroform/methanol mixtures, was tested on the two different chloroplast membrane systems: envolope and thylakoid membranes. Combining the use of classical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry analyses, this procedure enabled identification of hydrophobic proteins. The propensity of hydrophobic proteins to partition in chloroform/methanol mixtures was directly correlated with the number of amino acid residues/number of putative transmembrane regions (Res/TM ratio). Regardless of the particular case of some lipid-interacting proteins, chloroform/methanol extractions allowed enrichment of hydrophobic proteins and exclusion of hydrophilic proteins from both membrane systems, thus demonstrating the versatility of the procedure.

  12. Proteomic Analysis to Identify Tightly-Bound Cell Wall Protein in Rice Calli.

    PubMed

    Cho, Won Kyong; Hyun, Tae Kyung; Kumar, Dhinesh; Rim, Yeonggil; Chen, Xiong Yan; Jo, Yeonhwa; Kim, Suwha; Lee, Keun Woo; Park, Zee-Yong; Lucas, William J; Kim, Jae-Yean

    2015-08-01

    Rice is a model plant widely used for basic and applied research programs. Plant cell wall proteins play key roles in a broad range of biological processes. However, presently, knowledge on the rice cell wall proteome is rudimentary in nature. In the present study, the tightly-bound cell wall proteome of rice callus cultured cells using sequential extraction protocols was developed using mass spectrometry and bioinformatics methods, leading to the identification of 1568 candidate proteins. Based on bioinformatics analyses, 389 classical rice cell wall proteins, possessing a signal peptide, and 334 putative non-classical cell wall proteins, lacking a signal peptide, were identified. By combining previously established rice cell wall protein databases with current data for the classical rice cell wall proteins, a comprehensive rice cell wall proteome, comprised of 496 proteins, was constructed. A comparative analysis of the rice and Arabidopsis cell wall proteomes revealed a high level of homology, suggesting a predominant conservation between monocot and eudicot cell wall proteins. This study importantly increased information on cell wall proteins, which serves for future functional analyses of these identified rice cell wall proteins.

  13. Proteomic Analysis to Identify Tightly-Bound Cell Wall Protein in Rice Calli

    PubMed Central

    Cho, Won Kyong; Hyun, Tae Kyung; Kumar, Dhinesh; Rim, Yeonggil; Chen, Xiong Yan; Jo, Yeonhwa; Kim, Suwha; Lee, Keun Woo; Park, Zee-Yong; Lucas, William J.; Kim, Jae-Yean

    2015-01-01

    Rice is a model plant widely used for basic and applied research programs. Plant cell wall proteins play key roles in a broad range of biological processes. However, presently, knowledge on the rice cell wall proteome is rudimentary in nature. In the present study, the tightly-bound cell wall proteome of rice callus cultured cells using sequential extraction protocols was developed using mass spectrometry and bioinformatics methods, leading to the identification of 1568 candidate proteins. Based on bioinformatics analyses, 389 classical rice cell wall proteins, possessing a signal peptide, and 334 putative non-classical cell wall proteins, lacking a signal peptide, were identified. By combining previously established rice cell wall protein databases with current data for the classical rice cell wall proteins, a comprehensive rice cell wall proteome, comprised of 496 proteins, was constructed. A comparative analysis of the rice and Arabidopsis cell wall proteomes revealed a high level of homology, suggesting a predominant conservation between monocot and eudicot cell wall proteins. This study importantly increased information on cell wall proteins, which serves for future functional analyses of these identified rice cell wall proteins. PMID:26194822

  14. The use of proteomics in identifying differentially expressed serum proteins in humans with type 2 diabetes

    PubMed Central

    Sundsten, Tea; Eberhardson, Michael; Göransson, Michael; Bergsten, Peter

    2006-01-01

    Background The aim of the study was to optimize protocols for finding and identifying serum proteins that are differentially expressed in persons with normal glucose tolerance (NGT) compared to individuals with type 2 diabetes mellitus (T2DM). Serum from persons with NGT and persons with T2DM was profiled using ProteinChip arrays and time-of-flight mass spectra were generated by surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Results Mass spectra from NGT- and T2DM-groups were compared. Fifteen proteins ranging from 5 to 79 kDa were differentially expressed (p < 0.05). Five of these proteins showed decreased and ten showed increased serum levels in individuals with T2DM. To be able to identify the proteins, the complexity of the sample was reduced by fractionation approaches. Subsequently, the purified fractions containing biomarkers were separated by one-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in two identical lanes. Protein bands of the first lane were excised and subjected to passive elution to recapture the biomarkers on ProteinChip arrays. The corresponding bands of the second lane were subjected to peptide-mass fingerprinting (PMF). Using this approach four of the differentially expressed proteins were identified as apolipoprotein C3 (9.4 kDa), transthyretin (13.9 kDa), albumin (66 kDa) and transferrin (79 kDa). Whereas apolipoprotein C3 and transthyretin were up-regulated, albumin and transferrin were down-regulated in T2DM. Conclusion Protocols for protein profiling by SELDI-TOF MS and protein identification by fractionation, SDS-PAGE and PMF were optimized for serum from humans with T2DM. With these protocols differentially expressed proteins were discovered and identified when serum from NGT- and T2DM-individuals was analyzed. PMID:17163994

  15. HVint: A Strategy for Identifying Novel Protein-Protein Interactions in Herpes Simplex Virus Type 1*

    PubMed Central

    Hernandez, Anna; Buch, Anna; Sodeik, Beate; Cristea, Ileana Mihaela

    2016-01-01

    Human herpesviruses are widespread human pathogens with a remarkable impact on worldwide public health. Despite intense decades of research, the molecular details in many aspects of their function remain to be fully characterized. To unravel the details of how these viruses operate, a thorough understanding of the relationships between the involved components is key. Here, we present HVint, a novel protein-protein intraviral interaction resource for herpes simplex virus type 1 (HSV-1) integrating data from five external sources. To assess each interaction, we used a scoring scheme that takes into consideration aspects such as the type of detection method and the number of lines of evidence. The coverage of the initial interactome was further increased using evolutionary information, by importing interactions reported for other human herpesviruses. These latter interactions constitute, therefore, computational predictions for potential novel interactions in HSV-1. An independent experimental analysis was performed to confirm a subset of our predicted interactions. This subset covers proteins that contribute to nuclear egress and primary envelopment events, including VP26, pUL31, pUL40, and the recently characterized pUL32 and pUL21. Our findings support a coordinated crosstalk between VP26 and proteins such as pUL31, pUS9, and the CSVC complex, contributing to the development of a model describing the nuclear egress and primary envelopment pathways of newly synthesized HSV-1 capsids. The results are also consistent with recent findings on the involvement of pUL32 in capsid maturation and early tegumentation events. Further, they open the door to new hypotheses on virus-specific regulators of pUS9-dependent transport. To make this repository of interactions readily accessible for the scientific community, we also developed a user-friendly and interactive web interface. Our approach demonstrates the power of computational predictions to assist in the design of

  16. Novel proteins identified in the insoluble byssal matrix of the freshwater zebra mussel.

    PubMed

    Gantayet, Arpita; Rees, David J; Sone, Eli D

    2014-04-01

    The freshwater zebra mussel, Dreissena polymorpha, is an invasive, biofouling species that adheres to a variety of substrates underwater, using a proteinaceous anchor called the byssus. The byssus consists of a number of threads with adhesive plaques at the tips. It contains the unusual amino acid 3, 4-dihydroxyphenylalanine (DOPA), which is believed to play an important role in adhesion, in addition to providing structural integrity to the byssus through cross-linking. Extensive DOPA cross-linking, however, renders the zebra mussel byssus highly resistant to protein extraction, and therefore limits byssal protein identification. We report here on the identification of seven novel byssal proteins in the insoluble byssal matrix following protein extraction from induced, freshly secreted byssal threads with minimal cross-linking. These proteins were identified by LC-MS/MS analysis of tryptic digests of the matrix proteins by spectrum matching against a zebra mussel cDNA library of genes unique to the mussel foot, the organ that secretes the byssus. All seven proteins were present in both the plaque and thread. Comparisons of the protein sequences revealed common features of zebra mussel byssal proteins, and several recurring sequence motifs. Although their sequences are unique, many of the proteins display similarities to marine mussel byssal proteins, as well as to adhesive and structural proteins from other species. The large expansion of the byssal proteome reported here represents an important step towards understanding zebra mussel adhesion.

  17. Egg-in-Cube: Design and Fabrication of a Novel Artificial Eggshell with Functionalized Surface

    PubMed Central

    Huang, Wenjing; Arai, Fumihito; Kawahara, Tomohiro

    2015-01-01

    An eggshell is a porous microstructure that regulates the passage of gases to allow respiration. The chick embryo and its circulatory system enclosed by the eggshell has become an important model for biomedical research such as the control of angiogenesis, cancer therapy, and drug delivery test, because the use of embryo is ethically acceptable and it is inexpensive and small. However, chick embryo and extra-embryonic blood vessels cannot be accessed freely and has poor observability because the eggshell is tough and cannot be seen through, which limits its application. In this study, a novel artificial eggshell with functionalized surface is proposed, which allows the total amount of oxygen to pass into the egg for the chick embryo culturing and has high observability and accessibility for embryo manipulation. First, a 40-mm enclosed cubic-shaped eggshell consisting of a membrane structure and a rigid frame structure is designed, and then the threshold of the membrane thickness suitable for the embryo survival is figured out according to the oxygen-permeability of the membrane structure. The designed artificial eggshell was actually fabricated by using polydimethylsiloxane (PDMS) and polycarbonate (PC) in the current study. Using the fabricated eggshell, chick embryo and extra-embryonic blood vessels can be observed from multiple directions. To test the effectiveness of the design, the cubic eggshells were used to culture chick embryos and survivability was confirmed when PDMS membranes with adequate oxygen permeability were used. Since the surface of the eggshell is transparent, chick embryo tissue development could be observed during the culture period. Additionally, the chick embryo tissues could be accessed and manipulated from outside the cubic eggshell, by using mechanical tools without breakage of the eggshell. The proposed “Egg-in-Cube” with functionalized surface has great potential to serve as a promising platform for biomedical research. PMID

  18. Condition-dependent strategies of eggshell pigmentation: an experimental study of Japanese quail (Coturnix coturnix japonica).

    PubMed

    Duval, Camille; Cassey, Phillip; Miksík, Ivan; Reynolds, S James; Spencer, Karen A

    2013-02-15

    A relationship has been suggested between eggshell colour and female body condition based on the opposing antioxidant properties of the two main eggshell pigments: the antioxidant biliverdin (blue-green) and the pro-oxidant protoporphyrin (brown). We hypothesized that experimentally food-restricted females with low antioxidant capacity would deposit more protoporphyrin and less biliverdin in their eggshells, resulting in eggshells of reduced brightness but increased colour intensity. Two eggs were collected at the beginning and two at the end of a 2 week period from each of 24 female Japanese quails that were either food restricted or receiving ad libitum food (i.e. controls) during that time. Reflectance spectra were recorded and analysed using spectral shape descriptors, chromatic and achromatic contrasts were computed accounting for avian visual sensitivities, and eggshell pigments were quantified. We examined both spot and background pigmentation and found no significant effect of food restriction on eggshell reflectance. However, food-restricted females in lower body condition increased the deposition of protoporphyrin and decreased the amount of biliverdin invested in their eggshells. We hypothesize that in species laying brown-spotted eggshells, females modulate eggshell pigment investment in response to their body condition. According to this hypothesis, we predict that females maintain eggshell colour to limit visible changes that could be detected by predators and thereby conceal their eggs, although this work has yet to be conducted. We suggest that further experimental work on egg camouflage under different environmental conditions will elaborate on the process of pigment deposition and the physiological costs to females of laying heavily pigmented eggshells.

  19. Eggshell structure in Caiman latirostris eggs improves embryo survival during nest inundation.

    PubMed

    Cedillo-Leal, César; Simoncini, Melina S; Leiva, Pamela M L; Larriera, Alejandro; Lang, Jeffrey W; Piña, Carlos I

    2017-05-17

    Egg inundation often results in poor hatching success in crocodylians. However, how tolerant eggs are to submergence, and/or how eggshell ultrastructure may affect embryo survival when inundated, are not well understood. In this study, our objective was to determine if embryo survival in Caiman latirostris is affected by eggshell surface roughness, when eggs are submerged under water. Tolerance to inundation was tested early (day 30) versus late (day 60) in development, using eight clutches (four per time treatments), subdivided into four groups: (N = 9 per clutch per treatment; 9 × 4 = 36 eggs per group). 'Rough' eggshell represented the natural, unmodified eggshell surface structure. 'Smooth' eggshell surface structure was created by mechanically sanding the natural rough surface to remove surface columnar elements and secondary layer features, e.g. irregularities that result in 'roughness'. When inundated by submerging eggs under water for 10 h at day 30, 'smooth' eggshell structure resulted in more than twice as many dead embryos (16 versus 6, smooth versus rough; N = 36), and fewer than half as many healthy embryos (6 versus 13, smooth versus rough, respectively; N = 36). By contrast, at day 60, inundation resulted in very low hatching success, regardless of eggshell surface structure. Only two hatchlings survived the inundation, notably in the untreated group with intact, rough eggshells. Inundation produced a high rate of malformations (58% at day 30), but did not affect hatchling size. Our results indicate that eggshell roughness enhances embryo survival when eggs are inundated early in development, but not late in development. Apparently, the natural surface 'roughness' entraps air bubbles at the eggshell surface during inundation, thereby facilitating gas exchange through the eggshell even when the egg is submerged under water. © 2017 The Author(s).

  20. Salmonella penetration through eggshells of chickens of different genetic backgrounds.

    PubMed

    Rathgeber, Bruce M; McCarron, Paige; Budgell, Krista L

    2013-09-01

    Eggs have been identified as a source of salmonellosis, making the transmission of Salmonella to eggs of great concern to the poultry industry. The goal of this experiment was to determine the ability of Salmonella to penetrate the eggshell of 5 different breeds of noncommercial chicken, Barred Plymouth Rock, White Leghorn, Brown Leghorn, Fayoumi, and Light Sussex, and 1 commercial Lohmann LSL-Lite. Egg weight, breaking force, shell weight, and shell thickness measurements were taken for 30 eggs per breed. A 1 cm in diameter hole was cut out from the narrow end of 30 additional eggs per breed. The shells were filled with plate count agar containing tetracycline and 0.1% 2,3,5-triphenyl terazolium chloride and sealed with paraffin wax. Agar-filled eggs were submerged for 1 min in an overnight culture of tetracycline-resistant Salmonella Heidelberg and incubated at 37°C for 40 h. Eggs were candled and visual colonies were counted and reported as cfu per egg and cfu per gram of shell. The SAS mixed model was used to evaluate differences between breeds for egg quality characteristics and the number of cfu per egg and per gram of shell. Commercial layers (62.6 g) and Barred Plymouth Rock (61.5 g) produced the largest eggs, whereas Fayoumi (47.1 g) produced the smallest (P < 0.05). Force to break the shell was lowest (P < 0.05) for Barred Plymouth Rock (3.6 kg) and greatest for the commercial (4.4 kg), White Leghorn (4.4 kg), and Fayoumi (4.2 kg). Bacteria penetrating the shell was lowest (P < 0.05) for Barred Plymouth Rock (10.7 cfu/g) and highest for Light Sussex (27.7 cfu/g) and Brown Leghorn (27.2 cfu/g), with other breeds intermediate. These results indicate that there are breed-specific influences on the ability of an egg to resist Salmonella, which cannot be explained by shell quality measurements. Further investigations are warranted to determine the contributing factors to shell penetration by bacteria. This study highlights the value in maintaining heritage

  1. The sodium channel gene family is specifically expressed in hen uterus and associated with eggshell quality traits

    PubMed Central

    2013-01-01

    Background Eggshell quality is important for the poultry industry. During eggshell formation a mass of inorganic minerals is deposited. The Sodium Channel (SCNN1) gene family plays an essential role in cation transportation. The objective of this study was to investigate the pattern of expression of members of the SCNN1 gene family, their variation and their effects on eggshell quality. Result The highest expression of SCNN1a, SCNN1b, and SCNN1g genes were in the active uterus during eggshell mineralization, while SCNN1d showed its highest expression level in the quiescent uterus (no egg present). Nineteen candidate SNPs from the four genes were genotyped in a population of 338 White Leghorn layers. Association analysis between SNPs (haplotypes/diplotypes) and eggshell traits was performed. Among seven significant SNPs, five SNPs were associated with eggshell strength, eggshell thickness, eggshell percentage or/and egg weight, while the other two SNPs within SCNN1d were only associated with eggshell percentage. These SNPs had a 0.25-6.99% contribution to phenotypic variance, depending on the trait. In haplotype analysis, SCNN1b and SCNN1d were associated with egg weight. The SCNN1b and SCNN1g were significantly associated with eggshell weight while only SCNN1g explained 2.04% of phenotypic variance. All the alleles of the members of SCNN1 gene family were associated with eggshell percentage and eggshell thickness, and others members had an association with eggshell strength except for SCNN1a. The contribution of different haplotypes of the SCNN1 gene family to eggshell phenotypic variance ranged from 0.09% to 5.74%. Conclusions Our study indicated that the SCNN1 gene family showed tissue expression specificity and was significantly associated with eggshell traits in chicken. This study provides evidence that genetic variation in members of the sodium channel can influence eggshell quality. PMID:24059973

  2. Sequestosome 1/p62, a Scaffolding Protein, Is a Newly Identified Partner of IRS-1 Protein*

    PubMed Central

    Geetha, Thangiah; Zheng, Chen; Vishwaprakash, Nilmini; Broderick, Tom L.; Babu, Jeganathan Ramesh

    2012-01-01

    Defects in the insulin-signaling pathway may lead to the development of skeletal muscle insulin resistance, which is one of the earliest abnormalities detected in individuals with the metabolic syndrome and predisposes them to develop type 2 diabetes. Previous studies have shown that deletion of the mouse sequestosome 1/p62 gene results in mature-onset obesity that progresses to insulin and leptin resistance and, ultimately, type 2 diabetes. Sequestosome 1/p62 is involved in receptor-mediated signal transduction and functions as an intracellular signal modulator or adaptor protein. Insulin receptor substrate-1 (IRS-1) plays a central role in transducing the insulin signal via phosphorylation, protein-protein interactions, and protein modifications. Mapping studies demonstrated that the SH2 domain at the amino terminus of sequestosome 1/p62 interacts with IRS-1 upon insulin stimulation. Further, IRS-1 interacts with p62 through its YMXM motifs at Tyr-608, Tyr-628, and/or Tyr-658 in a manner similar to its interaction with p85 of phosphoinositol 3-kinase. Overexpression of p62 increased phosphorylation of Akt, GLUT4 translocation, and glucose uptake, providing evidence that p62 participates in the insulin-signaling pathway through its interactions with IRS-1. PMID:22761437

  3. Sequestosome 1/p62, a scaffolding protein, is a newly identified partner of IRS-1 protein.

    PubMed

    Geetha, Thangiah; Zheng, Chen; Vishwaprakash, Nilmini; Broderick, Tom L; Babu, Jeganathan Ramesh

    2012-08-24

    Defects in the insulin-signaling pathway may lead to the development of skeletal muscle insulin resistance, which is one of the earliest abnormalities detected in individuals with the metabolic syndrome and predisposes them to develop type 2 diabetes. Previous studies have shown that deletion of the mouse sequestosome 1/p62 gene results in mature-onset obesity that progresses to insulin and leptin resistance and, ultimately, type 2 diabetes. Sequestosome 1/p62 is involved in receptor-mediated signal transduction and functions as an intracellular signal modulator or adaptor protein. Insulin receptor substrate-1 (IRS-1) plays a central role in transducing the insulin signal via phosphorylation, protein-protein interactions, and protein modifications. Mapping studies demonstrated that the SH(2) domain at the amino terminus of sequestosome 1/p62 interacts with IRS-1 upon insulin stimulation. Further, IRS-1 interacts with p62 through its YMXM motifs at Tyr-608, Tyr-628, and/or Tyr-658 in a manner similar to its interaction with p85 of phosphoinositol 3-kinase. Overexpression of p62 increased phosphorylation of Akt, GLUT4 translocation, and glucose uptake, providing evidence that p62 participates in the insulin-signaling pathway through its interactions with IRS-1.

  4. Unique secreted–surface protein complex of Lactobacillus rhamnosus, identified by phage display

    PubMed Central

    Gagic, Dragana; Wen, Wesley; Collett, Michael A; Rakonjac, Jasna

    2013-01-01

    Proteins are the most diverse structures on bacterial surfaces; hence, they are candidates for species- and strain-specific interactions of bacteria with the host, environment, and other microorganisms. Genomics has decoded thousands of bacterial surface and secreted proteins, yet the function of most cannot be predicted because of the enormous variability and a lack of experimental data that would allow deduction of function through homology. Here, we used phage display to identify a pair of interacting extracellular proteins in the probiotic bacterium Lactobacillus rhamnosus HN001. A secreted protein, SpcA, containing two bacterial immunoglobulin-like domains type 3 (Big-3) and a domain distantly related to plant pathogen response domain 1 (PR-1-like) was identified by screening of an L. rhamnosus HN001 library using HN001 cells as bait. The SpcA-“docking” protein, SpcB, was in turn detected by another phage display library screening, using purified SpcA as bait. SpcB is a 3275-residue cell-surface protein that contains general features of large glycosylated Serine-rich adhesins/fibrils from gram-positive bacteria, including the hallmark signal sequence motif KxYKxGKxW. Both proteins are encoded by genes within a L. rhamnosus-unique gene cluster that distinguishes this species from other lactobacilli. To our knowledge, this is the first example of a secreted-docking protein pair identified in lactobacilli. PMID:23233310

  5. Proteomic analysis of mouse mammary terminal end buds identifies axonal growth cone proteins.

    PubMed

    Morris, Joanna S; Davies, Claire R; Griffiths, Matthew R; Page, Martin J; Bruce, James A; Patel, Thakor; Herath, Athula; Gusterson, Barry A

    2004-06-01

    Ductal morphogenesis in the mouse mammary gland occurs mainly postnatally and is driven by specialized structures at the ends of the developing ducts, the terminal end buds (TEBs), which later regress once ductal growth is complete. To identify proteins that are specifically associated with migration of TEBs we developed a novel method of isolating TEBs, which eliminated the mammary stroma. The protein expression profile of the TEBs was then compared with that of isolates taken from the 4th inguinal mammary gland of adult virgin mice using two-dimensional (2-D) gel electrophoresis and mass spectrometry (MS) analysis (matrix-assisted laser desorption/ionization and quadrupole time of flight). Following construction of an integrated protein expression database, 44 protein features which showed differential expression levels between the two sets were chosen for MS analysis. Of these, 24 gave protein annotations whereas the other 20 produced unidentified peptides. Fourteen unequivocal proteins were identified from these 24, whereas the remaining 10 matched more than one protein within a single 2-D gel feature. Several of the identified proteins were associated with the cytoskeleton and have previously been reported in axonal growth cones, suggesting that they may influence cell shape and motility within the advancing TEBs, in a similar fashion to migrating axons.

  6. Method for early detection of infectious mononucleosis by identifying Inmono proteins

    DOEpatents

    Willard, Karen E.

    1984-01-01

    Early detection of infectious mononucleosis is carried out using a sample of human blood by isolating and identifying the presence of Inmono proteins in the sample from a two-dimensional protein map with the proteins being characterized by having isoelectric banding as measured in urea of about -16 to -17 with respect to certain isoelectric point standards and molecular mass of about 70 to 75 K daltons as measured in the presence of sodium dodecylsulfate containing polyacrylamide gels, the presence of the Inmono proteins being correlated with the existence of infectious mononucleosis.

  7. Dissecting protein function: an efficient protocol for identifying separation-of-function mutations that encode structurally stable proteins.

    PubMed

    Lubin, Johnathan W; Rao, Timsi; Mandell, Edward K; Wuttke, Deborah S; Lundblad, Victoria

    2013-03-01

    Mutations that confer the loss of a single biochemical property (separation-of-function mutations) can often uncover a previously unknown role for a protein in a particular biological process. However, most mutations are identified based on loss-of-function phenotypes, which cannot differentiate between separation-of-function alleles vs. mutations that encode unstable/unfolded proteins. An alternative approach is to use overexpression dominant-negative (ODN) phenotypes to identify mutant proteins that disrupt function in an otherwise wild-type strain when overexpressed. This is based on the assumption that such mutant proteins retain an overall structure that is comparable to that of the wild-type protein and are able to compete with the endogenous protein (Herskowitz 1987). To test this, the in vivo phenotypes of mutations in the Est3 telomerase subunit from Saccharomyces cerevisiae were compared with the in vitro secondary structure of these mutant proteins as analyzed by circular-dichroism spectroscopy, which demonstrates that ODN is a more sensitive assessment of protein stability than the commonly used method of monitoring protein levels from extracts. Reverse mutagenesis of EST3, which targeted different categories of amino acids, also showed that mutating highly conserved charged residues to the oppositely charged amino acid had an increased likelihood of generating a severely defective est3(-) mutation, which nevertheless encoded a structurally stable protein. These results suggest that charge-swap mutagenesis directed at a limited subset of highly conserved charged residues, combined with ODN screening to eliminate partially unfolded proteins, may provide a widely applicable and efficient strategy for generating separation-of-function mutations.

  8. Nutritional Supplement of Hatchery Eggshell Membrane Improves Poultry Performance and Provides Resistance against Endotoxin Stress

    PubMed Central

    Makkar, S. K.; Rath, N. C.; Packialakshmi, B.; Zhou, Z. Y.; Huff, G. R.; Donoghue, A. M.

    2016-01-01

    Eggshells are significant part of hatchery waste which consist of calcium carbonate crust, membranes, and proteins and peptides of embryonic origins along with other entrapped contaminants including microbes. We hypothesized that using this product as a nutritional additive in poultry diet may confer better immunity to the chickens in the paradigm of mammalian milk that enhances immunity. Therefore, we investigated the effect of hatchery eggshell membranes (HESM) as a short term feed supplement on growth performance and immunity of chickens under bacterial lipopolysaccharide (LPS) challenged condition. Three studies were conducted to find the effect of HESM supplement on post hatch chickens. In the first study, the chickens were fed either a control diet or diets containing 0.5% whey protein or HESM as supplement and evaluated at 5 weeks of age using growth, hematology, clinical chemistry, plasma immunoglobulins, and corticosterone as variables. The second and third studies were done to compare the effects of LPS on control and HESM fed birds at 5 weeks of age following at 4 and 24 h of treatment where the HESM was also sterilized with ethanol to deplete bacterial factors. HESM supplement caused weight gain in 2 experiments and decreased blood corticosterone concentrations. While LPS caused a significant loss in body weight at 24 h following its administration, the HESM supplemented birds showed significantly less body weight loss compared with the control fed birds. The WBC, heterophil/lymphocyte ratio, and the levels of IgG were low in chickens fed diets with HESM supplement compared with control diet group. LPS challenge increased the expression of pro-inflammatory cytokine gene IL-6 but the HESM fed birds showed its effect curtailed, also, which also, favored the up-regulation of anti-inflammatory genes compared with control diet fed chickens. Post hatch supplementation of HESM appears to improve performance, modulate immunity, and increase resistance of

  9. Unique motifs identify PIG-A proteins from glycosyltransferases of the GT4 family

    PubMed Central

    2008-01-01

    Background The first step of GPI anchor biosynthesis is catalyzed by PIG-A, an enzyme that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to phosphatidylinositol. This protein is present in all eukaryotic organisms ranging from protozoa to higher mammals, as part of a larger complex of five to six 'accessory' proteins whose individual roles in the glycosyltransferase reaction are as yet unclear. The PIG-A gene has been shown to be an essential gene in various eukaryotes. In humans, mutations in the protein have been associated with paroxysomal noctural hemoglobuinuria. The corresponding PIG-A gene has also been recently identified in the genome of many archaeabacteria although genes of the accessory proteins have not been discovered in them. The present study explores the evolution of PIG-A and the phylogenetic relationship between this protein and other glycosyltransferases. Results In this paper we show that out of the twelve conserved motifs identified by us eleven are exclusively present in PIG-A and, therefore, can be used as markers to identify PIG-A from newly sequenced genomes. Three of these motifs are absent in the primitive eukaryote, G. lamblia. Sequence analyses show that seven of these conserved motifs are present in prokaryote and archaeal counterparts in rudimentary forms and can be used to differentiate PIG-A proteins from glycosyltransferases. Using partial least square regression analysis and data involving presence or absence of motifs in a range of PIG-A and glycosyltransferases we show that (i) PIG-A may have evolved from prokaryotic glycosyltransferases and lipopolysaccharide synthases, members of the GT4 family of glycosyltransferases and (ii) it is possible to uniquely classify PIG-A proteins versus glycosyltransferases. Conclusion Besides identifying unique motifs and showing that PIG-A protein from G. lamblia and some putative PIG-A proteins from archaebacteria are evolutionarily closer to glycosyltransferases, these studies

  10. Construction of a Comprehensive Protein-Protein Interaction Map for Vitiligo Disease to Identify Key Regulatory Elements: A Systemic Approach.

    PubMed

    Malhotra, Anvita Gupta; Jha, Mohit; Singh, Sudha; Pandey, Khushhali M

    2017-03-13

    Vitiligo is an idiopathic disorder characterized by depigmented patches on the skin due to progressive loss of melanocytes. Several genetic, immunological, and pathophysiological investigations have established vitiligo as a polygenetic disorder with multifactorial etiology. However, no definite model explaining the interplay between these causative factors has been established hitherto. Therefore, we studied the disorder at the system level to identify the key proteins involved by exploring their molecular connectivity in terms of topological parameters. The existing research data helped us in collating 215 proteins involved in vitiligo onset or progression. Interaction study of these proteins leads to a comprehensive vitiligo map with 4845 protein nodes linked with 107,416 edges. Based on centrality measures, a backbone network with 500 nodes has been derived. This has presented a clear overview of the proteins and processes involved and the crosstalk between them. Clustering backbone proteins revealed densely connected regions inferring major molecular interaction modules essential for vitiligo. Finally, a list of top order proteins that play a key role in the disease pathomechanism has been formulated. This includes SUMO2, ESR1, COPS5, MYC, SMAD3, and Cullin proteins. While this list is in fair agreement with the available literature, it also introduces new candidate proteins that can be further explored. A subnetwork of 64 vitiligo core proteins was built by analyzing the backbone and seed protein networks. Our finding suggests that the topology, along with functional clustering, provides a deep insight into the behavior of proteins. This in turn aids in the illustration of disease condition and discovery of significant proteins involved in vitiligo.

  11. Juvenile hormone-binding proteins of Melanoplus bivittatus identified by EFDA photoaffinity labeling

    SciTech Connect

    Winder, B.S.

    1988-01-01

    Proteins that bind juvenile hormone in the hemolymph and fat body of the grasshopper, Melanoplus bivittatus were identified by photoaffinity labeling with radiolabeled epoxyfarnesyl diazoacetate ({sup 3}H-EFDA), and were characterized by electrophoretic analysis. A protocol was developed which allowed detection of {sup 3}H-EFDA that was covalently linked to proteins upon exposure to ultraviolet light at 254 nm. Quantification of protein-linked {sup 3}H-EFDA by liquid scintillation spectrometry took advantage of the differential solubility of unlinked {sup 3}H-EFDA in toluene alone, and of the protein-linked {sup 3}H-EFDA in toluene plus the detergent, Triton X-100. Competition between EFDA and juvenile hormone (JH) for binding to JH-specific binding sites was measured by hydroxyapatite protein binding assays in the presence of radiolabeled JH or EFDA and competing non-radiolabeled hormone. The protein-linked EFDA was detected on fluorograms of SDS or nondenaturing polyacrylamide gels (PAGE), and by liquid scintillation spectrometry of membranes to which the proteins had been electrophoretically transferred. Proteins which specifically bound JH were identified by photolabeling proteins in the presence and absence of nonlabeled JH-III.

  12. A high-throughput method to examine protein-nucleotide interactions identifies targets of the bacterial transcriptional regulatory protein fur.

    PubMed

    Yu, Chunxiao; Lopez, Carlos A; Hu, Han; Xia, Yu; Freedman, David S; Reddington, Alexander P; Daaboul, George G; Unlü, M Selim; Genco, Caroline Attardo

    2014-01-01

    The Ferric uptake regulatory protein (Fur) is a transcriptional regulatory protein that functions to control gene transcription in response to iron in a number of pathogenic bacteria. In this study, we applied a label-free, quantitative and high-throughput analysis method, Interferometric Reflectance Imaging Sensor (IRIS), to rapidly characterize Fur-DNA interactions in vitro with predicted Fur binding sequences in the genome of Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea. IRIS can easily be applied to examine multiple protein-protein, protein-nucleotide and nucleotide-nucleotide complexes simultaneously and demonstrated here that seventy percent of the predicted Fur boxes in promoter regions of iron-induced genes bound to Fur in vitro with a range of affinities as observed using this microarray screening technology. Combining binding data with mRNA expression levels in a gonococcal fur mutant strain allowed us to identify five new gonococcal genes under Fur-mediated direct regulation.

  13. Proteomic profiling of the mitochondrial ribosome identifies Atp25 as a composite mitochondrial precursor protein.

    PubMed

    Woellhaf, Michael W; Sommer, Frederik; Schroda, Michael; Herrmann, Johannes M

    2016-10-15

    Whereas the structure and function of cytosolic ribosomes are well characterized, we only have a limited understanding of the mitochondrial translation apparatus. Using SILAC-based proteomic profiling, we identified 13 proteins that cofractionated with the mitochondrial ribosome, most of which play a role in translation or ribosomal biogenesis. One of these proteins is a homologue of the bacterial ribosome-silencing factor (Rsf). This protein is generated from the composite precursor protein Atp25 upon internal cleavage by the matrix processing peptidase MPP, and in this respect, it differs from all other characterized mitochondrial proteins of baker's yeast. We observed that cytosolic expression of Rsf, but not of noncleaved Atp25 protein, is toxic. Our results suggest that eukaryotic cells face the challenge of avoiding negative interference from the biogenesis of their two distinct translation machineries.

  14. Identifying protein complexes in PPI network using non-cooperative sequential game.

    PubMed

    Maulik, Ujjwal; Basu, Srinka; Ray, Sumanta

    2017-08-21

    Identifying protein complexes from protein-protein interaction (PPI) network is an important and challenging task in computational biology as it helps in better understanding of cellular mechanisms in various organisms. In this paper we propose a noncooperative sequential game based model for protein complex detection from PPI network. The key hypothesis is that protein complex formation is driven by mechanism that eventually optimizes the number of interactions within the complex leading to dense subgraph. The hypothesis is drawn from the observed network property named small world. The proposed multi-player game model translates the hypothesis into the game strategies. The Nash equilibrium of the game corresponds to a network partition where each protein either belong to a complex or form a singleton cluster. We further propose an algorithm to find the Nash equilibrium of the sequential game. The exhaustive experiment on synthetic benchmark and real life yeast networks evaluates the structural as well as biological significance of the network partitions.

  15. Identifying secondary structures in proteins using NMR chemical shift 3D correlation maps

    NASA Astrophysics Data System (ADS)

    Kumari, Amrita; Dorai, Kavita

    2013-06-01

    NMR chemical shifts are accurate indicators of molecular environment and have been extensively used as aids in protein structure determination. This work focuses on creating empirical 3D correlation maps of backbone chemical shift nuclei for use as identifiers of secondary structure elements in proteins. A correlated database of backbone nuclei chemical shifts was constructed from experimental structural data gathered from entries in the Protein Data Bank (PDB) as well as isotropic chemical shift values from the RefDB database. Rigorous statistical analysis of the maps led to the conclusion that specific correlations between triplets of backbone chemical shifts are best able to differentiate between different secondary structures such as α-helices, β-strands and turns. The method is compared with similar techniques that use NMR chemical shift information as aids in biomolecular structure determination and performs well in tests done on experimental data determined for different types of proteins, including large multi-domain proteins and membrane proteins.

  16. 9 CFR 147.13 - Procedure for bacteriological culturing of eggshells for colon bacilli organisms.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... culturing of eggshells for colon bacilli organisms. 147.13 Section 147.13 Animals and Animal Products ANIMAL... bacteriological culturing of eggshells for colon bacilli organisms. Proper precautions to avoid environmental... conclusion of the presence of colon bacilli organisms. (Approved by the Office of Management and Budget...

  17. Ultrastructural Alterations in the Eggshell Gland Epithelium of the Mallard Duck After Chronic Exposure to DDT.

    DTIC Science & Technology

    1978-03-01

    This study was conducted to determine what effects the chronic ingestion of DDT would have on the ultrastructure of the eggshell gland of the mallard...endoplasmic reticulum. Since the type II cells are responsible for the transport of calcium, alterations in these cells indicate that decreased calcium transport may be responsible for DDT-induced eggshell thinning. (Author)

  18. Sorption mechanism of Cd(II) from water solution onto chicken eggshell

    NASA Astrophysics Data System (ADS)

    Flores-Cano, Jose Valente; Leyva-Ramos, Roberto; Mendoza-Barron, Jovita; Guerrero-Coronado, Rosa María; Aragón-Piña, Antonio; Labrada-Delgado, Gladis Judith

    2013-07-01

    The mechanism and capacity of eggshell for sorbing Cd(II) from aqueous solution was examined in detail. The eggshell was characterized by several techniques. The eggshell was mainly composed of Calcite (CaCO3). The surface charge distribution was determined by acid-base titration and the point of zero charge (PZC) of the eggshell was found to be 11.4. The sorption equilibrium data were obtained in a batch adsorber, and the adsorption isotherm of Langmuir fitted the data quite well. The sorption capacity of eggshell increased while raising the pH from 4 to 6, this tendency was attributed to the electrostatic interaction between the Cd2+ in solution and the surface of the eggshell. Furthermore, the sorption capacity was augmented by increasing the temperature from 15 to 35 °C because the sorption was endothermic. The sorption of Cd(II) occurred mainly onto the calcareous layer of the eggshell, but slightly on the membrane layer. It was demonstrated that the sorption of Cd(II) was not reversible, and the main sorption mechanisms were precipitation and ion exchange. The precipitation of (Cd,Ca)CO3 on the surface of the eggshell was corroborated by SEM and XRD analysis.

  19. Combined Use of Electron and Light Microscopy Techniques Reveals False Secondary Shell Units in Megaloolithidae Eggshells.

    PubMed

    Moreno-Azanza, Miguel; Bauluz, Blanca; Canudo, José Ignacio; Gasca, José Manuel; Torcida Fernández-Baldor, Fidel

    2016-01-01

    Abnormalities in the histo- and ultrastructure of the amniote eggshell are often related to diverse factors, such as ambient stress during egg formation, pathologies altering the physiology of the egg-laying females, or evolutionarily selected modifications of the eggshell structure that vary the physical properties of the egg, for example increasing its strength so as to avoid fracture during incubation. When dealing with fossil materials, all the above hypotheses are plausible, but a detailed taphonomical study has to be performed to rule out the possibility that secondary processes of recrystallization have occurred during fossilization. Traditional analyses, such as optical microscopy inspection and cathodoluminescence, have proven not to be enough to understand the taphonomic story of some eggshells. Recently, electron backscatter diffraction has been used, in combination with other techniques, to better understand the alteration of fossil eggshells. Here we present a combined study using scanning electron microscopy, optical microscopy, cathodoluminescence and electron backscatter diffraction of eggshell fragments assigned to Megaloolithus cf. siruguei from the Upper Cretaceous outcrops of the Cameros Basin. We focus our study on the presence of secondary shell units that mimic most aspects of the ultrastructure of the eggshell mammillae, but grow far from the inner surface of the eggshell. We call these structures extra-spherulites, describe their crystal structure and demonstrate their secondary origin. Our study has important implications for the interpretation of secondary shell units as biological or pathological structures. Thus, electron backscatter diffraction complements other microscope techniques as a useful tool for understanding taphonomical alterations in fossil eggshells.

  20. Mineralization of clapper rail eggshell from a contaminated salt marsh system.

    PubMed

    Rodriguez-Navarro, A B; Gaines, K F; Romanek, C S; Masson, G R

    2002-11-01

    The effect of contamination on eggshell mineralization has been studied for clapper rails (Rallus longirostris) inhabiting a contaminated salt marsh in coastal Georgia. To assess the impact of contaminants, the thickness, microstructure (crystal orientation), mineral composition, and chemistry of shell material were analyzed from a contaminated site and a nearby reference site using optical microscopy, X-ray diffraction, inductively coupled plasma mass spectrometry, and gas chromatography with electron capture detector. Eggshells from the contaminated site were generally thinner than those from the reference site. Also, eggshells from the contaminated site were abnormally brittle and contained anomalous microstructural attributes. The combination of reduced shell thickness and anomalous microstructure resulted in weaker eggshells, which in turn could pose a significant threat to the reproductive success of the affected population.PCB concentrations in eggshells were at background levels in both sites. Eggshells from the contaminated site had higher concentrations of heavy metals, specifically mercury, than the reference site. The structural changes observed in eggshells may be related to the concentration of specific metals ( e.g., Mg, Cu, Zn, Pb, and Hg) in shell, however, statistical analyses indicated that metals only explained a small portion of the observed variation in properties ( i.e., thickness, crystal orientation). Further analysis is required to better constrain the factors leading to unusually weak eggshells in the contaminated site.

  1. 9 CFR 147.13 - Procedure for bacteriological culturing of eggshells for colon bacilli organisms.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... culturing of eggshells for colon bacilli organisms. 147.13 Section 147.13 Animals and Animal Products ANIMAL... bacteriological culturing of eggshells for colon bacilli organisms. Proper precautions to avoid environmental... conclusion of the presence of colon bacilli organisms. (Approved by the Office of Management and Budget...

  2. 9 CFR 147.13 - Procedure for bacteriological culturing of eggshells for colon bacilli organisms.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... culturing of eggshells for colon bacilli organisms. 147.13 Section 147.13 Animals and Animal Products ANIMAL... bacteriological culturing of eggshells for colon bacilli organisms. Proper precautions to avoid environmental... conclusion of the presence of colon bacilli organisms. (Approved by the Office of Management and Budget...

  3. 9 CFR 147.13 - Procedure for bacteriological culturing of eggshells for colon bacilli organisms.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... culturing of eggshells for colon bacilli organisms. 147.13 Section 147.13 Animals and Animal Products ANIMAL... bacteriological culturing of eggshells for colon bacilli organisms. Proper precautions to avoid environmental... conclusion of the presence of colon bacilli organisms. (Approved by the Office of Management and Budget...

  4. Trace-element interactions in Rook Corvus frugilegus eggshells along an urbanisation gradient.

    PubMed

    Orłowski, Grzegorz; Kasprzykowski, Zbigniew; Dobicki, Wojciech; Pokorny, Przemysław; Wuczyński, Andrzej; Polechoński, Ryszard; Mazgajski, Tomasz D

    2014-11-01

    Concentrations of seven trace elements [arsenic (As), chromium (Cr), nickel (Ni), lead (Pb), copper (Cu), zinc (Zn), and cadmium (Cd)] in the eggshells of Rooks Corvus frugilegus, a focal bird species of Eurasian agricultural environments, are increased above background levels and exceed levels of toxicological concern. The concentrations of Cr, Ni, Pb, Cu, and Zn are greater in eggshells from urban rookeries (large cities) compared with rural areas (small towns and villages) suggesting an urbanisation gradient effect among eggs laid by females. In the present study, the investigators assessed whether the pattern of relationships among the seven trace elements in eggshells change along an urbanisation/pollution gradient. Surprisingly, we found that eggshells with the greatest contaminant burden, i.e., from urban rookeries, showed far fewer significant relationships (n = 4) than eggshells from villages (n = 10), small towns (n = 6), or rural areas (n = 8). In most cases, the relationships were positive. As was an exception: Its concentration was negatively correlated with Ni and Cd levels in eggshells from small town rookeries (where As levels were the highest), whereas eggshells from villages (with a lower As level) showed positive relationships between As and Cd. Our findings suggest that at low to intermediate levels, interactions between the trace elements in Rook eggshells are of a synergistic character and appear to operate as parallel coaccumulation. A habitat-specific excess of some elements (primarily Cr, Ni, Cu, As) suggests their more competitively selective sequestration.

  5. Incubation reduces microbial growth on eggshells and the opportunity for trans-shell infection.

    Treesearch

    Mark I. Cook; Steven R. Beissinger; Gary A. Toranzos; Wayne J. Arendt

    2005-01-01

    Avian eggshells harbour microbes shortly after laying, and under appropriate ambient conditions they can multiply rapidly, penetrate through shell pores, infect egg contents and cause embryo mortality. We experimentally examined how incubation affects bacterial processes on the eggshells of pearl-eyed thrashers Margarops fuscatus nesting in tropical montane and lowland...

  6. Urine proteins identified by two-dimensional differential gel electrophoresis facilitate the differential diagnoses of scrapie.

    PubMed

    Lamoureux, Lise; Simon, Sharon L R; Plews, Margot; Ruddat, Viola; Brunet, Simone; Graham, Catherine; Czub, Stefanie; Knox, J David

    2013-01-01

    The difficulty in developing a diagnostic assay for Creutzfeldt - Jakob disease (CJD) and other transmissible spongiform encephalopathies (TSEs) stems in part from the fact that the infectious agent is an aberrantly folded form of an endogenous cellular protein. This precludes the use of the powerful gene based technologies currently applied to the direct detection of other infectious agents. To circumvent this problem our research objective has been to identify a set of proteins exhibiting characteristic differential abundance in response to TSE infection. The objective of the present study was to assess the disease specificity of differentially abundant urine proteins able to identify scrapie infected mice. Two-dimensional differential gel electrophoresis was used to analyze longitudinal collections of urine samples from both prion-infected mice and a transgenic mouse model of Alzheimer's disease. The introduction of fluorescent dyes, that allow multiple samples to be co-resolved and visualized on one two dimensional gel, have increased the accuracy of this methodology for the discovery of robust protein biomarkers for disease. The accuracy of a small panel of differentially abundant proteins to correctly classify an independent naïve sample set was determined. The results demonstrated that at the time of clinical presentation the differential abundance of urine proteins were capable of identifying the prion infected mice with 87% sensitivity and 93% specificity. The identity of the diagnostic differentially abundant proteins was investigated by mass spectrometry.

  7. Proteomics informed by transcriptomics identifies novel secreted proteins in Dermacentor andersoni saliva

    SciTech Connect

    Mudenda, Lwiindi; Aguilar Pierle, Sebastian; Turse, Joshua E.; Scoles, Glen A.; Purvine, Samuel O.; Nicora, Carrie D.; Clauss, Therese RW; Ueti, Massaro W.; Brown, Wendy C.; Brayton, Kelly A.

    2014-08-07

    Dermacentor andersoni, known as the Rocky Mountain wood tick, is found in the western United States and transmits pathogens that cause diseases of veterinary and public health importance including Rocky Mountain spotted fever, tularemia, Colorado tick fever and bovine anaplasmosis. Tick saliva is known to modulate both innate and acquired immune responses, enabling ticks to feed for several days without detection. During feeding ticks subvert host defences such as hemostasis and inflammation, which would otherwise result in coagulation, wound repair and rejection of the tick. Molecular characterization of the proteins and pharmacological molecules secreted in tick saliva offers an opportunity to develop tick vaccines as an alternative to the use of acaricides, as well as new anti-inflammatory drugs. We performed proteomics informed by transcriptomics to identify D. andersoni saliva proteins that are secreted during feeding. The transcript data generated a database of 21,797 consensus sequences, which we used to identify 677 proteins secreted in the saliva of D. andersoni ticks fed for 2 and 5 days, following proteomic investigations of whole saliva using mass spectrometry. Salivary gland transcript levels of unfed ticks were compared with 2 and 5 day fed ticks to identify genes upregulated early during tick feeding. We cross-referenced the proteomic data with the transcriptomic data to identify 157 proteins of interest for immunomodulation and blood feeding. Proteins of unknown function as well as known immunomodulators were identified.

  8. Differentially expressed proteins underlying childhood cortical dysplasia with epilepsy identified by iTRAQ proteomic profiling

    PubMed Central

    Liu, Shiyong; Liu, Yi; Yang, Yixuan; Yang, Hui; Chen, Yangmei; Chen, Lifen

    2017-01-01

    Cortical dysplasia accounts for at least 14% of epilepsy cases, and is mostly seen in children. However, the understanding of molecular mechanisms and pathogenesis underlying cortical dysplasia is limited. The aim of this cross-sectional study is to identify potential key molecules in the mechanisms of cortical dysplasia by screening the proteins expressed in brain tissues of childhood cortical dysplasia patients with epilepsy using isobaric tags for relative and absolute quantitation-based tandem mass spectrometry compared to controls, and several differentially expressed proteins that are not reported to be associated with cortical dysplasia previously were selected for validation using real-time polymerase chain reaction, immunoblotting and immunohistochemistry. 153 out of 3340 proteins were identified differentially expressed between childhood cortical dysplasia patients and controls. And FSCN1, CRMP1, NDRG1, DPYSL5, MAP4, and FABP3 were selected for validation and identified to be increased in childhood cortical dysplasia patients, while PRDX6 and PSAP were identified decreased. This is the first report on differentially expressed proteins in childhood cortical dysplasia. We identified differential expression of FSCN1, CRMP1, NDRG1, DPYSL5, MAP4, FABP3, PRDX6 and PSAP in childhood cortical dysplasia patients, these proteins are involved in various processes and have various function. These results may provide new directions or targets for the research of childhood cortical dysplasia, and may be helpful in revealing molecular mechanisms and pathogenesis and/or pathophysiology of childhood cortical dysplasia if further investigated. PMID:28222113

  9. Novel method for identifying sequence-specific DNA-binding proteins.

    PubMed

    Levens, D; Howley, P M

    1985-09-01

    We developed a general method for the enrichment and identification of sequence-specific DNA-binding proteins. A well-characterized protein-DNA interaction is used to isolate from crude cellular extracts or fractions thereof proteins which bind to specific DNA sequences; the method is based solely on this binding property of the proteins. The DNA sequence of interest, cloned adjacent to the lac operator DNA segment is incubated with a lac repressor-beta-galactosidase fusion protein which retains full operator and inducer binding properties. The DNA fragment bound to the lac repressor-beta-galactosidase fusion protein is precipitated by the addition of affinity-purified anti-beta-galactosidase immobilized on beads. This forms an affinity matrix for any proteins which might interact specifically with the DNA sequence cloned adjacent to the lac operator. When incubated with cellular extracts in the presence of excess competitor DNA, any protein(s) which specifically binds to the cloned DNA sequence of interest can be cleanly precipitated. When isopropyl-beta-D-thiogalactopyranoside is added, the lac repressor releases the bound DNA, and thus the protein-DNA complex consisting of the specific restriction fragment and any specific binding protein(s) is released, permitting the identification of the protein by standard biochemical techniques. We demonstrate the utility of this method with the lambda repressor, another well-characterized DNA-binding protein, as a model. In addition, with crude preparations of the yeast mitochondrial RNA polymerase, we identified a 70,000-molecular-weight peptide which binds specifically to the promoter region of the yeast mitochondrial 14S rRNA gene.

  10. ApicoAMP: the first computational model for identifying apicoplast-targeted transmembrane proteins in Apicomplexa.

    PubMed

    Cilingir, Gokcen; Lau, Audrey O T; Broschat, Shira L

    2013-12-01

    Computational identification of apicoplast-targeted proteins is important in drug target determination for diseases such as malaria. While there are established methods for identifying proteins with a bipartite signal in multiple species of Apicomplexa, not all apicoplast-targeted proteins possess this bipartite signature. The publication of recent experimental findings of apicoplast membrane proteins, called transmembrane proteins, that do not possess a bipartite signal has made it feasible to devise a machine learning approach for identifying this new class of apicoplast-targeted proteins computationally. In this work, we develop a method for predicting apicoplast-targeted transmembrane proteins for multiple species of Apicomplexa, whereby several classifiers trained on different feature sets and based on different algorithms are evaluated and combined in an ensemble classification model to obtain the best expected performance. The feature sets considered are the hydrophobicity and composition characteristics of amino acids over transmembrane domains, the existence of short sequence motifs over cytosolically disposed regions, and Gene Ontology (GO) terms associated with given proteins. Our model, ApicoAMP, is an ensemble classification model that combines decisions of classifiers following the majority vote principle. ApicoAMP is trained on a set of proteins from 11 apicomplexan species and achieves 91% overall expected accuracy. ApicoAMP is the first computational model capable of identifying apicoplast-targeted transmembrane proteins in Apicomplexa. The ApicoAMP prediction software is available at http://code.google.com/p/apicoamp/ and http://bcb.eecs.wsu.edu. © 2013 Elsevier B.V. All rights reserved.

  11. Proteomics using mammospheres as a model system to identify proteins deregulated in breast cancer stem cells.

    PubMed

    Li, G; Zhao, F; Cui, Y

    2013-03-01

    Breast cancer stem cells (BCSC) exist within many types of breast cancers, functioning to initiate tumorigenesis and augment its progression. The protein profile associated with BCSC has yet to be extensively studied. Mammospheres have been widely employed as a model system to study BCSC. We used proteomics on the MCF-7 breast cancer cell line to compare protein expression in mammosphere-derived cells to that of parental monolayer cells. We identified 34 differentially expressed proteins, seven of which were overexpressed, with the remaining downregulated in mammosphere-derived cells. These differentially expressed proteins include those involved in cell metabolism such as GAPDH and fatty acid synthase, stress response proteins like Hsp27 and FKBP4, and signal transduction related proteins like GIPC1. The expression of breast cancer tumorigenesis and progression-promoting proteins GAPDH and FKBP4 were validated through western blotting. These two proteins are especially recognized for their role in breast cancer resistance to current chemotherapies. The data generated by mammosphere proteomics suggest that this system can identify novel targets for breast cancer stem cells and may provide insights into novel therapy of breast cancer.

  12. Newly identified RNAs of raspberry leaf blotch virus encoding a related group of proteins.

    PubMed

    Lu, Yuwen; McGavin, Wendy; Cock, Peter J A; Schnettler, Esther; Yan, Fei; Chen, Jianping; MacFarlane, Stuart

    2015-11-01

    Members of the genus Emaravirus, including Raspberry leaf blotch virus (RLBV), are enveloped plant viruses with segmented genomes of negative-strand RNA, although the complete genome complement for any of these viruses is not yet clear. Currently, wheat mosaic virus has the largest emaravirus genome comprising eight RNAs. Previously, we identified five genomic RNAs for RLBV; here, we identify a further three RNAs (RNA6-8). RNA6-8 encode proteins that have clear homologies to one another, but not to any other emaravirus proteins. The proteins self-interacted in yeast two-hybrid and bimolecular fluorescence complementation (BiFC) experiments, and the P8 protein interacted with the virus nucleocapsid protein (P3) using BiFC. Expression of two of the proteins (P6 and P7) using potato virus X led to an increase in virus titre and symptom severity, suggesting that these proteins may play a role in RLBV pathogenicity; however, using two different tests, RNA silencing suppression activity was not detected for any of the RLBV proteins encoded by RNA2-8.

  13. A PQL (protein quantity loci) analysis of mature pea seed proteins identifies loci determining seed protein composition.

    PubMed

    Bourgeois, Michael; Jacquin, Françoise; Cassecuelle, Florence; Savois, Vincent; Belghazi, Maya; Aubert, Grégoire; Quillien, Laurence; Huart, Myriam; Marget, Pascal; Burstin, Judith

    2011-05-01

    Legume seeds are a major source of dietary proteins for humans and animals. Deciphering the genetic control of their accumulation is thus of primary significance towards their improvement. At first, we analysed the genetic variability of the pea seed proteome of three genotypes over 3 years of cultivation. This revealed that seed protein composition variability was under predominant genetic control, with as much as 60% of the spots varying quantitatively among the three genotypes. Then, by combining proteomic and quantitative trait loci (QTL) mapping approaches, we uncovered the genetic architecture of seed proteome variability. Protein quantity loci (PQL) were searched for 525 spots detected on 2-D gels obtained for 157 recombinant inbred lines. Most protein quantity loci mapped in clusters, suggesting that the accumulation of the major storage protein families was under the control of a limited number of loci. While convicilin accumulation was mainly under the control of cis-regulatory regions, vicilins and legumins were controlled by both cis- and trans-regulatory regions. Some loci controlled both seed protein composition and protein content and a locus on LGIIa appears to be a major regulator of protein composition and of protein in vitro digestibility. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Long-term decline in the thickness of eggshells of thrushes, Turdus spp., in Britain

    PubMed Central

    Green, R. E.

    1998-01-01

    The thickness of eggshells of four species of thrush, Turdus spp., was estimated by an index based on the mass and linear dimensions of blown eggs in museum collections from Britain. Shell thickness was also measured directly for two species and was highly correlated with the index. Widespread declines in eggshell thickness since the nineteenth century were found in all species. There have been no previous reports of trends in eggshell thickness of this long duration and large spatial scale. The cause of the declines is unknown, but, for three of the four species, eggshell thinning began before the introduction of the organochlorine pesticide DDT, which caused eggshell thinning in predatory and fish-eating birds from 1947 onwards. The effect of acid deposition on the availability of calcium-rich prey is a plausible explanation.

  15. A 700-year record of mercury in avian eggshells of Guangjin Island, South China Sea.

    PubMed

    Xu, Li-Qiang; Liu, Xiao-Dong; Sun, Li-guang; Chen, Qian-Qian; Yan, Hong; Liu, Yi; Luo, Yu-Han; Huang, Jing

    2011-04-01

    Ancient eggshells over the past 700 years were extracted from an ornithogenic sediment profile on Guangjin Island, South China Sea. Based on SEM and nitrogen isotope analyses, we determined that neither post-depositional processes nor seabirds' dietary changes had a large influence on eggshell Hg levels. The historical change of Hg in these eggshells was reconstructed. Eggshell Hg was a marker for past Hg deposition in marine environment. The eggshell Hg showed three small peaks at around 1300AD, 1600 AD and 1700-1750AD and rapid increase since 1800 AD. Before 1970 AD the Hg deposition in the Xisha area had global distribution characteristics, with increased Hg emissions due to global anthropogenic activities in industrial times. However, after 1970 AD, a further sharp increase up to present day occurred, implying that the Hg production center had gradually shifted from Europe and America to Asia.

  16. Identifying proteins in zebrafish embryos using spectral libraries generated from dissected adult organs and tissues.

    PubMed

    van der Plas-Duivesteijn, Suzanne J; Mohammed, Yassene; Dalebout, Hans; Meijer, Annemarie; Botermans, Anouk; Hoogendijk, Jordy L; Henneman, Alex A; Deelder, André M; Spaink, Herman P; Palmblad, Magnus

    2014-03-07

    Spectral libraries provide a sensitive and accurate method for identifying peptides from tandem mass spectra, complementary to searching genome-derived databases or sequencing de novo. Their application requires comprehensive libraries including peptides from low-abundant proteins. Here we describe a method for constructing such libraries using biological differentiation to "fractionate" the proteome by harvesting adult organs and tissues and build comprehensive libraries for identifying proteins in zebrafish (Danio rerio) embryos and larvae (an important and widely used model system). Hierarchical clustering using direct comparison of spectra was used to prioritize organ selection. The resulting and publicly available library covers 14,164 proteins, significantly improved the number of peptide-spectrum matches in zebrafish developmental stages, and can be used on data from different instruments and laboratories. The library contains information on tissue and organ expression of these proteins and is also applicable for adult experiments. The approach itself is not limited to zebrafish but would work for any model system.

  17. Genetically encoded protein photocrosslinker with a transferable mass spectrometry-identifiable label

    PubMed Central

    Yang, Yi; Song, Haiping; He, Dan; Zhang, Shuai; Dai, Shizhong; Lin, Shixian; Meng, Rong; Wang, Chu; Chen, Peng R.

    2016-01-01

    Coupling photocrosslinking reagents with mass spectrometry has become a powerful tool for studying protein–protein interactions in living systems, but it still suffers from high rates of false-positive identifications as well as the lack of information on interaction interface due to the challenges in deciphering crosslinking peptides. Here we develop a genetically encoded photo-affinity unnatural amino acid that introduces a mass spectrometry-identifiable label (MS-label) to the captured prey proteins after photocrosslinking and prey–bait separation. This strategy, termed IMAPP (In-situ cleavage and MS-label transfer After Protein Photocrosslinking), enables direct identification of photo-captured substrate peptides that are difficult to uncover by conventional genetically encoded photocrosslinkers. Taking advantage of the MS-label, the IMAPP strategy significantly enhances the confidence for identifying protein–protein interactions and enables simultaneous mapping of the binding interface under living conditions. PMID:27460181

  18. Substrates of the Arabidopsis thaliana Protein Isoaspartyl Methyltransferase 1 Identified Using Phage Display and Biopanning*

    PubMed Central

    Chen, Tingsu; Nayak, Nihar; Majee, Susmita Maitra; Lowenson, Jonathan; Schäfermeyer, Kim R.; Eliopoulos, Alyssa C.; Lloyd, Taylor D.; Dinkins, Randy; Perry, Sharyn E.; Forsthoefel, Nancy R.; Clarke, Steven G.; Vernon, Daniel M.; Zhou, Zhaohui Sunny; Rejtar, Tomas; Downie, A. Bruce

    2010-01-01

    The role of protein isoaspartyl methyltransferase (PIMT) in repairing a wide assortment of damaged proteins in a host of organisms has been inferred from the affinity of the enzyme for isoaspartyl residues in a plethora of amino acid contexts. The identification of PIMT target proteins in plant seeds, where the enzyme is highly active and proteome long-lived, has been hindered by large amounts of isoaspartate-containing storage proteins. Mature seed phage display libraries circumvented this problem. Inclusion of the PIMT co-substrate, S-adenosylmethionine (AdoMet), during panning permitted PIMT to retain aged phage in greater numbers than controls lacking co-substrate or when PIMT protein binding was poisoned with S-adenosyl homocysteine. After four rounds, phage titer plateaued in AdoMet-containing pans, whereas titer declined in both controls. This strategy identified 17 in-frame PIMT target proteins, including a cupin-family protein similar to those identified previously using on-blot methylation. All recovered phage had at least one susceptible Asp or Asn residue. Five targets were recovered independently. Two in-frame targets were produced in Escherichia coli as recombinant proteins and shown by on-blot methylation to acquire isoAsp, becoming a PIMT target. Both gained isoAsp rapidly in solution upon thermal insult. Mutant analysis of plants deficient in any of three in-frame PIMT targets resulted in demonstrable phenotypes. An over-representation of clones encoding proteins involved in protein production suggests that the translational apparatus comprises a subgroup for which PIMT-mediated repair is vital for orthodox seed longevity. Impaired PIMT activity would hinder protein function in these targets, possibly resulting in poor seed performance. PMID:20870712

  19. EST mining and functional expression assays identify extracellular effector proteins from the plant pathogen Phytophthora.

    PubMed

    Torto, Trudy A; Li, Shuang; Styer, Allison; Huitema, Edgar; Testa, Antonino; Gow, Neil A R; van West, Pieter; Kamoun, Sophien

    2003-07-01

    Plant pathogenic microbes have the remarkable ability to manipulate biochemical, physiological, and morphological processes in their host plants. These manipulations are achieved through a diverse array of effector molecules that can either promote infection or trigger defense responses. We describe a general functional genomics approach aimed at identifying extracellular effector proteins from plant pathogenic microorganisms by combining data mining of expressed sequence tags (ESTs) with virus-based high-throughput functional expression assays in plants. PexFinder, an algorithm for automated identification of extracellular proteins from EST data sets, was developed and applied to 2147 ESTs from the oomycete plant pathogen Phytophthora infestans. The program identified 261 ESTs (12.2%) corresponding to a set of 142 nonredundant Pex (Phytophthora extracellular protein) cDNAs. Of these, 78 (55%) Pex cDNAs were novel with no significant matches in public databases. Validation of PexFinder was performed using proteomic analysis of secreted protein of P. infestans. To identify which of the Pex cDNAs encode effector proteins that manipulate plant processes, high-throughput functional expression assays in plants were performed on 63 of the identified cDNAs using an Agrobacterium tumefaciens binary vector carrying the potato virus X (PVX) genome. This led to the discovery of two novel necrosis-inducing cDNAs, crn1 and crn2, encoding extracellular proteins that belong to a large and complex protein family in Phytophthora. Further characterization of the crn genes indicated that they are both expressed in P. infestans during colonization of the host plant tomato and that crn2 induced defense-response genes in tomato. Our results indicate that combining data mining using PexFinder with PVX-based functional assays can facilitate the discovery of novel pathogen effector proteins. In principle, this strategy can be applied to a variety of eukaryotic plant pathogens, including

  20. Protein functional links in Trypanosoma brucei, identified by gene fusion analysis

    PubMed Central

    2011-01-01

    Background Domain or gene fusion analysis is a bioinformatics method for detecting gene fusions in one organism by comparing its genome to that of other organisms. The occurrence of gene fusions suggests that the two original genes that participated in the fusion are functionally linked, i.e. their gene products interact either as part of a multi-subunit protein complex, or in a metabolic pathway. Gene fusion analysis has been used to identify protein functional links in prokaryotes as well as in eukaryotic model organisms, such as yeast and Drosophila. Results In this study we have extended this approach to include a number of recently sequenced protists, four of which are pathogenic, to identify fusion linked proteins in Trypanosoma brucei, the causative agent of African sleeping sickness. We have also examined the evolution of the gene fusion events identified, to determine whether they can be attributed to fusion or fission, by looking at the conservation of the fused genes and of the individual component genes across the major eukaryotic and prokaryotic lineages. We find relatively limited occurrence of gene fusions/fissions within the protist lineages examined. Our results point to two trypanosome-specific gene fissions, which have recently been experimentally confirmed, one fusion involving proteins involved in the same metabolic pathway, as well as two novel putative functional links between fusion-linked protein pairs. Conclusions This is the first study of protein functional links in T. brucei identified by gene fusion analysis. We have used strict thresholds and only discuss results which are highly likely to be genuine and which either have already been or can be experimentally verified. We discuss the possible impact of the identification of these novel putative protein-protein interactions, to the development of new trypanosome therapeutic drugs. PMID:21729286

  1. SCMMTP: identifying and characterizing membrane transport proteins using propensity scores of dipeptides

    PubMed Central

    2015-01-01

    Background Identifying putative membrane transport proteins (MTPs) and understanding the transport mechanisms involved remain important challenges for the advancement of structural and functional genomics. However, the transporter characters are mainly acquired from MTP crystal structures which are hard to crystalize. Therefore, it is desirable to develop bioinformatics tools for the effective large-scale analysis of available sequences to identify novel transporters and characterize such transporters. Results This work proposes a novel method (SCMMTP) based on the scoring card method (SCM) using dipeptide composition to identify and characterize MTPs from an existing dataset containing 900 MTPs and 660 non-MTPs which are separated into a training dataset consisting 1,380 proteins and an independent dataset consisting 180 proteins. The SCMMTP produced estimating propensity scores for amino acids and dipeptides as MTPs. The SCMMTP training and test accuracy levels respectively reached 83.81% and 76.11%. The test accuracy of support vector machine (SVM) using a complicated classification method with a low possibility for biological interpretation and position-specific substitution matrix (PSSM) as a protein feature is 80.56%, thus SCMMTP is comparable to SVM-PSSM. To identify MTPs, SCMMTP is applied to three datasets including: 1) human transmembrane proteins, 2) a photosynthetic protein dataset, and 3) a human protein database. MTPs showing α-helix rich structure is agreed with previous studies. The MTPs used residues with low hydration energy. It is hypothesized that, after filtering substrates, the hydrated water molecules need to be released from the pore regions. Conclusions SCMMTP yields estimating propensity scores for amino acids and dipeptides as MTPs, which can be used to identify novel MTPs and characterize transport mechanisms for use in further experiments. Availability http://iclab.life.nctu.edu.tw/iclab_webtools/SCMMTP/ PMID:26677931

  2. The effect of developmental stage on eggshell thickness variation in endangered falcons.

    PubMed

    Castilla, Aurora M; Herrel, Anthony; Robles, Hugo; Malone, Jim; Negro, Juan José

    2010-05-01

    We compared eggshell thickness of hatched eggs with that of non-developed eggs in endangered falcon taxa to explore the effect of embryo development on eggshell thinning. To our knowledge, this has never been examined before in falcons, despite the fact that eggshell thinning due to pollutants and environmental contamination is often considered the most common cause of egg failure in falcons. Because of the endangered nature of these birds, and the difficulty in gaining access to the nests and their eggs, there is a large gap in our knowledge regarding eggshell thickness variation and the factors affecting it. We used a linear mixed-effects (LME) model to explore the variation in eggshell thickness (n=335 eggs) in relation to the developmental stage of the eggs, but also in relation to the falcon taxa, the laying sequence and the study zone. Female identity (n=69) and clutch identity (n=98) were also included in the LME model. Our results are consistent with the prediction that eggshell thickness decreases during incubation because of the important effect of calcium uptake by the embryo during development. Our results also show that eggs laid later in the sequence had significantly thinner eggshells. In this study, we provide the first quantitative data on eggshell thickness variation of hatched eggs in different falcon taxa that were not subjected to contamination or food limitation (i.e., bred under captive conditions). Because eggshell thickness strongly influences survival and because the species examined in this study are endangered, our data represent a valuable control for future studies on the effects of pollution on eggshells from wild populations and thus are an important contribution to the conservation of falcons. (c) 2010 Elsevier GmbH. All rights reserved.

  3. A spatial simulation approach to account for protein structure when identifying non-random somatic mutations

    PubMed Central

    2014-01-01

    Background Current research suggests that a small set of “driver” mutations are responsible for tumorigenesis while a larger body of “passenger” mutations occur in the tumor but do not progress the disease. Due to recent pharmacological successes in treating cancers caused by driver mutations, a variety of methodologies that attempt to identify such mutations have been developed. Based on the hypothesis that driver mutations tend to cluster in key regions of the protein, the development of cluster identification algorithms has become critical. Results We have developed a novel methodology, SpacePAC (Spatial Protein Amino acid Clustering), that identifies mutational clustering by considering the protein tertiary structure directly in 3D space. By combining the mutational data in the Catalogue of Somatic Mutations in Cancer (COSMIC) and the spatial information in the Protein Data Bank (PDB), SpacePAC is able to identify novel mutation clusters in many proteins such as FGFR3 and CHRM2. In addition, SpacePAC is better able to localize the most significant mutational hotspots as demonstrated in the cases of BRAF and ALK. The R package is available on Bioconductor at: http://www.bioconductor.org/packages/release/bioc/html/SpacePAC.html. Conclusion SpacePAC adds a valuable tool to the identification of mutational clusters while considering protein tertiary structure. PMID:24990767

  4. Proteomic analysis identifies differentially expressed proteins after red propolis treatment in Hep-2 cells.

    PubMed

    Frozza, Caroline Olivieri da Silva; Ribeiro, Tanara da Silva; Gambato, Gabriela; Menti, Caroline; Moura, Sidnei; Pinto, Paulo Marcos; Staats, Charley Christian; Padilha, Francine Ferreira; Begnini, Karine Rech; de Leon, Priscila Marques Moura; Borsuk, Sibele; Savegnago, Lucielli; Dellagostin, Odir; Collares, Tiago; Seixas, Fabiana Kömmling; Henriques, João Antonio Pêgas; Roesch-Ely, Mariana

    2014-01-01

    Here we investigated alterations in the protein profile of Hep-2 treated with red propolis using two-dimensional electrophoresis associated to mass spectrometry and apoptotic rates of cells treated with and without red propolis extracts through TUNEL and Annexin-V assays. A total of 325 spots were manually excised from the two-dimensional gel electrophoresis and 177 proteins were identified using LC-MS-MS. Among all proteins identified that presented differential expression, most were down-regulated in presence of red propolis extract at a concentration of 120 μg/mL (IC50): GRP78, PRDX2, LDHB, VIM and TUBA1A. Only two up-regulated proteins were identified in this study in the non-cytotoxic (6 μg/mL) red propolis treated group: RPLP0 and RAD23B. TUNEL staining assay showed a markedly increase in the mid- to late-stage apoptosis of Hep-2 cells induced by red propolis at concentrations of 60 and 120 μg/mL when compared with non-treated cells. The increase of late apoptosis was confirmed by in situ Annexin-V analysis in which red propolis extract induced late apoptosis in a dose-dependent manner. The differences in tumor cell protein profiles warrant further investigations including isolation of major bioactive compounds of red propolis in different cell lines using proteomics and molecular tests to validate the protein expression here observed. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Endothelial cell palmitoylproteomics identifies novel lipid modified targets and potential substrates for protein acyl transferases

    PubMed Central

    Marin, Ethan P.; Derakhshan, Behrad; Lam, TuKiet T.; Davalos, Alberto; Sessa, William C.

    2012-01-01

    Rationale Protein S-palmitoylation is the post-translational attachment of a saturated 16-carbon palmitic acid to a cysteine side chain via a thioester bond. Palmitoylation can affect protein localization, trafficking, stability, and function. The extent and roles of palmitoylation in endothelial cell (EC) biology is not well understood, in part due to technological limits on palmitoylprotein detection. Objective To develop a method using acyl-biotinyl exchange (ABE) technology coupled with mass spectrometry to globally isolate and identify palmitoylproteins in EC. Methods and Results More than 150 putative palmitoyl proteins were identified in EC using ABE and mass spectrometry. Among the novel palmitoylproteins identified is superoxide dismutase 1 (SOD1), an intensively studied enzyme that protects all cells from oxidative damage. Mutation of cysteine 6 prevents palmitoylation, leads to reduction in SOD1 activity in vivo and in vitro, and inhibits nuclear localization, thereby supporting a functional role for SOD1 palmitoylation. Moreover, we used ABE to search for substrates of particular protein acyl transferases in EC. We found that palmitoylation of the cell adhesion protein PECAM1 is dependent on the protein acyl transferase ZDHHC21. We show that knockdown of ZDHHC21 leads to reduced levels of PECAM1 at the cell surface. Conclusions Our data demonstrate the utility of EC palmitoylproteomics to reveal new insights into the role of this important post-translational lipid modification in EC biology. PMID:22496122

  6. Quantitative Assessment of In-solution Digestion Efficiency Identifies Optimal Protocols for Unbiased Protein Analysis*

    PubMed Central

    León, Ileana R.; Schwämmle, Veit; Jensen, Ole N.; Sprenger, Richard R.

    2013-01-01

    The majority of mass spectrometry-based protein quantification studies uses peptide-centric analytical methods and thus strongly relies on efficient and unbiased protein digestion protocols for sample preparation. We present a novel objective approach to assess protein digestion efficiency using a combination of qualitative and quantitative liquid chromatography-tandem MS methods and statistical data analysis. In contrast to previous studies we employed both standard qualitative as well as data-independent quantitative workflows to systematically assess trypsin digestion efficiency and bias using mitochondrial protein fractions. We evaluated nine trypsin-based digestion protocols, based on standard in-solution or on spin filter-aided digestion, including new optimized protocols. We investigated various reagents for protein solubilization and denaturation (dodecyl sulfate, deoxycholate, urea), several trypsin digestion conditions (buffer, RapiGest, deoxycholate, urea), and two methods for removal of detergents before analysis of peptides (acid precipitation or phase separation with ethyl acetate). Our data-independent quantitative liquid chromatography-tandem MS workflow quantified over 3700 distinct peptides with 96% completeness between all protocols and replicates, with an average 40% protein sequence coverage and an average of 11 peptides identified per protein. Systematic quantitative and statistical analysis of physicochemical parameters demonstrated that deoxycholate-assisted in-solution digestion combined with phase transfer allows for efficient, unbiased generation and recovery of peptides from all protein classes, including membrane proteins. This deoxycholate-assisted protocol was also optimal for spin filter-aided digestions as compared with existing methods. PMID:23792921

  7. Identifying Schistosoma japonicum excretory/secretory proteins and their interactions with host immune system.

    PubMed

    Liao, Qi; Yuan, Xiongying; Xiao, Hui; Liu, Changning; Lv, Zhiyue; Zhao, Yi; Wu, Zhongdao

    2011-01-01

    Schistosoma japonicum is a major infectious agent of schistosomiasis. It has been reported that large number of proteins excreted and secreted by S. japonicum during its life cycle are important for its infection and survival in definitive hosts. These proteins can be used as ideal candidates for vaccines or drug targets. In this work, we analyzed the protein sequences of S. japonicum and found that compared with other proteins in S. japonicum, excretory/secretory (ES) proteins are generally longer, more likely to be stable and enzyme, more likely to contain immune-related binding peptides and more likely to be involved in regulation and metabolism processes. Based on the sequence difference between ES and non-ES proteins, we trained a support vector machine (SVM) with much higher accuracy than existing approaches. Using this SVM, we identified 191 new ES proteins in S. japonicum, and further predicted 7 potential interactions between these ES proteins and human immune proteins. Our results are useful to understand the pathogenesis of schistosomiasis and can serve as a new resource for vaccine or drug targets discovery for anti-schistosome.

  8. Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast.

    PubMed

    Huang, Mingtao; Bai, Yunpeng; Sjostrom, Staffan L; Hallström, Björn M; Liu, Zihe; Petranovic, Dina; Uhlén, Mathias; Joensson, Haakan N; Andersson-Svahn, Helene; Nielsen, Jens

    2015-08-25

    There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant α-amylase. Efficient secretion was genetically stable in the selected clones. We performed whole-genome sequencing of the eight clones and identified 330 mutations in total. Gene ontology analysis of mutated genes revealed many biological processes, including some that have not been identified before in the context of protein secretion. Mutated genes identified in this study can be potentially used for reverse metabolic engineering, with the objective to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could broadly facilitate design of novel cell factories.

  9. Outer membrane proteins can be simply identified using secondary structure element alignment

    PubMed Central

    2011-01-01

    Background Outer membrane proteins (OMPs) are frequently found in the outer membranes of gram-negative bacteria, mitochondria and chloroplasts and have been found to play diverse functional roles. Computational discrimination of OMPs from globular proteins and other types of membrane proteins is helpful to accelerate new genome annotation and drug discovery. Results Based on the observation that almost all OMPs consist of antiparallel β-strands in a barrel shape and that their secondary structure arrangements differ from those of other types of proteins, we propose a simple method called SSEA-OMP to identify OMPs using secondary structure element alignment. Through intensive benchmark experiments, the proposed SSEA-OMP method is better than some well-established OMP detection methods. Conclusions The major advantage of SSEA-OMP is its good prediction performance considering its simplicity. The web server implements the method is freely accessible at http://protein.cau.edu.cn/SSEA-OMP/index.html. PMID:21414186

  10. Incorporating hidden Markov models for identifying protein kinase-specific phosphorylation sites.

    PubMed

    Huang, Hsien-Da; Lee, Tzong-Yi; Tzeng, Shih-Wei; Wu, Li-Cheng; Horng, Jorng-Tzong; Tsou, Ann-Ping; Huang, Kuan-Tsae

    2005-07-30

    Protein phosphorylation, which is an important mechanism in posttranslational modification, affects essential cellular processes such as metabolism, cell signaling, differentiation, and membrane transportation. Proteins are phosphorylated by a variety of protein kinases. In this investigation, we develop a novel tool to computationally predict catalytic kinase-specific phosphorylation sites. The known phosphorylation sites from public domain data sources are categorized by their annotated protein kinases. Based on the concepts of profile Hidden Markov Models (HMM), computational models are trained from the kinase-specific groups of phosphorylation sites. After evaluating the trained models, we select the model with highest accuracy in each kinase-specific group and provide a Web-based prediction tool for identifying protein phosphorylation sites. The main contribution here is that we have developed a kinase-specific phosphorylation site prediction tool with both high sensitivity and specificity.

  11. Proteomic approach to identify champagne wine proteins as modified by Botrytis cinerea infection.

    PubMed

    Cilindre, Clara; Jégou, Sandrine; Hovasse, Agnès; Schaeffer, Christine; Castro, Antonio J; Clément, Christophe; Van Dorsselaer, Alain; Jeandet, Philippe; Marchal, Richard

    2008-03-01

    The presence of the fungal pathogen, Botrytis cinerea, in the vineyard causes reductions in both quality and quantity of grapes and wine. Because proteins are involved in the foam stabilization of sparkling wines, we have undertaken, for the first time, a thorough proteomic analysis of two champagne base wines prepared with either healthy or botrytized Chardonnay grapes, using two-dimensional electrophoresis (2DE) coupled with immunodetection and tandem mass spectrometry. Most of the identified proteins were from grape origin: invertase and pathogenesis-related (PR) proteins. The disappearance of numerous grape proteins was observed in the botrytized wine, suggesting that they were probably degraded or even repressed or the result of a differential expression of grape proteins upon fungal infection. On the other hand, two pectinolytic enzymes secreted by B. cinerea were found in the botrytized wine.

  12. An Improved Method for Identifying Specific DNA-Protein-Binding Sites In Vitro.

    PubMed

    Wang, Liangyan; Lu, Huizhi; Wang, Yunguang; Yang, Su; Xu, Hong; Cheng, Kaiying; Zhao, Ye; Tian, Bing; Hua, Yuejin

    2017-03-01

    Binding of proteins to specific DNA sequences is essential for a variety of cellular processes such as DNA replication, transcription and responses to external stimuli. Chromatin immunoprecipitation is widely used for determining intracellular DNA fragments bound by a specific protein. However, the subsequent specific or accurate DNA-protein-binding sequence is usually determined by DNA footprinting. Here, we report an alternative method for identifying specific sites of DNA-protein-binding (designated SSDP) in vitro. This technique is mainly dependent on antibody-antigen immunity, simple and convenient, while radioactive isotope labeling and optimization of partial degradation by deoxyribonuclease (DNase) are avoided. As an example, the specific binding sequence of a target promoter by DdrO (a DNA damage response protein from Deinococcus radiodurans) in vitro was determined by the developed method. The central sequence of the binding site could be easily located using this technique.

  13. The first identified nucleocytoplasmic shuttling herpesviral capsid protein: herpes simplex virus type 1 VP19C.

    PubMed

    Zhao, Lei; Zheng, Chunfu

    2012-01-01

    VP19C is a structural protein of herpes simplex virus type 1 viral particle, which is essential for assembly of the capsid. In this study, a nuclear export signal (NES) of VP19C is for the first time identified and mapped to amino acid residues 342 to 351. Furthermore, VP19C is demonstrated to shuttle between the nucleus and the cytoplasm through the NES in a chromosomal region maintenance 1 (CRM1)-dependent manner involving RanGTP hydrolysis. This makes VP19C the first herpesviral capsid protein with nucleocytoplasmic shuttling property and adds it to the list of HSV-1 nucleocytoplasmic shuttling proteins.

  14. Identifying relationships between unrelated pharmaceutical target proteins on the basis of shared active compounds.

    PubMed

    Miljković, Filip; Kunimoto, Ryo; Bajorath, Jürgen

    2017-08-01

    Computational exploration of small-molecule-based relationships between target proteins from different families. Target annotations of drugs and other bioactive compounds were systematically analyzed on the basis of high-confidence activity data. A total of 286 novel chemical links were established between distantly related or unrelated target proteins. These relationships involved a total of 1859 bioactive compounds including 147 drugs and 141 targets. Computational analysis of large amounts of compounds and activity data has revealed unexpected relationships between diverse target proteins on the basis of compounds they share. These relationships are relevant for drug discovery efforts. Target pairs that we have identified and associated compound information are made freely available.

  15. Altered protein profile in chronic myeloid leukemia chronic phase identified by a comparative proteomic study.

    PubMed

    Pizzatti, Luciana; Sá, Lílian Ayres; de Souza, Jamison Menezes; Bisch, Paulo Mascarello; Abdelhay, Eliana

    2006-05-01

    Chronic myeloid leukemia is a hematological disorder in which the Ph chromosome is a marker of the disease, detected virtually in all cases. The chimeric transcripts encode a 210-kDa chimeric protein with altered tyrosine kinase activity, responsible for the disease phenotype. In this work, we tried to identify which are the molecular changes common to chronic phase patients, those that represent the chronic phase molecular phenotype. To address this problem we analyzed through a comparative proteomic approach, several CML bone marrow cells protein profile from patients in chronic phase and healthy bone marrow donors. From these results, we identified 31 differentially expressed proteins. Among these proteins, we pointed out c-Myc binding protein 1, 53BP1, Mdm4, OSBP-related protein 3 and Mortalin as putative candidates to BCR-ABL targets in chronic phase. Moreover, we describe for the first time the cytoplasmic protein map from bone marrow cells that helped in the elucidation of the changes we were looking for.

  16. Several Novel Nuclear Envelope Transmembrane Proteins Identified in Skeletal Muscle Have Cytoskeletal Associations*

    PubMed Central

    Wilkie, Gavin S.; Korfali, Nadia; Swanson, Selene K.; Malik, Poonam; Srsen, Vlastimil; Batrakou, Dzmitry G.; de las Heras, Jose; Zuleger, Nikolaj; Kerr, Alastair R. W.; Florens, Laurence; Schirmer, Eric C.

    2011-01-01

    Nuclear envelopes from liver and a neuroblastoma cell line have previously been analyzed by proteomics; however, most diseases associated with the nuclear envelope affect muscle. To determine whether muscle has unique nuclear envelope proteins, rat skeletal muscle nuclear envelopes were prepared and analyzed by multidimensional protein identification technology. Many novel muscle-specific proteins were identified that did not appear in previous nuclear envelope data sets. Nuclear envelope residence was confirmed for 11 of these by expression of fusion proteins and by antibody staining of muscle tissue cryosections. Moreover, transcript levels for several of the newly identified nuclear envelope transmembrane proteins increased during muscle differentiation using mouse and human in vitro model systems. Some of these proteins tracked with microtubules at the nuclear surface in interphase cells and accumulated at the base of the microtubule spindle in mitotic cells, suggesting they may associate with complexes that connect the nucleus to the cytoskeleton. The finding of tissue-specific proteins in the skeletal muscle nuclear envelope proteome argues the importance of analyzing nuclear envelopes from all tissues linked to disease and suggests that general investigation of tissue differences in organellar proteomes might yield critical insights. PMID:20876400

  17. Comparative proteomic analysis of horseweed (Conyza canadensis) biotypes identifies candidate proteins for glyphosate resistance

    PubMed Central

    González-Torralva, Fidel; Brown, Adrian P.; Chivasa, Stephen

    2017-01-01

    Emergence of glyphosate-resistant horseweed (Conyza canadensis) biotypes is an example of how unrelenting use of a single mode of action herbicide in agricultural weed control drives genetic adaptation in targeted species. While in other weeds glyphosate resistance arose from target site mutation or target gene amplification, the resistance mechanism in horseweed uses neither of these, being instead linked to reduced herbicide uptake and/or translocation. The molecular components underpinning horseweed glyphosate-resistance remain unknown. Here, we used an in vitro leaf disc system for comparative analysis of proteins extracted from control and glyphosate-treated tissues of glyphosate-resistant and glyphosate-susceptible biotypes. Analysis of shikimic acid accumulation, ABC-transporter gene expression, and cell death were used to select a suitable glyphosate concentration and sampling time for enriching proteins pivotal to glyphosate resistance. Protein gel analysis and mass spectrometry identified mainly chloroplast proteins differentially expressed between the biotypes before and after glyphosate treatment. Chloroplasts are the organelles in which the shikimate pathway, which is targeted by glyphosate, is located. Calvin cycle enzymes and proteins of unknown function were among the proteins identified. Our study provides candidate proteins that could be pivotal in engendering resistance and implicates chloroplasts as the primary sites driving glyphosate-resistance in horseweed. PMID:28198407

  18. Staphylococcus aureus surface proteins involved in adaptation to oxacillin identified using a novel cell shaving approach.

    PubMed

    Solis, Nestor; Parker, Benjamin L; Kwong, Stephen M; Robinson, Gareth; Firth, Neville; Cordwell, Stuart J

    2014-06-06

    Staphylococcus aureus is a Gram-positive pathogen responsible for a variety of infections, and some strains are resistant to virtually all classes of antibiotics. Cell shaving proteomics using a novel probability scoring algorithm to compare the surfaceomes of the methicillin-resistant, laboratory-adapted S. aureus COL strain with a COL strain in vitro adapted to high levels of oxacillin (APT). APT displayed altered cell morphology compared with COL and increased aggregation in biofilm assays. Increased resistance to β-lactam antibiotics was observed, but adaptation to oxacillin did not confer multidrug resistance. Analysis of the S. aureus COL and APT surfaceomes identified 150 proteins at a threshold determined by the scoring algorithm. Proteins unique to APT included the LytR-CpsA-Psr (LCP) domain-containing MsrR and SACOL2302. Quantitative RT-PCR showed increased expression of sacol2302 in APT grown with oxacillin (>6-fold compared with COL). Overexpression of sacol2302 in COL to levels consistent with APT (+ oxacillin) did not influence biofilm formation or β-lactam resistance. Proteomics using iTRAQ and LC-MS/MS identified 1323 proteins (∼50% of the theoretical S. aureus proteome), and cluster analysis demonstrated elevated APT abundances of LCP proteins, capsule and peptidoglycan biosynthesis proteins, and proteins involved in wall remodelling. Adaptation to oxacillin also induced urease proteins, which maintained culture pH compared to COL. These results show that S. aureus modifies surface architecture in response to antibiotic adaptation.

  19. A Coordinated Proteomic Approach for Identifying Proteins that Interact with the E. coli Ribosomal Protein S12

    PubMed Central

    Strader, Michael Brad; Hervey, William Judson; Costantino, Nina; Fujigaki, Suwako; Chen, Cai Yun; Akal-Strader, Ayca; Ihunnah, Chibueze A.; Makusky, Anthony J.; Court, Donald L.; Markey, Sanford P.; Kowalak, Jeffrey A.

    2013-01-01

    The bacterial ribosomal protein S12 contains a universally conserved D88 residue on a loop region thought to be critically involved in translation due to its proximal location to the A site of the 30S subunit. While D88 mutants are lethal this residue has been found to be post-translationally modified to β-methylthioaspartic acid, a post-translational modification (PTM) identified in S12 orthologs from several phylogenetically distinct bacteria. In a previous report focused on characterizing this PTM, our results provided evidence that this conserved loop region might be involved in forming proteins-protein interactions (1). To follow-up on this study, the D88 containing loop was probed to identify candidate binders employing a two-step complementary affinity purification strategy. The first involved an endogenously expressed S12 protein containing a C-terminal tag for capturing S12 binding partners. The second strategy utilized a synthetic biotinylated peptide representing the D88 conserved loop region for capturing S12 loop interaction partners. Captured proteins from both approaches were detected by utilizing SDS-PAGE and one-dimensional liquid chromatography tandem mass spectrometry. The results presented in this report revealed proteins that form direct interactions with the 30S subunit and elucidated which are likely to interact with S12. In addition, we provide evidence that two proteins involved in regulating ribosome and/or mRNA transcript levels under stress conditions, RNase R and Hfq, form direct interactions with the S12 conserved loop suggesting that it is likely part of a protein binding interface. PMID:23305560

  20. Carboxylator: incorporating solvent-accessible surface area for identifying protein carboxylation sites

    NASA Astrophysics Data System (ADS)

    Lu, Cheng-Tsung; Chen, Shu-An; Bretaña, Neil Arvin; Cheng, Tzu-Hsiu; Lee, Tzong-Yi

    2011-10-01

    In proteins, glutamate (Glu) residues are transformed into γ-carboxyglutamate (Gla) residues in a process called carboxylation. The process of protein carboxylation catalyzed by γ-glutamyl carboxylase is deemed to be important due to its involvement in biological processes such as blood clotting cascade and bone growth. There is an increasing interest within the scientific community to identify protein carboxylation sites. However, experimental identification of carboxylation sites via mass spectrometry-based methods is observed to be expensive, time-consuming, and labor-intensive. Thus, we were motivated to design a computational method for identifying protein carboxylation sites. This work aims to investigate the protein carboxylation by considering the composition of amino acids that surround modification sites. With the implication of a modified residue prefers to be accessible on the surface of a protein, the solvent-accessible surface area (ASA) around carboxylation sites is also investigated. Radial basis function network is then employed to build a predictive model using various features for identifying carboxylation sites. Based on a five-fold cross-validation evaluation, a predictive model trained using the combined features of amino acid sequence (AA20D), amino acid composition, and ASA, yields the highest accuracy at 0.874. Furthermore, an independent test done involving data not included in the cross-validation process indicates that in silico identification is a feasible means of preliminary analysis. Additionally, the predictive method presented in this work is implemented as Carboxylator (http://csb.cse.yzu.edu.tw/Carboxylator/), a web-based tool for identifying carboxylated proteins with modification sites in order to help users in investigating γ-glutamyl carboxylation.

  1. Protein profiles of hatchery egg shell membrane.

    PubMed

    Rath, N C; Liyanage, R; Makkar, S K; Lay, J O

    2016-01-01

    Eggshells which consist largely of calcareous outer shell and shell membranes, constitute a significant part of poultry hatchery waste. The shell membranes (ESM) not only contain proteins that originate from egg whites but also from the developing embryos and different contaminants of microbial and environmental origins. As feed supplements, during post hatch growth, the hatchery egg shell membranes (HESM) have shown potential for imparting resistance of chickens to endotoxin stress and exert positive health effects. Considering that these effects are mediated by the bioactive proteins and peptides present in the membrane, the objective of the study was to identify the protein profiles of hatchery eggshell membranes (HESM). Hatchery egg shell membranes were extracted with acidified methanol and a guanidine hydrochloride buffer then subjected to reduction/alkylation, and trypsin digestion. The methanol extract was additionally analyzed by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). The tryptic digests were analyzed by liquid chromatography and tandem mass spectrometry (LC-MS-MS) to identify the proteins. Our results showed the presence of several proteins that are inherent and abundant in egg white such as, ovalbumin, ovotransferrin, ovocleidin-116, and lysozyme, and several proteins associated with cytoskeletal, cell signaling, antimicrobial, and catalytic functions involving carbohydrate, nucleic acid, and protein metabolisms. There were some blood derived proteins most likely originating from the embryos and several other proteins identified with different aerobic, anaerobic, gram positive, gram negative, soil, and marine bacterial species some commensals and others zoonotic. The variety of bioactive proteins, particularly the cell signaling and enzymatic proteins along with the diverse microbial proteins, make the HESM suitable for nutritional and biological application to improve post hatch immunity of poultry.

  2. vProtein: Identifying Optimal Amino Acid Complements from Plant-Based Foods

    PubMed Central

    Woolf, Peter J.; Fu, Leeann L.; Basu, Avik

    2011-01-01

    Background Indispensible amino acids (IAAs) are used by the body in different proportions. Most animal-based foods provide these IAAs in roughly the needed proportions, but many plant-based foods provide different proportions of IAAs. To explore how these plant-based foods can be better used in human nutrition, we have created the computational tool vProtein to identify optimal food complements to satisfy human protein needs. Methods vProtein uses 1251 plant-based foods listed in the United States Department of Agriculture standard release 22 database to determine the quantity of each food or pair of foods required to satisfy human IAA needs as determined by the 2005 daily recommended intake. The quantity of food in a pair is found using a linear programming approach that minimizes total calories, total excess IAAs, or the total weight of the combination. Results For single foods, vProtein identifies foods with particularly balanced IAA patterns such as wheat germ, quinoa, and cauliflower. vProtein also identifies foods with particularly unbalanced IAA patterns such as macadamia nuts, degermed corn products, and wakame seaweed. Although less useful alone, some unbalanced foods provide unusually good complements, such as Brazil nuts to legumes. Interestingly, vProtein finds no statistically significant bias toward grain/legume pairings for protein complementation. These analyses suggest that pairings of plant-based foods should be based on the individual foods themselves instead of based on broader food group-food group pairings. Overall, the most efficient pairings include sweet corn/tomatoes, apple/coconut, and sweet corn/cherry. The top pairings also highlight the utility of less common protein sources such as the seaweeds laver and spirulina, pumpkin leaves, and lambsquarters. From a public health perspective, many of the food pairings represent novel, low cost food sources to combat malnutrition. Full analysis results are available online at http

  3. vProtein: identifying optimal amino acid complements from plant-based foods.

    PubMed

    Woolf, Peter J; Fu, Leeann L; Basu, Avik

    2011-04-22

    Indispensible amino acids (IAAs) are used by the body in different proportions. Most animal-based foods provide these IAAs in roughly the needed proportions, but many plant-based foods provide different proportions of IAAs. To explore how these plant-based foods can be better used in human nutrition, we have created the computational tool vProtein to identify optimal food complements to satisfy human protein needs. vProtein uses 1251 plant-based foods listed in the United States Department of Agriculture standard release 22 database to determine the quantity of each food or pair of foods required to satisfy human IAA needs as determined by the 2005 daily recommended intake. The quantity of food in a pair is found using a linear programming approach that minimizes total calories, total excess IAAs, or the total weight of the combination. For single foods, vProtein identifies foods with particularly balanced IAA patterns such as wheat germ, quinoa, and cauliflower. vProtein also identifies foods with particularly unbalanced IAA patterns such as macadamia nuts, degermed corn products, and wakame seaweed. Although less useful alone, some unbalanced foods provide unusually good complements, such as Brazil nuts to legumes. Interestingly, vProtein finds no statistically significant bias toward grain/legume pairings for protein complementation. These analyses suggest that pairings of plant-based foods should be based on the individual foods themselves instead of based on broader food group-food group pairings. Overall, the most efficient pairings include sweet corn/tomatoes, apple/coconut, and sweet corn/cherry. The top pairings also highlight the utility of less common protein sources such as the seaweeds laver and spirulina, pumpkin leaves, and lambsquarters. From a public health perspective, many of the food pairings represent novel, low cost food sources to combat malnutrition. Full analysis results are available online at http://www.foodwiki.com/vprotein.

  4. Coevolutionary Analysis Identifies Protein–Protein Interaction Sites between HIV-1 Reverse Transcriptase and Integrase

    PubMed Central

    Hetti Arachchilage, Madara; Piontkivska, Helen

    2016-01-01

    The replication of human immunodeficiency virus-1 (HIV-1) requires reverse transcription of the viral RNA genome and integration of newly synthesized pro-viral DNA into the host genome. This is mediated by the viral proteins reverse transcriptase (RT) and integrase (IN). The formation and stabilization of the pre-integration complex (PIC), which is an essential step for reverse transcription, nuclear import, chromatin targeting, and subsequent integration, involves direct and indirect modes of interaction between RT and IN proteins. While epitope-based treatments targeting IN–viral DNA and IN–RT complexes appear to be a promising combination for an anti-HIV treatment, the mechanisms of IN-RT interactions within the PIC are not well understood due to the transient nature of the protein complex and the intrinsic flexibility of its components. Here, we identify potentially interacting regions between the IN and RT proteins within the PIC through the coevolutionary analysis of amino acid sequences of the two proteins. Our results show that specific regions in the two proteins have strong coevolutionary signatures, suggesting that these regions either experience direct and prolonged interactions between them that require high affinity and/or specificity or that the regions are involved in interactions mediated by dynamic conformational changes and, hence, may involve both direct and indirect interactions. Other regions were found to exhibit weak, but positive correlations, implying interactions that are likely transient and/or have low affinity. We identified a series of specific regions of potential interactions between the IN and RT proteins (e.g., specific peptide regions within the C-terminal domain of IN were identified as potentially interacting with the Connection domain of RT). Coevolutionary analysis can serve as an important step in predicting potential interactions, thus informing experimental studies. These studies can be integrated with structural data

  5. Unique secreted-surface protein complex of Lactobacillus rhamnosus, identified by phage display.

    PubMed

    Gagic, Dragana; Wen, Wesley; Collett, Michael A; Rakonjac, Jasna

    2013-02-01

    Proteins are the most diverse structures on bacterial surfaces; hence, they are candidates for species- and strain-specific interactions of bacteria with the host, environment, and other microorganisms. Genomics has decoded thousands of bacterial surface and secreted proteins, yet the function of most cannot be predicted because of the enormous variability and a lack of experimental data that would allow deduction of function through homology. Here, we used phage display to identify a pair of interacting extracellular proteins in the probiotic bacterium Lactobacillus rhamnosus HN001. A secreted protein, SpcA, containing two bacterial immunoglobulin-like domains type 3 (Big-3) and a domain distantly related to plant pathogen response domain 1 (PR-1-like) was identified by screening of an L. rhamnosus HN001 library using HN001 cells as bait. The SpcA-"docking" protein, SpcB, was in turn detected by another phage display library screening, using purified SpcA as bait. SpcB is a 3275-residue cell-surface protein that contains general features of large glycosylated Serine-rich adhesins/fibrils from gram-positive bacteria, including the hallmark signal sequence motif KxYKxGKxW. Both proteins are encoded by genes within a L. rhamnosus-unique gene cluster that distinguishes this species from other lactobacilli. To our knowledge, this is the first example of a secreted-docking protein pair identified in lactobacilli. © 2012 The Authors. Published by Blackwell Publishing Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

  6. Stable-isotope analyses of dinosaur eggshells: Paleoenvironmental implications

    NASA Astrophysics Data System (ADS)

    Sarkar, A.; Bhattacharya, S. K.; Mohabey, D. M.

    1991-11-01

    Well-preserved clutches of dinosaur (sauropod) eggshells and skeletal remains have been discovered in the Upper Cretaceous Lameta limestones of the Kheda district, Gujarat, India, indicating a dinosaur nesting site. Oxygen-isotope analyses of the eggs show that the dinosaurs drank from a variety of freshwater bodies such as rivers and small evaporative pools, whereas the carbon-isotope values indicate that the reptiles were consuming plants that utilize the C3 photosynthetic pathway, e.g., small palms, shrubs, conifers, etc. Similar analyses of the host limestones suggest that they were deposited in a freshwater environment that provided the niche for large-scale breeding and nesting of the dinosaurs.

  7. Immobilization of lead in a Korean military shooting range soil using eggshell waste: an integrated mechanistic approach.

    PubMed

    Ahmad, Mahtab; Hashimoto, Yohey; Moon, Deok Hyun; Lee, Sang Soo; Ok, Yong Sik

    2012-03-30

    This study evaluated the effectiveness of eggshell and calcined eggshell on lead (Pb) immobilization in a shooting range soil. Destructive and non-destructive analytical techniques were employed to determine the mechanism of Pb immobilization. The 5% additions of eggshell and calcined eggshell significantly decreased the TCLP-Pb concentration by 68.8% due mainly to increasing soil pH. Eggshell and calcined-eggshell amendments decreased the exchangeable Pb fraction to ≈ 1% of the total Pb in the soil, while the carbonate-associated Pb fraction was increased to 40.0-47.1% at >15% application rates. The thermodynamic modeling on Pb speciation in the soil solution predicted the precipitation of Pb-hydroxide [Pb(OH)(2)] in soils amended with eggshell and calcined eggshell. The SEM-EDS, XAFS and elemental dot mapping revealed that Pb in soil amended with calcined eggshell was associated with Si and Ca, and may be immobilized by entrapping into calcium-silicate-hydrate. Comparatively, in the soil amended with eggshell, Pb was immobilized via formation of Pb-hydroxide or lanarkite [Pb(2)O(SO(4))]. Applications of amendments increased activities of alkaline phosphatase up to 3.7 times greater than in the control soil. The use of eggshell amendments may have potential as an integrated remediation strategy that enables Pb immobilization and soil biological restoration in shooting range soils. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Mass Spectrometry-Based Methods for Identifying Oxidized Proteins in Disease: Advances and Challenges

    PubMed Central

    Verrastro, Ivan; Pasha, Sabah; Tveen Jensen, Karina; Pitt, Andrew R.; Spickett, Corinne M.

    2015-01-01

    Many inflammatory diseases have an oxidative aetiology, which leads to oxidative damage to biomolecules, including proteins. It is now increasingly recognized that oxidative post-translational modifications (oxPTMs) of proteins affect cell signalling and behaviour, and can contribute to pathology. Moreover, oxidized proteins have potential as biomarkers for inflammatory diseases. Although many assays for generic protein oxidation and breakdown products of protein oxidation are available, only advanced tandem mass spectrometry approaches have the power to localize specific oxPTMs in identified proteins. While much work has been carried out using untargeted or discovery mass spectrometry approaches, identification of oxPTMs in disease has benefitted from the development of sophisticated targeted or semi-targeted scanning routines, combined with chemical labeling and enrichment approaches. Nevertheless, many potential pitfalls exist which can result in incorrect identifications. This review explains the limitations, advantages and challenges of all of these approaches to detecting oxidatively modified proteins, and provides an update on recent literature in which they have been used to detect and quantify protein oxidation in disease. PMID:25874603

  9. PCE-FR: A Novel Method for Identifying Overlapping Protein Complexes in Weighted Protein-Protein Interaction Networks Using Pseudo-Clique Extension Based on Fuzzy Relation.

    PubMed

    Cao, Buwen; Luo, Jiawei; Liang, Cheng; Wang, Shulin; Ding, Pingjian

    2016-10-01

    Identifying overlapping protein complexes in protein-protein interaction (PPI) networks can provide insight into cellular functional organization and thus elucidate underlying cellular mechanisms. Recently, various algorithms for protein complexes detection have been developed for PPI networks. However, majority of algorithms primarily depend on network topological feature and/or gene expression profile, failing to consider the inherent biological meanings between protein pairs. In this paper, we propose a novel method to detect protein complexes using pseudo-clique extension based on fuzzy relation (PCE-FR). Our algorithm operates in three stages: it first forms the nonoverlapping protein substructure based on fuzzy relation and then expands each substructure by adding neighbor proteins to maximize the cohesive score. Finally, highly overlapped candidate protein complexes are merged to form the final protein complex set. Particularly, our algorithm employs the biological significance hidden in protein pairs to construct edge weight for protein interaction networks. The experiment results show that our method can not only outperform classical algorithms such as CFinder, ClusterONE, CMC, RRW, HC-PIN, and ProRank +, but also achieve ideal overall performance in most of the yeast PPI datasets in terms of composite score consisting of precision, accuracy, and separation. We further apply our method to a human PPI network from the HPRD dataset and demonstrate it is very effective in detecting protein complexes compared to other algorithms.

  10. Microstructure, crystallography and diagenetic alteration in fossil ostrich eggshells from Upper Palaeolithic sites of Indian peninsular region.

    PubMed

    Jain, Sonal; Bajpai, Sunil; Kumar, Giriraj; Pruthi, Vikas

    2016-05-01

    Biominerals studies are of importance as they provide an understanding of natural evolutionary processes. In this study we have investigated the fossil ostrich eggshells using Scanning Electron Microscopy (SEM), X-ray Diffraction (XRD) and Electron Backscatter Diffraction (EBSD). SEM studies demonstrated the ultrastructure of fossil eggshells and formation of calcified cuticular layer. The presence of calcified cuticle layer in eggshell is the basis for ancient DNA studies as it contains preserved biomolecules. EBSD accentuates the crystallographic structure of the ostrich eggshells with sub-micrometer resolution. It is a non-destructive tool for evaluating the extent of diagenesis in a biomineral. EBSD analysis revealed the presence of dolomite in the eggshells. This research resulted in the complete recognition of the structure of ostrich eggshells as well as the nature and extent of diagenesis in these eggshells which is vital for genetic and paleoenvironmental studies. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Comparison of the total amount of eggshell pigments in Dongxiang brown-shelled eggs and Dongxiang blue-shelled eggs.

    PubMed

    Wang, X T; Zhao, C J; Li, J Y; Xu, G Y; Lian, L S; Wu, C X; Deng, X M

    2009-08-01

    Based on the knowledge of the heme bio-synthetic and metabolic pathway and the structures of biliverdin and protoporphyrin, experiments were carried out to compare the difference between the total quality of eggshell pigments in blue-shelled eggs and brown-shelled eggs from the same population (Dongxiang, China) and to analyze the correlation between the quantity of protoporphyrin and biliverdin in the 2 kinds of eggshells. It was found that there was no significant difference between the total quantity of eggshell pigments in Dongxiang blue-shelled eggs and Dongxiang brown-shelled eggs (P = 0.9006), and a highly significant positive correlation between the quantity of protoporphyrin and biliverdin in blue eggshells (P < 0.01) and a significant positive correlation between the quantity of protoporphyrin and biliverdin in brown eggshells (P < 0.05). These results suggested that eggshell protoporphyrin and eggshell biliverdin probably derived from common precursor material.

  12. Crystallization Optimum Solubility Screening: using crystallization results to identify the optimal buffer for protein crystal formation

    SciTech Connect

    Collins, Bernard; Stevens, Raymond C.; Page, Rebecca

    2005-12-01

    It is shown how protein crystallization results can be used to identify buffers that improve protein solubility and, in turn, crystallization success. An optimal solubility screen is described that uses the results of crystallization trials to identify buffers that improve protein solubility and, in turn, crystallization success. This screen is useful not only for standard crystallization experiments, but also can easily be implemented into any high-throughput structure-determination pipeline. As a proof of principle, the predicted novel-fold protein AF2059 from Archaeoglobus fulgidus, which was known to precipitate in most buffers and particularly during concentration experiments, was selected. Using the crystallization results of 192 independent crystallization trials, it was possible to identify a buffer containing 100 mM CHES pH 9.25 that significantly improves its solubility. After transferring AF2059 into this ‘optimum-solubility’ buffer, the protein was rescreened for crystal formation against these same 192 conditions. Instead of extensive precipitation, as observed initially, it was found that 24 separate conditions produced crystals and the exchange of AF2059 into CHES buffer significantly improved crystallization success. Fine-screen optimization of these conditions led to the production of a crystal suitable for high-resolution (2.2 Å) structure determination.

  13. Using distant supervised learning to identify protein subcellular localizations from full-text scientific articles.

    PubMed

    Zheng, Wu; Blake, Catherine

    2015-10-01

    Databases of curated biomedical knowledge, such as the protein-locations reflected in the UniProtKB database, provide an accurate and useful resource to researchers and decision makers. Our goal is to augment the manual efforts currently used to curate knowledge bases with automated approaches that leverage the increased availability of full-text scientific articles. This paper describes experiments that use distant supervised learning to identify protein subcellular localizations, which are important to understand protein function and to identify candidate drug targets. Experiments consider Swiss-Prot, the manually annotated subset of the UniProtKB protein knowledge base, and 43,000 full-text articles from the Journal of Biological Chemistry that contain just under 11.5 million sentences. The system achieves 0.81 precision and 0.49 recall at sentence level and an accuracy of 57% on held-out instances in a test set. Moreover, the approach identifies 8210 instances that are not in the UniProtKB knowledge base. Manual inspection of the 50 most likely relations showed that 41 (82%) were valid. These results have immediate benefit to researchers interested in protein function, and suggest that distant supervision should be explored to complement other manual data curation efforts.

  14. Screening of cell cycle fusion proteins to identify kinase signaling networks.

    PubMed

    Trojanowsky, Michelle; Vidovic, Dusica; Simanski, Scott; Penas, Clara; Schurer, Stephan; Ayad, Nagi G

    2015-01-01

    Kinase signaling networks are well-established mediators of cell cycle transitions. However, how kinases interact with the ubiquitin proteasome system (UPS) to elicit protein turnover is not fully understood. We sought a means of identifying kinase-substrate interactions to better understand signaling pathways controlling protein degradation. Our prior studies used a luciferase fusion protein to uncover kinase networks controlling protein turnover. In this study, we utilized a similar approach to identify pathways controlling the cell cycle protein p27(Kip1). We generated a p27(Kip1)-luciferase fusion and expressed it in cells incubated with compounds from a library of pharmacologically active compounds. We then compared the relative effects of the compounds on p27(Kip1)-luciferase fusion stabilization. This was combined with in silico kinome profiling to identify potential kinases inhibited by each compound. This approach effectively uncovered known kinases regulating p27(Kip1) turnover. Collectively, our studies suggest that this parallel screening approach is robust and can be applied to fully understand kinase-ubiquitin pathway interactions.

  15. Protein microarrays identify disease-specific anti-cytokine autoantibody profiles in the landscape of immunodeficiency

    PubMed Central

    Rosenberg, Jacob M.; Price, Jordan V.; Barcenas-Morales, Gabriela; Ceron-Gutierrez, Lourdes; Davies, Sophie; Kumararatne, Dinakantha S.; Döffinger, Rainer; Utz, Paul J.

    2015-01-01

    Background Anti-cytokine autoantibodies (ACAAs) are pathogenic in a handful of rare immunodeficiencies. However, the prevalence and significance of other ACAAs across immunodeficiencies have not yet been described. Objective We sought to profile ACAAs in a diverse cohort of serum samples from patients with immunodeficiency and assess the sensitivity and specificity of protein microarrays for ACAA identification and discovery. Methods Highly multiplexed protein microarrays were designed and fabricated. Blinded serum samples from a cohort of 58 patients with immunodeficiency and healthy control subjects were used to probe microarrays. Unsupervised hierarchical clustering was used to identify clusters of reactivity, and after unblinding, significance analysis of microarrays was used to identify disease-specific autoantibodies. A bead-based assay was used to validate protein microarray results. Blocking activity of serum containing ACAAs was measured in vitro. Results Protein microarrays were highly sensitive and specific for the detection of ACAAs in patients with autoimmune polyendocrine syndrome type I and pulmonary alveolar proteinosis, detecting ACAA levels consistent with those in the published literature. Protein microarray results were validated by using an independent bead-based assay. To confirm the functional significance of these ACAAs, we tested and confirmed the blocking activity of select ACAAs in vitro. Conclusion Protein microarrays are a powerful tool for ACAA detection and discovery, and they hold promise as a diagnostic for the evaluation and monitoring of clinical immunodeficiency. PMID:26365387

  16. Identifying Similar Patterns of Structural Flexibility in Proteins by Disorder Prediction and Dynamic Programming

    PubMed Central

    Petrovich, Aidan; Borne, Adam; Uversky, Vladimir N.; Xue, Bin

    2015-01-01

    Computational methods are prevailing in identifying protein intrinsic disorder. The results from predictors are often given as per-residue disorder scores. The scores describe the disorder propensity of amino acids of a protein and can be further represented as a disorder curve. Many proteins share similar patterns in their disorder curves. The similar patterns are often associated with similar functions and evolutionary origins. Therefore, finding and characterizing specific patterns of disorder curves provides a unique and attractive perspective of studying the function of intrinsically disordered proteins. In this study, we developed a new computational tool named IDalign using dynamic programming. This tool is able to identify similar patterns among disorder curves, as well as to present the distribution of intrinsic disorder in query proteins. The disorder-based information generated by IDalign is significantly different from the information retrieved from classical sequence alignments. This tool can also be used to infer functions of disordered regions and disordered proteins. The web server of IDalign is available at (http://labs.cas.usf.edu/bioinfo/service.html). PMID:26086829

  17. Identifying proteins that bind to specific RNAs - focus on simple repeat expansion diseases

    PubMed Central

    Jazurek, Magdalena; Ciesiolka, Adam; Starega-Roslan, Julia; Bilinska, Katarzyna; Krzyzosiak, Wlodzimierz J.

    2016-01-01

    RNA–protein complexes play a central role in the regulation of fundamental cellular processes, such as mRNA splicing, localization, translation and degradation. The misregulation of these interactions can cause a variety of human diseases, including cancer and neurodegenerative disorders. Recently, many strategies have been developed to comprehensively analyze these complex and highly dynamic RNA–protein networks. Extensive efforts have been made to purify in vivo-assembled RNA–protein complexes. In this review, we focused on commonly used RNA-centric approaches that involve mass spectrometry, which are powerful tools for identifying proteins bound to a given RNA. We present various RNA capture strategies that primarily depend on whether the RNA of interest is modified. Moreover, we briefly discuss the advantages and limitations of in vitro and in vivo approaches. Furthermore, we describe recent advances in quantitative proteomics as well as the methods that are most commonly used to validate robust mass spectrometry data. Finally, we present approaches that have successfully identified expanded repeat-binding proteins, which present abnormal RNA–protein interactions that result in the development of many neurological diseases. PMID:27625393

  18. A comprehensive analytical strategy to identify malondialdehyde-modified proteins and peptides.

    PubMed

    Weißer, Juliane; Ctortecka, Claudia; Busch, Clara J; Austin, Shane R; Nowikovsky, Karin; Uchida, Koji; Binder, Christoph J; Bennett, Keiryn L

    2017-03-01

    Mass spectrometric-based proteomics is a powerful tool to analyse post-translationally modified proteins. Carbonylation modifications that result from oxidative lipid breakdown are a class of post-translational modifications that are poorly charac-terised with respect to protein targets and function. This is partly due to the lack of dedicated mass spectrometry-based technologies to facilitate the analysis of these modifications. Here, we present a comprehensive approach to identify malondialdehyde-modified proteins and peptides. Malondialdehyde is amongst the most abundant of the lipid peroxidation products; and malondialdehyde-derived adducts on proteins have been implicated in cardiovascular diseases, neurodegenerative disorders and other clinical conditions. Our integrated approach targets three levels of the overall proteomic workflow: (i) sample preparation, by employing a targeted enrichment strategy; (ii) high-performance liquid chromatography, by using a gradient optimised for the separation of the modified peptides; and (iii) tandem mass spectrometry, by improving the spectral quality of very low-abundance peptides. By applying the optimised procedure to a whole cell lysate spiked with a low amount of malondialdehyde-modified proteins, we were able to identify up to 350 different modified peptides and localise the modification to a specific lysine residue. This methodology allows the comprehensive analysis of malondialdehyde-modified proteins.

  19. Peptides identified in soybean protein increase plasma cholesterol in mice on hypercholesterolemic diets

    USDA-ARS?s Scientific Manuscript database

    The in vitro micellar cholesterol displacement assay has been used to identify peptides that may potentially reduce cholesterol in vivo. We tested two of these peptides, LPYPR and WGAPSI, derived from soybean protein (SP) that have been reported to displace cholesterol from micelles by feeding them...

  20. Functional characterization of candidate effector proteins identified from the wheat scab fungus Fusarium graminearum

    USDA-ARS?s Scientific Manuscript database

    Fungal pathogens often produce certain small secreted cysteine-rich proteins (SSCPs) during pathogenesis that may function in triggering resistance or susceptibility in specific host plants. We have recently identified a total of 190 SSCPs encoded in the genome of the wheat scab fungus Fusarium gra...

  1. Proteomic profiling of human plasma exosomes identifies PPAR{gamma} as an exosome-associated protein

    SciTech Connect

    Looze, Christopher; Yui, David; Leung, Lester; Ingham, Matthew; Kaler, Maryann; Yao, Xianglan; Wu, Wells W.; Shen Rongfong; Daniels, Mathew P.; Levine, Stewart J.

    2009-01-16

    Exosomes are nanovesicles that are released from cells as a mechanism of cell-free intercellular communication. Only a limited number of proteins have been identified from the plasma exosome proteome. Here, we developed a multi-step fractionation scheme incorporating gel exclusion chromatography, rate zonal centrifugation through continuous sucrose gradients, and high-speed centrifugation to purify exosomes from human plasma. Exosome-associated proteins were separated by SDS-PAGE and 66 proteins were identified by LC-MS/MS, which included both cellular and extracellular proteins. Furthermore, we identified and characterized peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}), a nuclear receptor that regulates adipocyte differentiation and proliferation, as well as immune and inflammatory cell functions, as a novel component of plasma-derived exosomes. Given the important role of exosomes as intercellular messengers, the discovery of PPAR{gamma} as a component of human plasma exosomes identifies a potential new pathway for the paracrine transfer of nuclear receptors.

  2. Proteomics identifies differentially expressed proteins in neonatal murine thymus compared with adults

    PubMed Central

    2012-01-01

    Background The thymus is an immune organ essential for life and plays a crucial role in the development of T cells. It undergoes a fetal to adult developmental maturation process occurring in mouse during the postnatal months. The molecular modifications underlying these ontogenic changes are essentially unknown. Here we used a differential proteomic-based technique (2D-Difference Gel Electrophoresis) coupled with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry to search for key proteins in the postnatal development of the thymus. Eight different BALB/c mice were used in the study: four mice aged of 1 day (neonatal) and four mice aged of 60 days (adult). Protein samples derived from thymus were labeled and run in 2D-PAGE (Two-Dimensional Polyacrylamide Gel Electrophoresis). One whole-thymus tissue from each mouse was run on gels and each gel containing a pooled sample of the eight mice was run in parallel. The pooled sample was set as the internal pool, containing equal amount of each protein extract used in the experiment. Gels were matched and compared with Difference In-gel Analysis software. Differential spots were picked, in-gel digested and peptide mass fingerprints were obtained. Results Among the differentially regulated proteins in neonatal thymus group, 111 proteins were identified by mass spectrometry, of which 95 proteins were up-regulated and 16 proteins were down-regulated. The identified proteins belong to several functional categories, including cell proliferation, cycle and apoptosis, transcription regulation, signal transduction, nucleotide processing, proteolysis and translation, protein folding, metabolism, oxidoreduction, cytoskeleton, immune response, and embryonic development. The major interaction networks comprised of cellular function and maintenance, cellular assembly and organization, and metabolism were also identified by STRING analysis. Conclusions The demonstrated molecular changes are

  3. A proteomic approach to identify seminal plasma proteins in roosters (Gallus gallus domesticus).

    PubMed

    Marzoni, Margherita; Castillo, Annelisse; Sagona, Simona; Citti, Lorenzo; Rocchiccioli, Silvia; Romboli, Isabella; Felicioli, Antonio

    2013-08-01

    Considering the interest in avian semen processing and storage, the objective of this study was to identify the domestic fowl seminal plasma proteins using two-dimensional gel electrophoresis (2-DE) and mass spectrometry MS/MS. For three times in a 4-month period, seminal plasma was obtained from semen collected from four local male chickens (Gallus gallus domesticus) and prepared for two-dimensional polyacrylamide gel electrophoresis. A total of 83 spots were detected across all gels and analyzed by MALDI-TOF/TOF. Among these spots, 17 have been successfully identified. The most intensely stained spots were recognized as serum albumin, ovotransferrin, alpha-enolase, fatty acid binding protein, thioredoxin, trypsin inhibitor CITI-1 and gallinacin-9. From these proteins, two are characteristic of avian seminal plasma, the ovotransferrin and gallinacin-9, and one is specific of the Gallus species, the chicken trypsin inhibitor CITI-1. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Cell surface proteins Nasrat and Polehole stabilize the Torso-like extracellular determinant in Drosophila oogenesis

    PubMed Central

    Jiménez, Gerardo; González-Reyes, Acaimo; Casanova, Jordi

    2002-01-01

    Structural cell-surface and extracellular-matrix proteins modulate intercellular signaling events during development, but how this is achieved remains largely unknown. Here we identify a novel family of Drosophila proteins, Nasrat and Polehole, that coat the oocyte surface and play two roles: They mediate assembly of the eggshell, and act in the Torso RTK signaling pathway that specifies the terminal regions of the embryo. Nasrat and Polehole are essential for extracellular accumulation of Torso-like, a factor secreted during oogenesis that initiates Torso receptor activation. Stabilization of secreted factors by specialized pericellular proteins may be a general mechanism during signaling and developmental patterning. PMID:11959840

  5. Whole human genome proteogenomic mapping for ENCODE cell line data: identifying protein-coding regions.

    PubMed

    Khatun, Jainab; Yu, Yanbao; Wrobel, John A; Risk, Brian A; Gunawardena, Harsha P; Secrest, Ashley; Spitzer, Wendy J; Xie, Ling; Wang, Li; Chen, Xian; Giddings, Morgan C

    2013-02-28

    Proteogenomic mapping is an approach that uses mass spectrometry data from proteins to directly map protein-coding genes and could aid in locating translational regions in the human genome. In concert with the ENcyclopedia of DNA Elements (ENCODE) project, we applied proteogenomic mapping to produce proteogenomic tracks for the UCSC Genome Browser, to explore which putative translational regions may be missing from the human genome. We generated ~1 million high-resolution tandem mass (MS/MS) spectra for Tier 1 ENCODE cell lines K562 and GM12878 and mapped them against the UCSC hg19 human genome, and the GENCODE V7 annotated protein and transcript sets. We then compared the results from the three searches to identify the best-matching peptide for each MS/MS spectrum, thereby increasing the confidence of the putative new protein-coding regions found via the whole genome search. At a 1% false discovery rate, we identified 26,472, 24,406, and 13,128 peptides from the protein, transcript, and whole genome searches, respectively; of these, 481 were found solely via the whole genome search. The proteogenomic mapping data are available on the UCSC Genome Browser at http://genome.ucsc.edu/cgi-bin/hgTrackUi?db=hg19&g=wgEncodeUncBsuProt. The whole genome search revealed that ~4% of the uniquely mapping identified peptides were located outside GENCODE V7 annotated exons. The comparison of the results from the disparate searches also identified 15% more spectra than would have been found solely from a protein database search. Therefore, whole genome proteogenomic mapping is a complementary method for genome annotation when performed in conjunction with other searches.

  6. Distinct Host Tropism Protein Signatures to Identify Possible Zoonotic Influenza A Viruses.

    PubMed

    Eng, Christine L P; Tong, Joo Chuan; Tan, Tin Wee

    2016-01-01

    Zoonotic influenza A viruses constantly pose a health threat to humans as novel strains occasionally emerge from the avian population to cause human infections. Many past epidemic as well as pandemic strains have originated from avian species. While most viruses are restricted to their primary hosts, zoonotic strains can sometimes arise from mutations or reassortment, leading them to acquire the capability to escape host species barrier and successfully infect a new host. Phylogenetic analyses and genetic markers are useful in tracing the origins of zoonotic infections, but there are still no effective means to identify high risk strains prior to an outbreak. Here we show that distinct host tropism protein signatures can be used to identify possible zoonotic strains in avian species which have the potential to cause human infections. We have discovered that influenza A viruses can now be classified into avian, human, or zoonotic strains based on their host tropism protein signatures. Analysis of all influenza A viruses with complete proteome using the host tropism prediction system, based on machine learning classifications of avian and human viral proteins has uncovered distinct signatures of zoonotic strains as mosaics of avian and human viral proteins. This is in contrast with typical avian or human strains where they show mostly avian or human viral proteins in their signatures respectively. Moreover, we have found that zoonotic strains from the same influenza outbreaks carry similar host tropism protein signatures characteristic of a common ancestry. Our results demonstrate that the distinct host tropism protein signature in zoonotic strains may prove useful in influenza surveillance to rapidly identify potential high risk strains circulating in avian species, which may grant us the foresight in anticipating an impending influenza outbreak.

  7. Distinct Host Tropism Protein Signatures to Identify Possible Zoonotic Influenza A Viruses

    PubMed Central

    Eng, Christine L. P.; Tong, Joo Chuan; Tan, Tin Wee

    2016-01-01

    Zoonotic influenza A viruses constantly pose a health threat to humans as novel strains occasionally emerge from the avian population to cause human infections. Many past epidemic as well as pandemic strains have originated from avian species. While most viruses are restricted to their primary hosts, zoonotic strains can sometimes arise from mutations or reassortment, leading them to acquire the capability to escape host species barrier and successfully infect a new host. Phylogenetic analyses and genetic markers are useful in tracing the origins of zoonotic infections, but there are still no effective means to identify high risk strains prior to an outbreak. Here we show that distinct host tropism protein signatures can be used to identify possible zoonotic strains in avian species which have the potential to cause human infections. We have discovered that influenza A viruses can now be classified into avian, human, or zoonotic strains based on their host tropism protein signatures. Analysis of all influenza A viruses with complete proteome using the host tropism prediction system, based on machine learning classifications of avian and human viral proteins has uncovered distinct signatures of zoonotic strains as mosaics of avian and human viral proteins. This is in contrast with typical avian or human strains where they show mostly avian or human viral proteins in their signatures respectively. Moreover, we have found that zoonotic strains from the same influenza outbreaks carry similar host tropism protein signatures characteristic of a common ancestry. Our results demonstrate that the distinct host tropism protein signature in zoonotic strains may prove useful in influenza surveillance to rapidly identify potential high risk strains circulating in avian species, which may grant us the foresight in anticipating an impending influenza outbreak. PMID:26915079

  8. An Ensemble Method with Hybrid Features to Identify Extracellular Matrix Proteins

    PubMed Central

    Yang, Runtao; Zhang, Chengjin; Gao, Rui; Zhang, Lina

    2015-01-01

    The extracellular matrix (ECM) is a dynamic composite of secreted proteins that play important roles in numerous biological processes such as tissue morphogenesis, differentiation and homeostasis. Furthermore, various diseases are caused by the dysfunction of ECM proteins. Therefore, identifying these important ECM proteins may assist in understanding related biological processes and drug development. In view of the serious imbalance in the training dataset, a Random Forest-based ensemble method with hybrid features is developed in this paper to identify ECM proteins. Hybrid features are employed by incorporating sequence composition, physicochemical properties, evolutionary and structural information. The Information Gain Ratio and Incremental Feature Selection (IGR-IFS) methods are adopted to select the optimal features. Finally, the resulting predictor termed IECMP (Identify ECM Proteins) achieves an balanced accuracy of 86.4% using the 10-fold cross-validation on the training dataset, which is much higher than results obtained by other methods (ECMPRED: 71.0%, ECMPP: 77.8%). Moreover, when tested on a common independent dataset, our method also achieves significantly improved performance over ECMPP and ECMPRED. These results indicate that IECMP is an effective method for ECM protein prediction, which has a more balanced prediction capability for positive and negative samples. It is anticipated that the proposed method will provide significant information to fully decipher the molecular mechanisms of ECM-related biological processes and discover candidate drug targets. For public access, we develop a user-friendly web server for ECM protein identification that is freely accessible at http://iecmp.weka.cc. PMID:25680094

  9. Identifying Novel Cell Cycle Proteins in Apicomplexa Parasites through Co-Expression Decision Analysis

    PubMed Central

    Butler, Carrie L.; Lucas, Olivier; Wuchty, Stefan; Xue, Bin; Uversky, Vladimir N.; White, Michael

    2014-01-01

    Hypothetical proteins comprise roughly half of the predicted gene complement of Toxoplasma gondii and Plasmodium falciparum and represent the largest class of uniquely functioning proteins in these parasites. Following the idea that functional relationships can be informed by the timing of gene expression, we devised a strategy to identify the core set of apicomplexan cell division cycling genes with important roles in parasite division, which includes many uncharacterized proteins. We assembled an expanded list of orthologs from the T. gondii and P. falciparum genome sequences (2781 putative orthologs), compared their mRNA profiles during synchronous replication, and sorted the resulting set of dual cell cycle regulated orthologs (744 total) into protein pairs conserved across many eukaryotic families versus those unique to the Apicomplexa. The analysis identified more than 100 ortholog gene pairs with unknown function in T. gondii and P. falciparum that displayed co-conserved mRNA abundance, dynamics of cyclical expression and similar peak timing that spanned the complete division cycle in each parasite. The unknown cyclical mRNAs encoded a diverse set of proteins with a wide range of mass and showed a remarkable conservation in the internal organization of ordered versus disordered structural domains. A representative sample of cyclical unknown genes (16 total) was epitope tagged in T. gondii tachyzoites yielding the discovery of new protein constituents of the parasite inner membrane complex, key mitotic structures and invasion organelles. These results demonstrate the utility of using gene expression timing and dynamic profile to identify proteins with unique roles in Apicomplexa biology. PMID:24841368

  10. Identifying and quantitating conformational exchange in membrane proteins using site-directed spin labeling.

    PubMed

    Cafiso, David S

    2014-10-21

    Protein structures are not static but sample different conformations over a range of amplitudes and time scales. These fluctuations may involve relatively small changes in bond angles or quite large rearrangements in secondary structure and tertiary fold. The equilibrium between discrete structural substates on the microsecond to millisecond time scale is sometimes termed conformational exchange. Protein dynamics and conformational exchange are believed to provide the basis for many important activities, such as protein-protein and protein-ligand interactions, enzymatic activity and protein allostery; however, for many proteins, the dynamics and conformational exchange that lead to function are poorly defined. Spectroscopic methods, such as NMR, are among the most important methods to explore protein dynamics and conformational exchange; however, they are difficult to implement in some systems and with some types of exchange events. Site-directed spin labeling (SDSL) is an EPR based approach that is particularly well-suited to high molecular-weight systems such as membrane proteins. Because of the relatively fast time scale for EPR spectroscopy, it is an excellent method to examine exchange. Conformations that are in exchange are captured as distinct populations in the EPR spectrum, and this feature when combined with the use of methods that can shift the free energy of conformational substates allows one to identify regions of proteins that are in dynamic exchange. In addition, modern pulse EPR methods have the ability to examine conformational heterogeneity, resolve discrete protein states, and identify the substates in exchange. Protein crystallography has provided high-resolution models for a number of membrane proteins; but because of conformational exchange, these models do not always reflect the structures that are present when the protein is in a native bilayer environment. In the case of the Escherichia coli vitamin B12 transporter, BtuB, the energy

  11. HUGE: a database for human large proteins identified by Kazusa cDNA sequencing project.

    PubMed Central

    Suyama, M; Nagase, T; Ohara, O

    1999-01-01

    HUGE is a database for human large proteins newly identified by Kazusa cDNA project, which aims to predict protein primary structures from sequences of human large cDNAs (>4 kb). In particular, cDNA clones capable of coding for large proteins (>50 kDa) are current targets of the project. More than 700 sequences of human cDNAs (average size, 5.1 kb) have been determined to date and deposited in the public databases. Notable information implied from the cDNAs and the predicted protein sequences can be obtained through HUGE via the World Wide Web at URL http://www.kazusa.or.jp/huge PMID:9847221

  12. FunMod: A Cytoscape Plugin for Identifying Functional Modules in Undirected Protein–Protein Networks

    PubMed Central

    Natale, Massimo; Benso, Alfredo; Di Carlo, Stefano; Ficarra, Elisa

    2014-01-01

    The characterization of the interacting behaviors of complex biological systems is a primary objective in protein–protein network analysis and computational biology. In this paper we present FunMod, an innovative Cytoscape version 2.8 plugin that is able to mine undirected protein–protein networks and to infer sub-networks of interacting proteins intimately correlated with relevant biological pathways. This plugin may enable the discovery of new pathways involved in diseases. In order to describe the role of each protein within the relevant biological pathways, FunMod computes and scores three topological features of the identified sub-networks. By integrating the results from biological pathway clustering and topological network analysis, FunMod proved to be useful for the data interpretation and the generation of new hypotheses in two case studies. PMID:25153667

  13. Whole-genome screening identifies proteins localized to distinct nuclear bodies.

    PubMed

    Fong, Ka-Wing; Li, Yujing; Wang, Wenqi; Ma, Wenbin; Li, Kunpeng; Qi, Robert Z; Liu, Dan; Songyang, Zhou; Chen, Junjie

    2013-10-14

    The nucleus is a unique organelle that contains essential genetic materials in chromosome territories. The interchromatin space is composed of nuclear subcompartments, which are defined by several distinctive nuclear bodies believed to be factories of DNA or RNA processing and sites of transcriptional and/or posttranscriptional regulation. In this paper, we performed a genome-wide microscopy-based screening for proteins that form nuclear foci and characterized their localizations using markers of known nuclear bodies. In total, we identified 325 proteins localized to distinct nuclear bodies, including nucleoli (148), promyelocytic leukemia nuclear bodies (38), nuclear speckles (27), paraspeckles (24), Cajal bodies (17), Sam68 nuclear bodies (5), Polycomb bodies (2), and uncharacterized nuclear bodies (64). Functional validation revealed several proteins potentially involved in the assembly of Cajal bodies and paraspeckles. Together, these data establish the first atlas of human proteins in different nuclear bodies and provide key information for research on nuclear bodies.

  14. A machine learning approach to identify hydrogenosomal proteins in Trichomonas vaginalis.

    PubMed

    Burstein, David; Gould, Sven B; Zimorski, Verena; Kloesges, Thorsten; Kiosse, Fuat; Major, Peter; Martin, William F; Pupko, Tal; Dagan, Tal

    2012-02-01

    The protozoan parasite Trichomonas vaginalis is the causative agent of trichomoniasis, the most widespread nonviral sexually transmitted disease in humans. It possesses hydrogenosomes-anaerobic mitochondria that generate H(2), CO(2), and acetate from pyruvate while converting ADP to ATP via substrate-level phosphorylation. T. vaginalis hydrogenosomes lack a genome and translation machinery; hence, they import all their proteins from the cytosol. To date, however, only 30 imported proteins have been shown to localize to the organelle. A total of 226 nuclear-encoded proteins inferred from the genome sequence harbor a characteristic short N-terminal presequence, reminiscent of mitochondrial targeting peptides, which is thought to mediate hydrogenosomal targeting. Recent studies suggest, however, that the presequences might be less important than previously thought. We sought to identify new hydrogenosomal proteins within the 59,672 annotated open reading frames (ORFs) of T. vaginalis, independent of the N-terminal targeting signal, using a machine learning approach. Our training set included 57 gene and protein features determined for all 30 known hydrogenosomal proteins and 576 nonhydrogenosomal proteins. Several classifiers were trained on this set to yield an import score for all proteins encoded by T. vaginalis ORFs, predicting the likelihood of hydrogenosomal localization. The machine learning results were tested through immunofluorescence assay and immunodetection in isolated cell fractions of 14 protein predictions using hemagglutinin constructs expressed under the homologous SCSα promoter in transiently transformed T. vaginalis cells. Localization of 6 of the 10 top predicted hydrogenosome-localized proteins was confirmed, and two of these were found to lack an obvious N-terminal targeting signal.

  15. A yeast-based genetic screening to identify human proteins that increase homologous recombination.

    PubMed

    Collavoli, Anita; Comelli, Laura; Rainaldi, Giuseppe; Galli, Alvaro

    2008-05-01

    To identify new human proteins implicated in homologous recombination (HR), we set up 'a papillae assay' to screen a human cDNA library using the RS112 strain of Saccharomyces cerevisiae containing an intrachromosomal recombination substrate. We isolated 23 cDNAs, 11 coding for complete proteins and 12 for partially deleted proteins that increased HR when overexpressed in yeast. We characterized the effect induced by the overexpression of the complete human proteasome subunit beta 2, the partially deleted proteasome subunits alpha 3 and beta 8, the ribosomal protein L12, the brain abundant membrane signal protein (BASP1) and the human homologue to v-Ha-RAS (HRAS), which elevated HR by 2-6.5-fold over the control. We found that deletion of the RAD52 gene, which has a key role in most HR events, abolished the increase of HR induced by the proteasome subunits and HRAS; by contrast, the RAD52 deletion did not affect the high level of HR due to BASP1 and RPL12. This suggests that the proteins stimulated yeast HR via different mechanisms. Overexpression of the complete beta 2 human proteasome subunit or the partially deleted alpha 3 and beta 8 subunits increased methyl methanesulphonate (MMS) resistance much more in the rad52 Delta mutant than in the wild-type. Overexpression of RPL12 and BASP1 did not affect MMS resistance in both the wild-type and the rad52 Delta mutant, whereas HRAS decreased MMS resistance in the rad52 Delta mutant. The results indicate that these proteins may interfere with the pathway(s) involved in the repair of MMS-induced DNA damage. Finally, we provide further evidence that yeast is a helpful tool to identify human proteins that may have a regulatory role in HR.

  16. Controllability analysis of the directed human protein interaction network identifies disease genes and drug targets.

    PubMed

    Vinayagam, Arunachalam; Gibson, Travis E; Lee, Ho-Joon; Yilmazel, Bahar; Roesel, Charles; Hu, Yanhui; Kwon, Young; Sharma, Amitabh; Liu, Yang-Yu; Perrimon, Norbert; Barabási, Albert-László

    2016-05-03

    The protein-protein interaction (PPI) network is crucial for cellular information processing and decision-making. With suitable inputs, PPI networks drive the cells to diverse functional outcomes such as cell proliferation or cell death. Here, we characterize the structural controllability of a large directed human PPI network comprising 6,339 proteins and 34,813 interactions. This network allows us to classify proteins as "indispensable," "neutral," or "dispensable," which correlates to increasing, no effect, or decreasing the number of driver nodes in the network upon removal of that protein. We find that 21% of the proteins in the PPI network are indispensable. Interestingly, these indispensable proteins are the primary targets of disease-causing mutations, human viruses, and drugs, suggesting that altering a network's control property is critical for the transition between healthy and disease states. Furthermore, analyzing copy number alterations data from 1,547 cancer patients reveals that 56 genes that are frequently amplified or deleted in nine different cancers are indispensable. Among the 56 genes, 46 of them have not been previously associated with cancer. This suggests that controllability analysis is very useful in identifying novel disease genes and potential drug targets.

  17. High throughput atmospheric pressure plasma-induced graft polymerization for identifying protein-resistant surfaces.

    PubMed

    Gu, Minghao; Kilduff, James E; Belfort, Georges

    2012-02-01

    Three critical aspects of searching for and understanding how to find highly resistant surfaces to protein adhesion are addressed here with specific application to synthetic membrane filtration. They include the (i) discovery of a series of previously unreported monomers from a large library of monomers with high protein resistance and subsequent low fouling characteristics for membrane ultrafiltration of protein-containing fluids, (ii) development of a new approach to investigate protein-resistant mechanisms from structure-property relationships, and (iii) adaptation of a new surface modification method, called atmospheric pressure plasma-induced graft polymerization (APP), together with a high throughput platform (HTP), for low cost vacuum-free synthesis of anti-fouling membranes. Several new high-performing chemistries comprising two polyethylene glycol (PEG), two amines and one zwitterionic monomers were identified from a library (44 commercial monomers) of five different classes of monomers as strong protein-resistant monomers. Combining our analysis here, using the Hansen solubility parameters (HSP) approach, and data from the literature, we conclude that strong interactions with water (hydrogen bonding) and surface flexibility are necessary for producing the highest protein resistance. Superior protein-resistant surfaces and subsequent anti-fouling performance was obtained with the HTP-APP as compared with our earlier HTP-photo graft-induced polymerization (PGP).

  18. Genetic architecture dissection by genome-wide association analysis reveals avian eggshell ultrastructure traits

    PubMed Central

    Duan, Zhongyi; Sun, Congjiao; Shen, ManMan; Wang, Kehua; Yang, Ning; Zheng, Jiangxia; Xu, Guiyun

    2016-01-01

    The ultrastructure of an eggshell is considered the major determinant of eggshell quality, which has biological and economic significance for the avian and poultry industries. However, the interrelationships and genome-wide architecture of eggshell ultrastructure remain to be elucidated. Herein, we measured eggshell thickness (EST), effective layer thickness (ET), mammillary layer thickness (MT), and mammillary density (MD) and conducted genome-wide association studies in 927 F2 hens. The SNP-based heritabilities of eggshell ultrastructure traits were estimated to be 0.39, 0.36, 0.17 and 0.19 for EST, ET, MT and MD, respectively, and a total of 719, 784, 1 and 10 genome-wide significant SNPs were associated with EST, ET, MT and MD, respectively. ABCC9, ITPR2, KCNJ8 and WNK1, which are involved in ion transport, were suggested to be the key genes regulating EST and ET. ITM2C and KNDC1 likely affect MT and MD, respectively. Additionally, there were linear relationships between the chromosome lengths and the variance explained per chromosome for EST (R2 = 0.57) and ET (R2 = 0.67). In conclusion, the interrelationships and genetic architecture of eggshell ultrastructure traits revealed in this study are valuable for our understanding of the avian eggshell and contribute to research on a variety of other calcified shells. PMID:27456605

  19. Avian embryonic development does not change the stable isotope composition of the calcite eggshell.

    PubMed

    Maurer, G; Portugal, S J; Boomer, I; Cassey, P

    2011-01-01

    The avian embryo resorbs most of the calcium for bone formation from the calcite eggshell but the exact mechanisms of the resorption are unknown. The present study tested whether this process results in variable fractionation of the oxygen and carbon isotopes in shell calcium carbonate, which could provide a detailed insight into the temporal and spatial use of the eggshell by the developing embryo. Despite the uncertainty regarding changes in stable isotope composition of the eggshell across developmental stages or regions of the shell, eggshells are a popular resource for the analysis of historic and extant trophic relationships. To clarify how the stable isotope composition varies with embryonic development, the δ(13)C and δ(18)O content of the carbonate fraction in shells of black-headed gull (Larus ridibundus) eggs were sampled at four different stages of embryonic development and at five eggshell regions. No consistent relationship between the stable isotope composition of the eggshell and embryonic development, shell region or maculation was observed, although shell thickness decreased with development in all shell regions. By contrast, individual eggs differed significantly in isotope composition. These results establish that eggshells can be used to investigate a species' carbon and oxygen sources, regardless of the egg's developmental stage.

  20. Proteomic analysis identifies interleukin 11 regulated plasma membrane proteins in human endometrial epithelial cells in vitro

    PubMed Central

    2011-01-01

    Background During the peri-implantation period, the embryo adheres to an adequately prepared or receptive endometrial surface epithelium. Abnormal embryo adhesion to the endometrium results in embryo implantation failure and infertility. Endometrial epithelial cell plasma membrane proteins critical in regulating adhesion may potentially be infertility biomarkers or targets for treating infertility. Interleukin (IL) 11 regulates human endometrial epithelial cells (hEEC) adhesion. Its production is abnormal in women with infertility. The objective of the study was to identify IL11 regulated plasma membrane proteins in hEEC in vitro using a proteomic approach. Methods Using a 2D-differential in-gel electrophoresis (DIGE) electrophoresis combined with LCMS/MS mass spectrometry approach, we identified 20 unique plasma membrane proteins differentially regulated by IL11 in ECC-1 cells, a hEEC derived cell line. Two IL11 regulated proteins with known roles in cell adhesion, annexin A2 (ANXA2) and flotillin-1 (FLOT1), were validated by Western blot and immunocytochemistry in hEEC lines (ECC-1 and an additional cell line, Ishikawa) and primary hEEC. Flotilin-1 was further validated by immunohistochemistry in human endometrium throughout the menstrual cycle (n = 6-8/cycle). Results 2D-DIGE analysis identified 4 spots that were significantly different between control and IL11 treated group. Of these 4 spots, there were 20 proteins that were identified with LCMS/MS. Two proteins; ANXA2 and FLOT1 were chosen for further analyses and have found to be significantly up-regulated following IL11 treatment. Western blot analysis showed a 2-fold and a 2.5-fold increase of ANXA2 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. Similarly, a 1.8-fold and a 2.3/2.4-fold increase was also observed for FLOT1 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. In vitro, IL11 induced stronger ANXA2 expression on cell surface of primary hEEC and ECC-1 whilst

  1. Preliminary studies on immobilization of lipase using chicken eggshell

    NASA Astrophysics Data System (ADS)

    Salleh, S.; Serri, N. A.; Hena, S.; Tajarudin, H. A.

    2016-06-01

    A few advantages of enzyme immobilization are reusability of expensive enzyme, improvement of stability and activity compared to crude enzyme. Various organic components can be used as carrier for enzyme immobilization such as chicken eggshell. It can be used as a carrier for immobilization as its mineral component mostly contains of calcium carbonate. In the present study, Tributyrin method was used to test enzyme activity of Rhizomucour Miehei, Candida Antarctica and Candida Rugosa. Rhizomucour Miehei shows the highest enzyme activity (360.8 mol/min/mL lipase) and was used in further experiment. Experiment was continued to study incubation time for lipase immobilization on eggshell (1-4 hours) and reaction time of esterification of sugar ester (0-72 hours). Two hours incubation time for lipase immobilization was observed and gives the highest yield of sugar ester (78.13%). Fructose and stearic acid as substrate was used for the production of sugar ester. The highest percentage of sugar ester production was shown at 36 hours of reaction time.

  2. Dicofol (Kelthane?)-induced eggshell thinning in captive American kestrels

    USGS Publications Warehouse

    Clark, D.R.; Spann, J.W.; Bunck, C.M.

    1990-01-01

    Reproductive parameters of American kestrels (Falco sparverius) were measured through two breeding seasons. Exposure to Kelthane? (containing no DDT-related compounds) at dietary concentrations of 0 (contro!), 1, 3, 10 and 30 ug/g (wet weight) began in late November before, and continued through, the second season. Kelthane thinned eggshells and lowered the thickness index at dietary concentrations >3 ?g/g and it reduced shell weight at >10 ?g/g when comparisons were to concurrent controls. Kelthane reduced the thickness index at >3 ug/g and it reduced shell thickness and weight at >10 ug/g when comparisons were to the same birds during the previous season. All changes were dose-related. It was not previously known that as little as 3 ug/g dicofol could cause these effects. Kestrels resembled previously studied eastern screech-owls (Otus asio) in that 10 ug/ g reduced hatchability of eggs. Both these raptors showed eggshell changes without serious effects on production of young. Available data show dicofol only equal to or less effective than DDE as a shell-thinning agent. Also, DDE may have more impact than dicofol on such critical aspects of reproduction as egg hatchability and survivability of hatchlings. Field studies of dicofol residues in food chains and of residue concentrations in eggs vs. nesting success from areas of heavy dicofol use are needed to judge this chemical's ecological impact.

  3. Synthesis of nano-textured biocompatible scaffolds from chicken eggshells

    NASA Astrophysics Data System (ADS)

    Asghar, Waseem; Kim, Young-Tae; Ilyas, Azhar; Sankaran, Jeyantt; Wan, Yuan; Iqbal, Samir M.

    2012-11-01

    Cell adhesion, morphology and growth are influenced by surface topography at nano and micrometer scales. Nano-textured surfaces are prepared using photolithography, plasma etching and long polymer chemical etching which are cost prohibitive and require specialized equipment. This article demonstrates a simple approach to synthesize nano-textured scaffolds from chicken eggshells. Varieties of pattern are made on the eggshells like micro-needle forests and nanopores, giving very uniform nano-textures to the surfaces. The surfaces are characterized for chemical composition and crystal phase. The novel patterns are transferred to PDMS surfaces and the nano-textured PDMS surfaces are used to study the effect of texturing on human fibroblast cell growth and attachment. The effects of surface topographies, along with laminin coating on cell cultures, are also studied. We find an exciting phenomenon that the initial seeding density of the fibroblast cells affects the influence of the nano-texturing on cell growth. These nano-textured surfaces give 16 times more fibroblast growth when compared to flat PDMS surfaces. The novel nano-textured patterns also double the laminin adsorption on PDMS.

  4. Synthesis of nano-textured biocompatible scaffolds from chicken eggshells.

    PubMed

    Asghar, Waseem; Kim, Young-Tae; Ilyas, Azhar; Sankaran, Jeyantt; Wan, Yuan; Iqbal, Samir M

    2012-11-30

    Cell adhesion, morphology and growth are influenced by surface topography at nano and micrometer scales. Nano-textured surfaces are prepared using photolithography, plasma etching and long polymer chemical etching which are cost prohibitive and require specialized equipment. This article demonstrates a simple approach to synthesize nano-textured scaffolds from chicken eggshells. Varieties of pattern are made on the eggshells like micro-needle forests and nanopores, giving very uniform nano-textures to the surfaces. The surfaces are characterized for chemical composition and crystal phase. The novel patterns are transferred to PDMS surfaces and the nano-textured PDMS surfaces are used to study the effect of texturing on human fibroblast cell growth and attachment. The effects of surface topographies, along with laminin coating on cell cultures, are also studied. We find an exciting phenomenon that the initial seeding density of the fibroblast cells affects the influence of the nano-texturing on cell growth. These nano-textured surfaces give 16 times more fibroblast growth when compared to flat PDMS surfaces. The novel nano-textured patterns also double the laminin adsorption on PDMS.

  5. Early life stress shapes female reproductive strategy through eggshell pigmentation in Japanese quail.

    PubMed

    Duval, Camille; Zimmer, Cédric; Mikšík, Ivan; Cassey, Phillip; Spencer, Karen A

    2014-11-01

    Physiological constraints on colouration have been widely reported; especially in birds, which trade-off antioxidant responses against colourful costly signals. One female extended phenotypic trait, which might also highlight important physiological trade-offs, is the pigmentation of their eggshells. In ground-nesting species, producing eggs that are visually undetectable by predators is the best camouflage strategy. However, the condition-dependence of eggshell pigmentation, and the pigments role in oxidative stress, may constrain females to trade-off between their antioxidant capacity and maximising the camouflage of their eggs when they deposit eggshell pigments. Developmental stress is one factor that influences female antioxidant capacity, and could lead to variations in eggshell pigmentation that might have crucial consequences on individual fitness if egg crypsis is compromised especially under stressful conditions. We investigated the interaction between developmental and breeding conditions with respect to eggshell pigmentation in Japanese quail. We studied 30 females that bred under both control and stressful conditions, and were exposed to pre- and/or post-natal stress, or neither. Pre- and post-natal stress independently influenced eggshell pigmentation strategies under stressful breeding conditions. Under stressful reproduction, eggshell protoporphyrin concentration and maculation were affected by pre-natal stress, whereas eggshell reflectance and biliverdin concentration were influenced by post-natal stress. These changes may reflect potential adaptive strategies shaped by developmental stress, but additional data on the benefit of egg crypsis in quail, combined with studies on the role of both pigments on chick survival, will help to clarify whether early life stress can enhance fitness through eggshell pigmentation when developmental and reproductive environments match.

  6. Identifying and Quantitating Conformational Exchange in Membrane Proteins Using Site-Directed Spin Labeling

    PubMed Central

    2015-01-01

    Conspectus Protein structures are not static but sample different conformations over a range of amplitudes and time scales. These fluctuations may involve relatively small changes in bond angles or quite large rearrangements in secondary structure and tertiary fold. The equilibrium between discrete structural substates on the microsecond to millisecond time scale is sometimes termed conformational exchange. Protein dynamics and conformational exchange are believed to provide the basis for many important activities, such as protein–protein and protein–ligand interactions, enzymatic activity and protein allostery; however, for many proteins, the dynamics and conformational exchange that lead to function are poorly defined. Spectroscopic methods, such as NMR, are among the most important methods to explore protein dynamics and conformational exchange; however, they are difficult to implement in some systems and with some types of exchange events. Site-directed spin labeling (SDSL) is an EPR based approach that is particularly well-suited to high molecular-weight systems such as membrane proteins. Because of the relatively fast time scale for EPR spectroscopy, it is an excellent method to examine exchange. Conformations that are in exchange are captured as distinct populations in the EPR spectrum, and this feature when combined with the use of methods that can shift the free energy of conformational substates allows one to identify regions of proteins that are in dynamic exchange. In addition, modern pulse EPR methods have the ability to examine conformational heterogeneity, resolve discrete protein states, and identify the substates in exchange. Protein crystallography has provided high-resolution models for a number of membrane proteins; but because of conformational exchange, these models do not always reflect the structures that are present when the protein is in a native bilayer environment. In the case of the Escherichia coli vitamin B12 transporter, Btu

  7. Identifying DNA-binding proteins using structural motifs and the electrostatic potential

    PubMed Central

    Shanahan, Hugh P.; Garcia, Mario A.; Jones, Susan; Thornton, Janet M.

    2004-01-01

    Robust methods to detect DNA-binding proteins from structures of unknown function are important for structural biology. This paper describes a method for identifying such proteins that (i) have a solvent accessible structural motif necessary for DNA-binding and (ii) a positive electrostatic potential in the region of the binding region. We focus on three structural motifs: helix–turn-helix (HTH), helix–hairpin–helix (HhH) and helix–loop–helix (HLH). We find that the combination of these variables detect 78% of proteins with an HTH motif, which is a substantial improvement over previous work based purely on structural templates and is comparable to more complex methods of identifying DNA-binding proteins. Similar true positive fractions are achieved for the HhH and HLH motifs. We see evidence of wide evolutionary diversity for DNA-binding proteins with an HTH motif, and much smaller diversity for those with an HhH or HLH motif. PMID:15356290

  8. A novel assay to identify the trafficking proteins that bind to specific vesicle populations

    PubMed Central

    Bentley, Marvin; Banker, Gary

    2016-01-01

    Here we describe a method capable of identifying interactions between candidate trafficking proteins and a defined vesicle population in intact cells. The assay involves the expression of an FKBP12-rapamycin–binding domain (FRB)–tagged candidate vesicle-binding protein that can be inducibly linked to an FKBP-tagged molecular motor. If the FRB-tagged candidate protein binds the labeled vesicles, then linking the FRB and FKBP domains recruits motors to the vesicles and causes a predictable, highly distinctive change in vesicle trafficking. We describe two versions of the assay: a general protocol for use in cells with a typical microtubule-organizing center and a specialized protocol designed to detect protein-vesicle interactions in cultured neurons. We have successfully used this assay to identify kinesins and Rabs that bind to a variety of different vesicle populations. In principle, this assay could be used to investigate interactions between any category of vesicle trafficking proteins and any vesicle population that can be specifically labeled. PMID:26621371

  9. Mass spectrometric approach for identifying putative plasma membrane proteins of Arabidopsis leaves associated with cold acclimation.

    PubMed

    Kawamura, Yukio; Uemura, Matsuo

    2003-10-01

    Although enhancement of freezing tolerance in plants during cold acclimation is closely associated with an increase in the cryostability of plasma membrane, the molecular mechanism for the increased cryostability of plasma membrane is still to be elucidated. In Arabidopsis, enhanced freezing tolerance was detectable after cold acclimation at 2 degrees C for as short as 1 day, and maximum freezing tolerance was attained after 1 week. To identify the plasma membrane proteins that change in quantity in response to cold acclimation, a highly purified plasma membrane fraction was isolated from leaves before and during cold acclimation, and the proteins in the fraction were separated with gel electrophoresis. We found that there were substantial changes in the protein profiles after as short as 1 day of cold acclimation. Subsequently, using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS), we identified 38 proteins that changed in quantity during cold acclimation. The proteins that changed in quantity during the first day of cold acclimation include those that are associated with membrane repair by membrane fusion, protection of the membrane against osmotic stress, enhancement of CO2 fixation, and proteolysis.

  10. Identifying paths of allosteric communication in the protein BirA through simulations

    NASA Astrophysics Data System (ADS)

    Custer, Gregory; Beckett, Dorothy; Matysiak, Silvina

    Biotin ligase/repressor (BirA) is a bifunctional enzyme which adenylates biotin and transfers the product, biotinyl-5'-AMP (bio-5'-AMP) to biotin carboxyl carrier protein (BCCP). In the absence of BCCP, bio-5'-AMP promotes the dimerization of BirA. In dimer form, the BirA.bio-5'-AMP complex is able to bind to the biotin operator and prevents further synthesis of biotin. The bio-5'-AMP binds away from the dimer interface, so it is acting as an allosteric activator. We perform all-atom molecular dynamics simulations with BirA to look at fluctuations within the protein at equilibrium. We simulate apoBirA, liganded BirA, as well as two mutants, M211A and V219A. In agreement with experimental observations, several loops of the protein become stabilized for the liganded BirA when compared to the apo protein. In addition, changes in the dimer interface are observed for the M211A and V219A mutations, which are located in the ligand binding region. Using inter-residue correlation coefficients and pair energies a communication network through the protein is constructed. With this network we have identified paths which have the potential to be important in allosteric activation of BirA. These paths and the methods we use to identify them will be presented.

  11. Interaction of Proteus mirabilis Urease Apoenzyme and Accessory Proteins Identified with Yeast Two-Hybrid Technology

    PubMed Central

    Heimer, Susan R.; Mobley, Harry L. T.

    2001-01-01

    Proteus mirabilis, a gram-negative bacterium associated with complicated urinary tract infections, produces a metalloenzyme urease which hydrolyzes urea to ammonia and carbon dioxide. The apourease is comprised of three structural subunits, UreA, UreB, and UreC, assembled as a homotrimer of individual UreABC heterotrimers (UreABC)3. To become catalytically active, apourease acquires divalent nickel ions through a poorly understood process involving four accessory proteins, UreD, UreE, UreF, and UreG. While homologues of UreD, UreF, and UreG have been copurified with apourease, it remains unclear specifically how these polypeptides associate with the apourease or each other. To identify interactions among P. mirabilis accessory proteins, in vitro immunoprecipitation and in vivo yeast two-hybrid assays were employed. A complex containing accessory protein UreD and structural protein UreC was isolated by immunoprecipitation and characterized with immunoblots. This association occurs independently of coaccessory proteins UreE, UreF, and UreG and structural protein UreA. In a yeast two-hybrid screen, UreD was found to directly interact in vivo with coaccessory protein UreF. Unique homomultimeric interactions of UreD and UreF were also detected in vivo. To substantiate the study of urease proteins with a yeast two-hybrid assay, previously described UreE dimers and homomultimeric UreA interactions among apourease trimers were confirmed in vivo. Similarly, a known structural interaction involving UreA and UreC was also verified. This report suggests that in vivo, P. mirabilis UreD may be important for recruitment of UreF to the apourease and that crucial homomultimeric associations occur among these accessory proteins. PMID:11157956

  12. A predicted protein interactome identifies conserved global networks and disease resistance subnetworks in maize

    PubMed Central

    Musungu, Bryan; Bhatnagar, Deepak; Brown, Robert L.; Fakhoury, Ahmad M.; Geisler, Matt

    2015-01-01

    Interactomes are genome-wide roadmaps of protein-protein interactions. They have been produced for humans, yeast, the fruit fly, and Arabidopsis thaliana and have become invaluable tools for generating and testing hypotheses. A predicted interactome for Zea mays (PiZeaM) is presented here as an aid to the research community for this valuable crop species. PiZeaM was built using a proven method of interologs (interacting orthologs) that were identified using both one-to-one and many-to-many orthology between genomes of maize and reference species. Where both maize orthologs occurred for an experimentally determined interaction in the reference species, we predicted a likely interaction in maize. A total of 49,026 unique interactions for 6004 maize proteins were predicted. These interactions are enriched for processes that are evolutionarily conserved, but include many otherwise poorly annotated proteins in maize. The predicted maize interactions were further analyzed by comparing annotation of interacting proteins, including different layers of ontology. A map of pairwise gene co-expression was also generated and compared to predicted interactions. Two global subnetworks were constructed for highly conserved interactions. These subnetworks showed clear clustering of proteins by function. Another subnetwork was created for disease response using a bait and prey strategy to capture interacting partners for proteins that respond to other organisms. Closer examination of this subnetwork revealed the connectivity between biotic and abiotic hormone stress pathways. We believe PiZeaM will provide a useful tool for the prediction of protein function and analysis of pathways for Z. mays researchers and is presented in this paper as a reference tool for the exploration of protein interactions in maize. PMID:26089837

  13. Protein Quantitative Trait Loci Identify Novel Candidates Modulating Cellular Response to Chemotherapy

    PubMed Central

    Gorsic, Lidija K.; Antao, Nirav N.; Wong, Shan S.; Chung, Sophie H.; Gill, Daniel F.; Im, Hae K.; Myers, Jamie L.; White, Kevin P.; Jones, Richard Baker; Dolan, M. Eileen

    2014-01-01

    Annotating and interpreting the results of genome-wide association studies (GWAS) remains challenging. Assigning function to genetic variants as expression quantitative trait loci is an expanding and useful approach, but focuses exclusively on mRNA rather than protein levels. Many variants remain without annotation. To address this problem, we measured the steady state abundance of 441 human signaling and transcription factor proteins from 68 Yoruba HapMap lymphoblastoid cell lines to identify novel relationships between inter-individual protein levels, genetic variants, and sensitivity to chemotherapeutic agents. Proteins were measured using micro-western and reverse phase protein arrays from three independent cell line thaws to permit mixed effect modeling of protein biological replicates. We observed enrichment of protein quantitative trait loci (pQTLs) for cellular sensitivity to two commonly used chemotherapeutics: cisplatin and paclitaxel. We functionally validated the target protein of a genome-wide significant trans-pQTL for its relevance in paclitaxel-induced apoptosis. GWAS overlap results of drug-induced apoptosis and cytotoxicity for paclitaxel and cisplatin revealed unique SNPs associated with the pharmacologic traits (at p<0.001). Interestingly, GWAS SNPs from various regions of the genome implicated the same target protein (p<0.0001) that correlated with drug induced cytotoxicity or apoptosis (p≤0.05). Two genes were functionally validated for association with drug response using siRNA: SMC1A with cisplatin response and ZNF569 with paclitaxel response. This work allows pharmacogenomic discovery to progress from the transcriptome to the proteome and offers potential for identification of new therapeutic targets. This approach, linking targeted proteomic data to variation in pharmacologic response, can be generalized to other studies evaluating genotype-phenotype relationships and provide insight into chemotherapeutic mechanisms. PMID:24699359

  14. Inflammatory and fibrotic proteins proteomically identified as key protein constituents in urine and stone matrix of patients with kidney calculi.

    PubMed

    Boonla, Chanchai; Tosukhowong, Piyaratana; Spittau, Björn; Schlosser, Andreas; Pimratana, Chaowat; Krieglstein, Kerstin

    2014-02-15

    To uncover whether urinary proteins are incorporated into stones, the proteomic profiles of kidney stones and urine collected from the same patients have to be explored. We employed 1D-PAGE and nanoHPLC-ESI-MS/MS to analyze the proteomes of kidney stone matrix (n=16), nephrolithiatic urine (n=14) and healthy urine (n=3). We identified 62, 66 and 22 proteins in stone matrix, nephrolithiatic urine and healthy urine, respectively. Inflammation- and fibrosis-associated proteins were frequently detected in the stone matrix and nephrolithiatic urine. Eighteen proteins were exclusively found in the stone matrix and nephrolithiatic urine, considered as candidate biomarkers for kidney stone formation. S100A8 and fibronectin, representatives of inflammation and fibrosis, respectively, were up-regulated in nephrolithiasis renal tissues. S100A8 was strongly expressed in infiltrated leukocytes. Fibronectin was over-expressed in renal tubular cells. S100A8 and fibronectin were immunologically confirmed to exist in nephrolithiatic urine and stone matrix, but in healthy urine they were undetectable. Conclusion, both kidney stones and urine obtained from the same patients greatly contained inflammatory and fibrotic proteins. S100A8 and fibronectin were up-regulated in stone-baring kidneys and nephrolithiatic urine. Therefore, inflammation and fibrosis are suggested to be involved in the formation of kidney calculi.

  15. A Review of Methods Used for Identifying Structural Changes in a Large Protein Complex

    PubMed Central

    Nadeau, Owen W.; Carlson, Gerald M.

    2013-01-01

    This chapter explores the structural responses of a massive, hetero-oligomeric protein complex to a single allosteric activator as probed by a wide range of chemical, biochemical, and biophysical approaches. Some of the approaches used are amenable only to large protein targets, whereas others push the limits of their utility. Some of the techniques focus on individual subunits, or portions thereof, while others examine the complex as a whole. Despite the absence of crystallographic data for the complex, the diverse techniques identify and implicate a small region of its catalytic subunit as the master allosteric activation switch for the entire complex. PMID:22052488

  16. Identifying protein phosphorylation sites with kinase substrate specificity on human viruses.

    PubMed

    Bretaña, Neil Arvin; Lu, Cheng-Tsung; Chiang, Chiu-Yun; Su, Min-Gang; Huang, Kai-Yao; Lee, Tzong-Yi; Weng, Shun-Long

    2012-01-01

    Viruses infect humans and progress inside the body leading to various diseases and complications. The phosphorylation of viral proteins catalyzed by host kinases plays crucial regulatory roles in enhancing replication and inhibition of normal host-cell functions. Due to its biological importance, there is a desire to identify the protein phosphorylation sites on human viruses. However, the use of mass spectrometry-based experiments is proven to be expensive and labor-intensive. Furthermore, previous studies which have identified phosphorylation sites in human viruses do not include the investigation of the responsible kinases. Thus, we are motivated to propose a new method to identify protein phosphorylation sites with its kinase substrate specificity on human viruses. The experimentally verified phosphorylation data were extracted from virPTM--a database containing 301 experimentally verified phosphorylation data on 104 human kinase-phosphorylated virus proteins. In an attempt to investigate kinase substrate specificities in viral protein phosphorylation sites, maximal dependence decomposition (MDD) is employed to cluster a large set of phosphorylation data into subgroups containing significantly conserved motifs. The experimental human phosphorylation sites are collected from Phospho.ELM, grouped according to its kinase annotation, and compared with the virus MDD clusters. This investigation identifies human kinases such as CK2, PKB, CDK, and MAPK as potential kinases for catalyzing virus protein substrates as confirmed by published literature. Profile hidden Markov model is then applied to learn a predictive model for each subgroup. A five-fold cross validation evaluation on the MDD-clustered HMMs yields an average accuracy of 84.93% for Serine, and 78.05% for Threonine. Furthermore, an independent testing data collected from UniProtKB and Phospho.ELM is used to make a comparison of predictive performance on three popular kinase-specific phosphorylation site

  17. A comparative hidden Markov model analysis pipeline identifies proteins characteristic of cereal-infecting fungi

    PubMed Central

    2013-01-01

    Background Fungal pathogens cause devastating losses in economically important cereal crops by utilising pathogen proteins to infect host plants. Secreted pathogen proteins are referred to as effectors and have thus far been identified by selecting small, cysteine-rich peptides from the secretome despite increasing evidence that not all effectors share these attributes. Results We take advantage of the availability of sequenced fungal genomes and present an unbiased method for finding putative pathogen proteins and secreted effectors in a query genome via comparative hidden Markov model analyses followed by unsupervised protein clustering. Our method returns experimentally validated fungal effectors in Stagonospora nodorum and Fusarium oxysporum as well as the N-terminal Y/F/WxC-motif from the barley powdery mildew pathogen. Application to the cereal pathogen Fusarium graminearum reveals a secreted phosphorylcholine phosphatase that is characteristic of hemibiotrophic and necrotrophic cereal pathogens and shares an ancient selection process with bacterial plant pathogens. Three F. graminearum protein clusters are found with an enriched secretion signal. One of these putative effector clusters contains proteins that share a [SG]-P-C-[KR]-P sequence motif in the N-terminal and show features not commonly associated with fungal effectors. This motif is conserved in secreted pathogenic Fusarium proteins and a prime candidate for functional testing. Conclusions Our pipeline has successfully uncovered conservation patterns, putative effectors and motifs of fungal pathogens that would have been overlooked by existing approaches that identify effectors as small, secreted, cysteine-rich peptides. It can be applied to any pathogenic proteome data, such as microbial pathogen data of plants and other organisms. PMID:24252298

  18. A Human Proteome Array Approach to Identifying Key Host Proteins Targeted by Toxoplasma Kinase ROP18.

    PubMed

    Yang, Zhaoshou; Hou, Yongheng; Hao, Taofang; Rho, Hee-Sool; Wan, Jun; Luan, Yizhao; Gao, Xin; Yao, Jianping; Pan, Aihua; Xie, Zhi; Qian, Jiang; Liao, Wanqin; Zhu, Heng; Zhou, Xingwang

    2017-03-01

    Toxoplasma kinase ROP18 is a key molecule responsible for the virulence of Toxoplasma gondii; however, the mechanisms by which ROP18 exerts parasite virulence via interaction with host proteins remain limited to a small number of identified substrates. To identify a broader array of ROP18 substrates, we successfully purified bioactive mature ROP18 and used it to probe a human proteome array. Sixty eight new putative host targets were identified. Functional annotation analysis suggested that these proteins have a variety of functions, including metabolic process, kinase activity and phosphorylation, cell growth, apoptosis and cell death, and immunity, indicating a pleiotropic role of ROP18 kinase. Among these proteins, four candidates, p53, p38, UBE2N, and Smad1, were further validated. We demonstrated that ROP18 targets p53, p38, UBE2N, and Smad1 for degradation. Importantly, we demonstrated that ROP18 phosphorylates Smad1 Ser-187 to trigger its proteasome-dependent degradation. Further functional characterization of the substrates of ROP18 may enhance understanding of the pathogenesis of Toxoplasma infection and provide new therapeutic targets. Similar strategies could be used to identify novel host targets for other microbial kinases functioning at the pathogen-host interface.

  19. ESR dating of teeth, bones and eggshells excavated at a Paleolithic site of Douara Cave, Syria

    NASA Astrophysics Data System (ADS)

    Kai, A.; Miki, T.; Ikeya, M.

    Teeth, bones and eggshells excavated at a Paleolithic site, Douara Cave in Syria were measured with electron spin resonance (ESR) in their natural state without destructive sample pre-treatment. The total dose of natural radiation (TD) was estimated using the ESR signals of CO 33- radicals ( g⊥ = 2.0024, g∥ = 1.9977) for teeth and bones and those of radicals ( g∥ = 2.0016, g⊥ = 1.9981) for eggshells. Microwave saturation behaviour and thermal annealing of eggshells are studied in detail.

  20. Strontium-90 in Canada goose eggshells: Non-fatal monitoring for contamination in wildlife

    SciTech Connect

    Rickard, W.H.; Eberhardt, L.E. )

    1989-08-01

    Strontium-90 as measured in eggshells taken form newly hatched eggs in 42 Canada goose nests. Higher-than-background levels were present in eggshells from only a few nests. The origin of the enhanced strontium-90 levels appears to be Hanford Site facilities. However, the amounts measured are too low to expect to see any deleterious health or reproductive effects on the goose population. Eggshells provide an efficient way to obtain biological samples for environmental monitoring without inducing goose mortality. 10 refs., 2 figs.

  1. Ultrastructure of the calcareous layer eggshell of the turtle Emys orbicularis (L.). Preliminary study.

    PubMed

    Mitrus, S

    1997-01-01

    The calcareous layer of Emys orbicularis eggshell was examined by scanning electron microscopy. The layer is composed of well distinguished shell units which consist of needle-like crystallites radiating outwards from the cores. The linear structures of the cores on the inner surface appear to be similar to those in the eggshell of Mauremys caspica. On the inner surface of shell units of eggs with dead embryos there are flat, conical depressions. This surface in specimens from unfertilized eggs is almost flat. It may suggest that a developing embryo derives calcium from the eggshell.

  2. Acute and chronic eggshell temperature manipulations during hatching term influence hatchability, broiler performance, and ascites incidence.

    PubMed

    Sozcu, A; Ipek, A

    2015-02-01

    The aim of the current study was to determine how a control temperature and acute and chronic high eggshell temperatures during the last three days of incubation, can affect hatchability, chick quality, and organ development on day of hatch as well as broiler performance and ascites incidence in later life. The eggshell temperature manipulations were applied during hatching term (days 19 to 21) as follows: control EST (37.3 to 38.0°C), acute high eggshell temperature manipulations (38.4- to 39.0°C for three hours daily) and chronic high eggshell temperature manipulations (38.4 to 39.0°C). The lowest hatchability and the highest cull chick rate were in the chronic high eggshell temperature manipulations group. Lower chick quality parameters correlated with lower chick weights and heavier residual yolk sac weights that were in the chronic high eggshell temperature manipulations group depending on hatch time. The live weights on the 1(st) day of the growing period were higher in the control and acute high eggshell temperature manipulations groups than the chronic high eggshell temperature manipulations group. At 6 wk of age, live weights of broilers were the highest in the control than in the acute and chronic high eggshell temperature manipulations groups. The total mortality was 2.5, 9.2, and 13.3%, the mortality due to ascites was 2.1, 8.3, and 12.9% in the control, acute ,and chronic high eggshell temperature manipulations groups, respectively. The right ventricular/total ventricular ratios for the control, acute and chronic high eggshell temperature manipulations groups were 0.22, 0.28, and 0.30%, respectively. In conclusion, short-term and long-term higher temperatures during the hatching term affect embryo development, incubation results, broiler performance, and ascites incidence. Although the acute high eggshell temperature manipulations did not affect the chick quality parameters at hatch, it negatively affected incubation results and broiler performance

  3. Controllability analysis of the directed human protein interaction network identifies disease genes and drug targets

    PubMed Central

    Vinayagam, Arunachalam; Gibson, Travis E.; Lee, Ho-Joon; Yilmazel, Bahar; Roesel, Charles; Hu, Yanhui; Kwon, Young; Sharma, Amitabh; Liu, Yang-Yu; Perrimon, Norbert; Barabási, Albert-László

    2016-01-01

    The protein–protein interaction (PPI) network is crucial for cellular information processing and decision-making. With suitable inputs, PPI networks drive the cells to diverse functional outcomes such as cell proliferation or cell death. Here, we characterize the structural controllability of a large directed human PPI network comprising 6,339 proteins and 34,813 interactions. This network allows us to classify proteins as “indispensable,” “neutral,” or “dispensable,” which correlates to increasing, no effect, or decreasing the number of driver nodes in the network upon removal of that protein. We find that 21% of the proteins in the PPI network are indispensable. Interestingly, these indispensable proteins are the primary targets of disease-causing mutations, human viruses, and drugs, suggesting that altering a network’s control property is critical for the transition between healthy and disease states. Furthermore, analyzing copy number alterations data from 1,547 cancer patients reveals that 56 genes that are frequently amplified or deleted in nine different cancers are indispensable. Among the 56 genes, 46 of them have not been previously associated with cancer. This suggests that controllability analysis is very useful in identifying novel disease genes and potential drug targets. PMID:27091990

  4. A biotin enrichment strategy identifies novel carbonylated amino acids in proteins from human plasma.

    PubMed

    Havelund, Jesper F; Wojdyla, Katarzyna; Davies, Michael J; Jensen, Ole N; Møller, Ian Max; Rogowska-Wrzesinska, Adelina

    2017-03-06

    Protein carbonylation is an irreversible protein oxidation correlated with oxidative stress, various diseases and ageing. Here we describe a peptide-centric approach for identification and characterisation of up to 14 different types of carbonylated amino acids in proteins. The modified residues are derivatised with biotin-hydrazide, enriched and characterised by tandem mass spectrometry. The strength of the method lies in an improved elution of biotinylated peptides from monomeric avidin resin using hot water (95°C) and increased sensitivity achieved by reduction of analyte losses during sample preparation and chromatography. For the first time MS/MS data analysis utilising diagnostic biotin fragment ions is used to pinpoint sites of biotin labelling and improve the confidence of carbonyl peptide assignments. We identified a total of 125 carbonylated residues in bovine serum albumin after extensive in vitro metal ion-catalysed oxidation. Furthermore, we assigned 133 carbonylated sites in 36 proteins in native human plasma protein samples. The optimised workflow enabled detection of 10 hitherto undetected types of carbonylated amino acids in proteins: aldehyde and ketone modifications of leucine, valine, alanine, isoleucine, glutamine, lysine and glutamic acid (+14Da), an oxidised form of methionine - aspartate semialdehyde (-32Da) - and decarboxylated glutamic acid and aspartic acid (-30Da).

  5. Novel protein kinase signaling systems regulating lifespan identified by small molecule library screening using Drosophila.

    PubMed

    Spindler, Stephen R; Li, Rui; Dhahbi, Joseph M; Yamakawa, Amy; Sauer, Frank

    2012-01-01

    Protein kinase signaling cascades control most aspects of cellular function. The ATP binding domains of signaling protein kinases are the targets of most available inhibitors. These domains are highly conserved from mammals to flies. Herein we describe screening of a library of small molecule inhibitors of protein kinases for their ability to increase Drosophila lifespan. We developed an assay system which allowed screening using the small amounts of materials normally present in commercial chemical libraries. The studies identified 17 inhibitors, the majority of which targeted tyrosine kinases associated with the epidermal growth factor receptor (EGFR), platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF) receptors, G-protein coupled receptor (GPCR), Janus kinase (JAK)/signal transducer and activator of transcription (STAT), the insulin and insulin-like growth factor (IGFI) receptors. Comparison of the protein kinase signaling effects of the inhibitors in vitro defined a consensus intracellular signaling profile which included decreased signaling by p38MAPK (p38), c-Jun N-terminal kinase (JNK) and protein kinase C (PKC). If confirmed, many of these kinases will be novel additions to the signaling cascades known to regulate metazoan longevity.

  6. Genome-wide screen of human bromodomain-containing proteins identifies Cecr2 as a novel DNA damage response protein.

    PubMed

    Lee, Seul-Ki; Park, Eun-Jung; Lee, Han-Sae; Lee, Ye Seul; Kwon, Jongbum

    2012-07-01

    The formation of γ-H2AX foci after DNA double strand breaks (DSBs) is crucial for the cellular response to this lethal DNA damage. We previously have shown that BRG1, a chromatin remodeling enzyme, facilitates DSB repair by stimulating γ-H2AX formation, and this function of BRG1 requires the binding of BRGI to acetylated histone H3 on γ-H2AX-containing nucleosomes using its bromodomain (BRD), a protein module that specifically recognizes acetyl-Lys moieties. We also have shown that the BRD of BRG1, when ectopically expressed in cells, functions as a dominant negative inhibitor of the BRG1 activity to stimulate γ-H2AX and DSB repair. Here, we found that BRDs from a select group of proteins have no such activity, suggesting that the γ-H2AX inhibition activity of BRG1 BRD is specific. This finding led us to search for more BRDs that exhibit γ-H2AX inhibition activity in the hope of finding additional BRD-containing proteins involved in DNA damage responses. We screened a total of 52 individual BRDs present in 38 human BRD-containing proteins, comprising 93% of all human BRDs. We identified the BRD of cat eye syndrome chromosome region candidate 2 (Cecr2), which recently was shown to form a novel chromatin remodeling complex with unknown cellular functions, as having a strong γ-H2AX inhibition activity. This activity of Cecr2 BRD is specific because it depends on the chromatin binding affinity of Cecr2 BRD. Small interfering RNA knockdown experiments showed that Cecr2 is important for γ-H2AX formation and DSB repair. Therefore, our genomewide screen identifies Cecr2 as a novel DNA damage response protein.

  7. Investigation of eggshell thickness and biochemical indicators of contaminant exposure in Great Blue Herons(Ardea herodias) from Mason Neck National Wildlife Refuge

    USGS Publications Warehouse

    Johnson, K.N.; Pinkney, A.E.; Melancon, M.J.; Hoffman, D.J.

    2001-01-01

    Mason Neck National Wildlife Refuge supports the largest great blue heron (Ardea herodias) rookery in the State of Virginia. The presence of bioaccumulative compounds such as polychlorinated biphenyls and DDT in fish collected from the Potomac River and tidal tributaries along the Refuge led to this study. The objective was to determine if there were any indications of pollutant-induced eggshell thinning or evidence of biochemical exposure to contaminants. We examined eggshell thickness and biomarkers of contaminant exposure in livers of embryos collected from the refuge and Coaches Island, a reference location in Chesapeake Bay. There was no evidence of eggshell thinning. Cytochrome P450 activity, measured as ethoxyresomfin-O-dealkylase (EROD) and benzyloxy-resorufin-O-dealkylase (BROD), was not significantly different in embryos from the two colonies. Biochemical indicators of oxidative stress can be reflected as changes in levels of reduced thiols, oxidized glutathione, and thiobarbituric reactive substances (TBARS). Although there were significant differences in the levels of reduced glutathione (GSH) and total thiol (TSH) activities in the embryo livers, there were no statistically significant differences in TBARS, protein-bound sulfhydryls (PBSH), oxidized glutathione (GSSG) and the ratio of GSSG to GSH. In fact, the concentrations of GSH and TSH were higher in the Mason Neck birds relative to Coaches Island. Under conditions of increased oxidative stress at least one or more of the following would be expected: decreased concentrations of reduced thiols (GSH and TSH), increased GSSG, and increased TBARS. In conclusion, we did not detect eggshell thinning or find evidence of a biochemical response to contaminant exposure in the Mason Neck great blue herons.

  8. Genome-wide RNAi screening identifies protein damage as a regulator of osmoprotective gene expression

    PubMed Central

    Lamitina, Todd; Huang, Chunyi George; Strange, Kevin

    2006-01-01

    The detection, stabilization, and repair of stress-induced damage are essential requirements for cellular life. All cells respond to osmotic stress-induced water loss with increased expression of genes that mediate accumulation of organic osmolytes, solutes that function as chemical chaperones and restore osmotic homeostasis. The signals and signaling mechanisms that regulate osmoprotective gene expression in animal cells are poorly understood. Here, we show that gpdh-1 and gpdh-2, genes that mediate the accumulation of the organic osmolyte glycerol, are essential for survival of the nematode Caenorhabditis elegans during osmotic stress. Expression of GFP driven by the gpdh-1 promoter (Pgpdh-1::GFP) is detected only during hypertonic stress but is not induced by other stressors. Using Pgpdh-1::GFP expression as a phenotype, we screened ≈16,000 genes by RNAi feeding and identified 122 that cause constitutive activation of gpdh-1 expression and glycerol accumulation. Many of these genes function to regulate protein translation and cotranslational protein folding and to target and degrade denatured proteins, suggesting that the accumulation of misfolded proteins functions as a signal to activate osmoprotective gene expression and organic osmolyte accumulation in animal cells. Consistent with this hypothesis, 73% of these protein-homeostasis genes have been shown to slow age-dependent protein aggregation in C. elegans. Because diverse environmental stressors and numerous disease states result in protein misfolding, mechanisms must exist that discriminate between osmotically induced and other forms of stress-induced protein damage. Our findings provide a foundation for understanding how these damage-selectivity mechanisms function. PMID:16880390

  9. SPECTRUS: A Dimensionality Reduction Approach for Identifying Dynamical Domains in Protein Complexes from Limited Structural Datasets.

    PubMed

    Ponzoni, Luca; Polles, Guido; Carnevale, Vincenzo; Micheletti, Cristian

    2015-08-04

    Identifying dynamical, quasi-rigid domains in proteins provides a powerful means for characterizing functionally oriented structural changes via a parsimonious set of degrees of freedom. In fact, the relative displacements of few dynamical domains usually suffice to rationalize the mechanics underpinning biological functionality in proteins and can even be exploited for structure determination or refinement purposes. Here we present SPECTRUS, a general scheme that, by solely using amino acid distance fluctuations, can pinpoint the innate quasi-rigid domains of single proteins or large complexes in a robust way. Consistent domains are usually obtained by using either a pair of representative structures or thousands of conformers. The functional insights offered by the approach are illustrated for biomolecular systems of very different size and complexity such as kinases, ion channels, and viral capsids. The decomposition tool is available as a software package and web server at spectrus.sissa.it.

  10. KinasePhos: a web tool for identifying protein kinase-specific phosphorylation sites.

    PubMed

    Huang, Hsien-Da; Lee, Tzong-Yi; Tzeng, Shih-Wei; Horng, Jorng-Tzong

    2005-07-01

    KinasePhos is a novel web server for computationally identifying catalytic kinase-specific phosphorylation sites. The known phosphorylation sites from public domain data sources are categorized by their annotated protein kinases. Based on the profile hidden Markov model, computational models are learned from the kinase-specific groups of the phosphorylation sites. After evaluating the learned models, the model with highest accuracy was selected from each kinase-specific group, for use in a web-based prediction tool for identifying protein phosphorylation sites. Therefore, this work developed a kinase-specific phosphorylation site prediction tool with both high sensitivity and specificity. The prediction tool is freely available at http://KinasePhos.mbc.nctu.edu.tw/.

  11. Changes in pigment, spectral transmission and element content of pink chicken eggshells with different pigment intensity during incubation

    PubMed Central

    Yu, Yue; Li, Zhanming

    2016-01-01

    Objective. The objective of this study was to investigate changes in pigment, spectral transmission and element content of chicken eggshells with different intensities of pink pigment during the incubation period. We also investigated the effects of the region (small pole, equator and large pole) and pink pigment intensity of the chicken eggshell on the percent transmission of light passing through the chicken eggshells. Method. Eggs of comparable weight from a meat-type breeder (Meihuang) were used, and divided based on three levels of pink pigment (light, medium and dark) in the eggshells. During the incubation (0–21 d), the values of the eggshell pigment (ΔE, L∗, a∗, b∗) were measured. The percent transmission of light for different regions and intensities of eggshell pigmentation was measured by using the visible wavelength range of 380–780 nm. Result. Three measured indicators of eggshell color, ΔE, L∗ and a∗, did not change significantly during incubation. Compared with other regions and pigment intensities, eggshell at the small pole and with light pigmentation intensity showed the highest percent transmission of light. The transmission value varied significantly (P < 0.001) with incubation time. The element analysis of eggshells with different levels of pink pigment showed that the potassium content of the eggshells for all pigment levels decreased significantly during incubation. Conclusion. In summary, pigment intensity and the region of the eggshell influenced the percent transmission of light of eggshell. Differences in the spectral characteristics of different eggshells may influence the effects of photostimulation during the incubation of eggs. All of these results will be applicable for perfecting the design of light intensity for lighted incubation to improve productivity. PMID:27019785

  12. Eggshell Biliverdin and Protoporphyrin Pigments in a Songbird: Are They Derived from Erythrocytes, Blood Plasma, or the Shell Gland?

    PubMed

    Hargitai, Rita; Boross, Nóra; Hámori, Susanne; Neuberger, Eszter; Nyiri, Zoltán

    Biliverdin and protoporphyrin pigments are deposited into the eggshell when the developing egg is in the shell gland. However, the site of synthesis of eggshell pigments is still uncertain, although it may influence the possible costs and potential functions of eggshell coloration in avian species. Eggshell pigments may be derived from red blood cells or be produced in other organs and then transferred to the shell gland, or they may be synthesized de novo in the shell gland. We studied in the canary (Serinus canaria) whether eggshell blue-green and brown pigmentations are associated with experimentally elevated anemia, female hematocrit level, immature erythrocyte percentage, and feces and plasma pigment levels during egg laying to find out the possible origin of eggshell pigments. We found no significant effects of hematocrit level or experimentally elevated anemia on intensity of eggshell blue-green and brown pigmentations; therefore, we consider it less likely that eggshell pigments are derived from erythrocytes. In addition, we found no significant associations between female feces biliverdin concentration during egg laying and intensity of eggshell blue-green pigmentation, suggesting that eggshell biliverdin may not originate from the spleen or liver. We found a negative association between plasma and feces protoporphyrin concentrations during egg laying and eggshell brown chroma. This result suggests that an increased production of protoporphyrin in the liver, which could have elevated plasma and feces protoporphyrin concentrations, could inhibit eggshell protoporphyrin pigmentation, probably through affecting enzymatic activities. We suggest that both pigments are produced de novo in the shell gland in the canary, but circulating pigment levels may influence shell gland pigment synthesis, thus connecting the physiological status of the female to eggshell coloration.

  13. Changes in pigment, spectral transmission and element content of pink chicken eggshells with different pigment intensity during incubation.

    PubMed

    Yu, Yue; Li, Zhanming; Pan, Jinming

    2016-01-01

    Objective. The objective of this study was to investigate changes in pigment, spectral transmission and element content of chicken eggshells with different intensities of pink pigment during the incubation period. We also investigated the effects of the region (small pole, equator and large pole) and pink pigment intensity of the chicken eggshell on the percent transmission of light passing through the chicken eggshells. Method. Eggs of comparable weight from a meat-type breeder (Meihuang) were used, and divided based on three levels of pink pigment (light, medium and dark) in the eggshells. During the incubation (0-21 d), the values of the eggshell pigment (ΔE, L (∗), a (∗), b (∗)) were measured. The percent transmission of light for different regions and intensities of eggshell pigmentation was measured by using the visible wavelength range of 380-780 nm. Result. Three measured indicators of eggshell color, ΔE, L (∗) and a (∗), did not change significantly during incubation. Compared with other regions and pigment intensities, eggshell at the small pole and with light pigmentation intensity showed the highest percent transmission of light. The transmission value varied significantly (P < 0.001) with incubation time. The element analysis of eggshells with different levels of pink pigment showed that the potassium content of the eggshells for all pigment levels decreased significantly during incubation. Conclusion. In summary, pigment intensity and the region of the eggshell influenced the percent transmission of light of eggshell. Differences in the spectral characteristics of different eggshells may influence the effects of photostimulation during the incubation of eggs. All of these results will be applicable for perfecting the design of light intensity for lighted incubation to improve productivity.

  14. Revealing divergent evolution, identifying circular permutations and detecting active-sites by protein structure comparison.

    PubMed

    Chen, Luonan; Wu, Ling-Yun; Wang, Yong; Zhang, Shihua; Zhang, Xiang-Sun

    2006-09-02

    Protein structure comparison is one of the most important problems in computational biology and plays a key role in protein structure prediction, fold family classification, motif finding, phylogenetic tree reconstruction and protein docking. We propose a novel method to compare the protein structures in an accurate and efficient manner. Such a method can be used to not only reveal divergent evolution, but also identify circular permutations and further detect active-sites. Specifically, we define the structure alignment as a multi-objective optimization problem, i.e., maximizing the number of aligned atoms and minimizing their root mean square distance. By controlling a single distance-related parameter, theoretically we can obtain a variety of optimal alignments corresponding to different optimal matching patterns, i.e., from a large matching portion to a small matching portion. The number of variables in our algorithm increases with the number of atoms of protein pairs in almost a linear manner. In addition to solid theoretical background, numerical experiments demonstrated significant improvement of our approach over the existing methods in terms of quality and efficiency. In particular, we show that divergent evolution, circular permutations and active-sites (or structural motifs) can be identified by our method. The software SAMO is available upon request from the authors, or from http://zhangroup.aporc.org/bioinfo/samo/ and http://intelligent.eic.osaka-sandai.ac.jp/chenen/samo.htm. A novel formulation is proposed to accurately align protein structures in the framework of multi-objective optimization, based on a sequence order-independent strategy. A fast and accurate algorithm based on the bipartite matching algorithm is developed by exploiting the special features. Convergence of computation is shown in experiments and is also theoretically proven.

  15. An RNAi Screen To Identify Protein Phosphatases That Function Within the Drosophila Circadian Clock.

    PubMed

    Agrawal, Parul; Hardin, Paul E

    2016-12-07

    Circadian clocks in eukaryotes keep time via cell-autonomous transcriptional feedback loops. A well-characterized example of such a transcriptional feedback loop is in Drosophila, where CLOCK-CYCLE (CLK-CYC) complexes activate transcription of period (per) and timeless (tim) genes, rising levels of PER-TIM complexes feed-back to repress CLK-CYC activity, and degradation of PER and TIM permits the next cycle of CLK-CYC transcription. The timing of CLK-CYC activation and PER-TIM repression is regulated posttranslationally, in part through rhythmic phosphorylation of CLK, PER, and TIM. Previous behavioral screens identified several kinases that control CLK, PER, and TIM levels, subcellular localization, and/or activity, but two phosphatases that function within the clock were identified through the analysis of candidate genes from other pathways or model systems. To identify phosphatases that play a role in the clock, we screened clock cell-specific RNA interference (RNAi) knockdowns of all annotated protein phosphatases and protein phosphatase regulators in Drosophila for altered activity rhythms. This screen identified 19 protein phosphatases that lengthened or shortened the circadian period by ≥1 hr (p ≤ 0.05 compared to controls) or were arrhythmic. Additional RNAi lines, transposon inserts, overexpression, and loss-of-function mutants were tested to independently confirm these RNAi phenotypes. Based on genetic validation and molecular analysis, 15 viable protein phosphatases remain for future studies. These candidates are expected to reveal novel features of the circadian timekeeping mechanism in Drosophila that are likely to be conserved in all animals including humans.

  16. An RNAi Screen To Identify Protein Phosphatases That Function Within the Drosophila Circadian Clock

    PubMed Central

    Agrawal, Parul; Hardin, Paul E.

    2016-01-01

    Circadian clocks in eukaryotes keep time via cell-autonomous transcriptional feedback loops. A well-characterized example of such a transcriptional feedback loop is in Drosophila, where CLOCK-CYCLE (CLK-CYC) complexes activate transcription of period (per) and timeless (tim) genes, rising levels of PER-TIM complexes feed-back to repress CLK-CYC activity, and degradation of PER and TIM permits the next cycle of CLK-CYC transcription. The timing of CLK-CYC activation and PER-TIM repression is regulated posttranslationally, in part through rhythmic phosphorylation of CLK, PER, and TIM. Previous behavioral screens identified several kinases that control CLK, PER, and TIM levels, subcellular localization, and/or activity, but two phosphatases that function within the clock were identified through the analysis of candidate genes from other pathways or model systems. To identify phosphatases that play a role in the clock, we screened clock cell-specific RNA interference (RNAi) knockdowns of all annotated protein phosphatases and protein phosphatase regulators in Drosophila for altered activity rhythms. This screen identified 19 protein phosphatases that lengthened or shortened the circadian period by ≥1 hr (p ≤ 0.05 compared to controls) or were arrhythmic. Additional RNAi lines, transposon inserts, overexpression, and loss-of-function mutants were tested to independently confirm these RNAi phenotypes. Based on genetic validation and molecular analysis, 15 viable protein phosphatases remain for future studies. These candidates are expected to reveal novel features of the circadian timekeeping mechanism in Drosophila that are likely to be conserved in all animals including humans. PMID:27784754

  17. Identifying Key Proteins in Hg Methylation Pathways of Desulfovibrio by Global Proteomics, Final Technical Report

    SciTech Connect

    Summers, Anne O.; Miller, Susan M.; Wall, Judy; Lipton, Mary

    2016-06-18

    Elemental mercury, Hg(0) is a contaminant at many DOE sites, especially at Oak Ridge National Laboratory (ORNL) where the spread of spilled Hg and its effects on microbial populations have been monitored for decades. To explore the microbial interactions with Hg, we have devised a global proteomic approach capable of directly detecting Hg-adducts of proteins. This technique developed in the facultative anaerobe, Escherichia coli, allows us to identify the proteins most vulnerable to acute exposure to organomercurials phenyl- and ethyl-mercury (as surrogates for the highly neurotoxic methyl-Hg) (Polacco, et al, 2011). We have found >300 such proteins in all metabolic functional groups and cellular compartments; most are highly conserved and can serve as markers for acute Hg exposure (Zink, et al. 2016, in preparation). We have also discovered that acute Hg exposure severely disrupts thiol, iron and redox homeostases, and electrolyte balance (LaVoie, et al., 2015) Thus, we proposed to bring these techniques to bear on the central problem of identifying the cellular proteins involved in bacterial uptake and methylation of mercury and its release from the cell.

  18. Identify High-Quality Protein Structural Models by Enhanced K-Means.

    PubMed

    Wu, Hongjie; Li, Haiou; Jiang, Min; Chen, Cheng; Lv, Qiang; Wu, Chuang

    2017-01-01

    Background. One critical issue in protein three-dimensional structure prediction using either ab initio or comparative modeling involves identification of high-quality protein structural models from generated decoys. Currently, clustering algorithms are widely used to identify near-native models; however, their performance is dependent upon different conformational decoys, and, for some algorithms, the accuracy declines when the decoy population increases. Results. Here, we proposed two enhanced K-means clustering algorithms capable of robustly identifying high-quality protein structural models. The first one employs the clustering algorithm SPICKER to determine the initial centroids for basic K-means clustering (SK-means), whereas the other employs squared distance to optimize the initial centroids (K-means++). Our results showed that SK-means and K-means++ were more robust as compared with SPICKER alone, detecting 33 (59%) and 42 (75%) of 56 targets, respectively, with template modeling scores better than or equal to those of SPICKER. Conclusions. We observed that the classic K-means algorithm showed a similar performance to that of SPICKER, which is a widely used algorithm for protein-structure identification. Both SK-means and K-means++ demonstrated substantial improvements relative to results from SPICKER and classical K-means.

  19. Identify High-Quality Protein Structural Models by Enhanced K-Means

    PubMed Central

    Li, Haiou; Chen, Cheng; Lv, Qiang; Wu, Chuang

    2017-01-01

    Background. One critical issue in protein three-dimensional structure prediction using either ab initio or comparative modeling involves identification of high-quality protein structural models from generated decoys. Currently, clustering algorithms are widely used to identify near-native models; however, their performance is dependent upon different conformational decoys, and, for some algorithms, the accuracy declines when the decoy population increases. Results. Here, we proposed two enhanced K-means clustering algorithms capable of robustly identifying high-quality protein structural models. The first one employs the clustering algorithm SPICKER to determine the initial centroids for basic K-means clustering (SK-means), whereas the other employs squared distance to optimize the initial centroids (K-means++). Our results showed that SK-means and K-means++ were more robust as compared with SPICKER alone, detecting 33 (59%) and 42 (75%) of 56 targets, respectively, with template modeling scores better than or equal to those of SPICKER. Conclusions. We observed that the classic K-means algorithm showed a similar performance to that of SPICKER, which is a widely used algorithm for protein-structure identification. Both SK-means and K-means++ demonstrated substantial improvements relative to results from SPICKER and classical K-means. PMID:28421198

  20. Effectively identifying compound-protein interactions by learning from positive and unlabeled examples.

    PubMed

    Cheng, Zhanzhan; Zhou, Shuigeng; Wang, Yang; Liu, Hui; Guan, Jihong; Chen, Yi-Ping Phoebe

    2016-05-18

    Prediction of compound-protein interactions (CPIs) is to find new compound-protein pairs where a protein is targeted by at least a compound, which is a crucial step in new drug design. Currently, a number of machine learning based methods have been developed to predict new CPIs in the literature. However, as there is not yet any publicly available set of validated negative CPIs, most existing machine learning based approaches use the unknown interactions (not validated CPIs) selected randomly as the negative examples to train classifiers for predicting new CPIs. Obviously, this is not quite reasonable and unavoidably impacts the CPI prediction performance. In this paper, we simply take the unknown CPIs as unlabeled examples, and propose a new method called PUCPI (the abbreviation of PU learning for Compound-Protein Interaction identification) that employs biased-SVM (Support Vector Machine) to predict CPIs using only positive and unlabeled examples. PU learning is a class of learning methods that leans from positive and unlabeled (PU) samples. To the best of our knowledge, this is the first work that identifies CPIs using only positive and unlabeled examples. We first collect known CPIs as positive examples and then randomly select compound-protein pairs not in the positive set as unlabeled examples. For each CPI/compound-protein pair, we extract protein domains as protein features and compound substructures as chemical features, then take the tensor product of the corresponding compound features and protein features as the feature vector of the CPI/compound-protein pair. After that, biased-SVM is employed to train classifiers on different datasets of CPIs and compound-protein pairs. Experiments over various datasets show that our method outperforms six typical classifiers, including random forest, L1- and L2-regularized logistic regression, naive Bayes, SVM and k-nearest neighbor (kNN), and three types of existing CPI prediction models. Source code, datasets and

  1. Application of M13 phage display for identifying immunogenic proteins from tick (Ixodes scapularis) saliva.

    PubMed

    Becker, Martin; Felsberger, André; Frenzel, André; Shattuck, Wendy M C; Dyer, Megan; Kügler, Jonas; Zantow, Jonas; Mather, Thomas N; Hust, Michael

    2015-05-30

    Ticks act as vectors for a large number of different pathogens, perhaps most notably Borrelia burgdorferi, the causative agent of Lyme disease. The most prominent tick vector in the United States is the blacklegged tick, Ixodes scapularis. Tick bites are of special public health concern since there are no vaccines available against most tick-transmitted pathogens. Based on the observation that certain non-natural host animals such as guinea pigs or humans can develop adaptive immune responses to tick bites, anti-tick vaccination is a potential approach to tackle health risks associated with tick bites. The aim of this study was to use an oligopeptide phage display strategy to identify immunogenic salivary gland proteins from I. scapularis that are recognized by human immune sera. Oligopeptide libraries were generated from salivary gland mRNA of 18 h fed nymphal I. scapularis. Eight immunogenic oligopeptides were selected using human immune sera. Three selected immunogenic oligopeptides were cloned and produced as recombinant proteins. The immunogenic character of an identified metalloprotease (MP1) was validated with human sera. This enzyme has been described previously and was hypothesized as immunogenic which was confirmed in this study. Interestingly, it also has close homologs in other Ixodes species. An immunogenic protein of I. scapularis was identified by oligopeptide phage display. MP1 is a potential candidate for vaccine development.

  2. Proteomics approach to identify unique xylem sap proteins in Pierce's disease-tolerant Vitis species.

    PubMed

    Basha, Sheikh M; Mazhar, Hifza; Vasanthaiah, Hemanth K N

    2010-03-01

    Pierce's disease (PD) is a destructive bacterial disease of grapes caused by Xylella fastidiosa which is xylem-confined. The tolerance level to this disease varies among Vitis species. Our research was aimed at identifying unique xylem sap proteins present in PD-tolerant Vitis species. The results showed wide variation in the xylem sap protein composition, where a set of polypeptides with pI between 4.5 and 4.7 and M(r) of 31 kDa were present in abundant amount in muscadine (Vitis rotundifolia, PD-tolerant), in reduced levels in Florida hybrid bunch (Vitis spp., PD-tolerant) and absent in bunch grapes (Vitis vinifera, PD-susceptible). Liquid chromatography/mass spectrometry/mass spectrometry analysis of these proteins revealed their similarity to beta-1, 3-glucanase, peroxidase, and a subunit of oxygen-evolving enhancer protein 1, which are known to play role in defense and oxygen generation. In addition, the amount of free amino acids and soluble sugars was found to be significantly lower in xylem sap of muscadine genotypes compared to V. vinifera genotypes, indicating that the higher nutritional value of bunch grape sap may be more suitable for Xylella growth. These data suggest that the presence of these unique proteins in xylem sap is vital for PD tolerance in muscadine and Florida hybrid bunch grapes.

  3. Identifying technical aliases in SELDI mass spectra of complex mixtures of proteins

    PubMed Central

    2013-01-01

    Background Biomarker discovery datasets created using mass spectrum protein profiling of complex mixtures of proteins contain many peaks that represent the same protein with different charge states. Correlated variables such as these can confound the statistical analyses of proteomic data. Previously we developed an algorithm that clustered mass spectrum peaks that were biologically or technically correlated. Here we demonstrate an algorithm that clusters correlated technical aliases only. Results In this paper, we propose a preprocessing algorithm that can be used for grouping technical aliases in mass spectrometry protein profiling data. The stringency of the variance allowed for clustering is customizable, thereby affecting the number of peaks that are clustered. Subsequent analysis of the clusters, instead of individual peaks, helps reduce difficulties associated with technically-correlated data, and can aid more efficient biomarker identification. Conclusions This software can be used to pre-process and thereby decrease the complexity of protein profiling proteomics data, thus simplifying the subsequent analysis of biomarkers by decreasing the number of tests. The software is also a practical tool for identifying which features to investigate further by purification, identification and confirmation. PMID:24010718

  4. Host Protein Biomarkers Identify Active Tuberculosis in HIV Uninfected and Co-infected Individuals

    PubMed Central

    Achkar, Jacqueline M.; Cortes, Laetitia; Croteau, Pascal; Yanofsky, Corey; Mentinova, Marija; Rajotte, Isabelle; Schirm, Michael; Zhou, Yiyong; Junqueira-Kipnis, Ana Paula; Kasprowicz, Victoria O.; Larsen, Michelle; Allard, René; Hunter, Joanna; Paramithiotis, Eustache

    2015-01-01

    Biomarkers for active tuberculosis (TB) are urgently needed to improve rapid TB diagnosis. The objective of this study was to identify serum protein expression changes associated with TB but not latent Mycobacterium tuberculosis infection (LTBI), uninfected states, or respiratory diseases other than TB (ORD). Serum samples from 209 HIV uninfected (HIV−) and co-infected (HIV+) individuals were studied. In the discovery phase samples were analyzed via liquid chromatography and mass spectrometry, and in the verification phase biologically independent samples were analyzed via a multiplex multiple reaction monitoring mass spectrometry (MRM-MS) assay. Compared to LTBI and ORD, host proteins were significantly differentially expressed in TB, and involved in the immune response, tissue repair, and lipid metabolism. Biomarker panels whose composition differed according to HIV status, and consisted of 8 host proteins in HIV− individuals (CD14, SEPP1, SELL, TNXB, LUM, PEPD, QSOX1, COMP, APOC1), or 10 host proteins in HIV+ individuals (CD14, SEPP1, PGLYRP2, PFN1, VASN, CPN2, TAGLN2, IGFBP6), respectively, distinguished TB from ORD with excellent accuracy (AUC = 0.96 for HIV− TB, 0.95 for HIV+ TB). These results warrant validation in larger studies but provide promise that host protein biomarkers could be the basis for a rapid, blood-based test for TB. PMID:26501113

  5. Potential serodiagnostic markers for Q fever identified in Coxiella burnetii by immunoproteomic and protein microarray approaches

    PubMed Central

    2012-01-01

    Background Coxiella burnetii is the etiological agent of Q fever. The clinical diagnosis of Q fever is mainly based on several serological tests. These tests all need Coxiella organisms which are difficult and hazardous to culture and purify. Results An immunoproteomic study of C. burnetii Xinqiao strain isolated in China was conducted with the sera from experimentally infected BALB/c mice and Q fever patients. Twenty of whole proteins of Xinqiao recognized by the infection sera were identified by mass spectrometry. Nineteen of the 20 proteins were successfully expressed in Escherichia coli and used to fabricate a microarray which was probed with Q fever patient sera. As a result, GroEL, YbgF, RplL, Mip, OmpH, Com1, and Dnak were recognized as major seroreactive antigens. The major seroreactive proteins were fabricated in a small microarray and further analyzed with the sera of patients with rickettsial spotted fever, Legionella pneumonia or streptococcal pneumonia. In this analysis, these proteins showed fewer cross-reactions with the tested sera. Conclusions Our results demonstrate that these 7 Coxiella proteins gave a modest sensitivity and specificity for recognizing of Q fever patient sera, suggesting that they are potential serodiagnostic markers for Q fever. PMID:22420424

  6. Eggshell Porosity Provides Insight on Evolution of Nesting in Dinosaurs.

    PubMed

    Tanaka, Kohei; Zelenitsky, Darla K; Therrien, François

    2015-01-01

    Knowledge about the types of nests built by dinosaurs can provide insight into the evolution of nesting and reproductive behaviors among archosaurs. However, the low preservation potential of their nesting materials and nesting structures means that most information can only be gleaned indirectly through comparison with extant archosaurs. Two general nest types are recognized among living archosaurs: 1) covered nests, in which eggs are incubated while fully covered by nesting material (as in crocodylians and megapodes), and 2) open nests, in which eggs are exposed in the nest and brooded (as in most birds). Previously, dinosaur nest types had been inferred by estimating the water vapor conductance (i.e., diffusive capacity) of their eggs, based on the premise that high conductance corresponds to covered nests and low conductance to open nests. However, a lack of statistical rigor and inconsistencies in this method render its application problematic and its validity questionable. As an alternative we propose a statistically rigorous approach to infer nest type based on large datasets of eggshell porosity and egg mass compiled for over 120 extant archosaur species and 29 archosaur extinct taxa/ootaxa. The presence of a strong correlation between eggshell porosity and nest type among extant archosaurs indicates that eggshell porosity can be used as a proxy for nest type, and thus discriminant analyses can help predict nest type in extinct taxa. Our results suggest that: 1) covered nests are likely the primitive condition for dinosaurs (and probably archosaurs), and 2) open nests first evolved among non-avian theropods more derived than Lourinhanosaurus and were likely widespread in non-avian maniraptorans, well before the appearance of birds. Although taphonomic evidence suggests that basal open nesters (i.e., oviraptorosaurs and troodontids) were potentially the first dinosaurs to brood their clutches, they still partially buried their eggs in sediment. Open nests

  7. Eggshell Porosity Provides Insight on Evolution of Nesting in Dinosaurs

    PubMed Central

    2015-01-01

    Knowledge about the types of nests built by dinosaurs can provide insight into the evolution of nesting and reproductive behaviors among archosaurs. However, the low preservation potential of their nesting materials and nesting structures means that most information can only be gleaned indirectly through comparison with extant archosaurs. Two general nest types are recognized among living archosaurs: 1) covered nests, in which eggs are incubated while fully covered by nesting material (as in crocodylians and megapodes), and 2) open nests, in which eggs are exposed in the nest and brooded (as in most birds). Previously, dinosaur nest types had been inferred by estimating the water vapor conductance (i.e., diffusive capacity) of their eggs, based on the premise that high conductance corresponds to covered nests and low conductance to open nests. However, a lack of statistical rigor and inconsistencies in this method render its application problematic and its validity questionable. As an alternative we propose a statistically rigorous approach to infer nest type based on large datasets of eggshell porosity and egg mass compiled for over 120 extant archosaur species and 29 archosaur extinct taxa/ootaxa. The presence of a strong correlation between eggshell porosity and nest type among extant archosaurs indicates that eggshell porosity can be used as a proxy for nest type, and thus discriminant analyses can help predict nest type in extinct taxa. Our results suggest that: 1) covered nests are likely the primitive condition for dinosaurs (and probably archosaurs), and 2) open nests first evolved among non-avian theropods more derived than Lourinhanosaurus and were likely widespread in non-avian maniraptorans, well before the appearance of birds. Although taphonomic evidence suggests that basal open nesters (i.e., oviraptorosaurs and troodontids) were potentially the first dinosaurs to brood their clutches, they still partially buried their eggs in sediment. Open nests

  8. Comparative Proteomics Identifies Host Immune System Proteins Affected by Infection with Mycobacterium bovis

    PubMed Central

    López, Vladimir; Villar, Margarita; Queirós, João; Vicente, Joaquín; Mateos-Hernández, Lourdes; Díez-Delgado, Iratxe; Contreras, Marinela; Alves, Paulo C.; Alberdi, Pilar; Gortázar, Christian; de la Fuente, José

    2016-01-01

    Mycobacteria of the Mycobacterium tuberculosis complex (MTBC) greatly impact human and animal health worldwide. The mycobacterial life cycle is complex, and the mechanisms resulting in pathogen infection and survival in host cells are not fully understood. Eurasian wild boar (Sus scrofa) are natural reservoir hosts for MTBC and a model for mycobacterial infection and tuberculosis (TB). In the wild boar TB model, mycobacterial infection affects the expression of innate and adaptive immune response genes in mandibular lymph nodes and oropharyngeal tonsils, and biomarkers have been proposed as correlates with resistance to natural infection. However, the mechanisms used by mycobacteria to manipulate host immune response are not fully characterized. Our hypothesis is that the immune system proteins under-represented in infected animals, when compared to uninfected controls, are used by mycobacteria to guarantee pathogen infection and transmission. To address this hypothesis, a comparative proteomics approach was used to compare host response between uninfected (TB-) and M. bovis-infected young (TB+) and adult animals with different infection status [TB lesions localized in the head (TB+) or affecting multiple organs (TB++)]. The results identified host immune system proteins that play an important role in host response to mycobacteria. Calcium binding protein A9, Heme peroxidase, Lactotransferrin, Cathelicidin and Peptidoglycan-recognition protein were under-represented in TB+ animals when compared to uninfected TB- controls, but protein levels were higher as infection progressed in TB++ animals when compared to TB- and/or TB+ adult wild boar. MHCI was the only protein over-represented in TB+ adult wild boar when compared to uninfected TB- controls. The results reported here suggest that M. bovis manipulates host immune response by reducing the production of immune system proteins. However, as infection progresses, wild boar immune response recovers to limit pathogen

  9. Comparative Proteomics Identifies Host Immune System Proteins Affected by Infection with Mycobacterium bovis.

    PubMed

    López, Vladimir; Villar, Margarita; Queirós, João; Vicente, Joaquín; Mateos-Hernández, Lourdes; Díez-Delgado, Iratxe; Contreras, Marinela; Alves, Paulo C; Alberdi, Pilar; Gortázar, Christian; de la Fuente, José

    2016-03-01

    Mycobacteria of the Mycobacterium tuberculosis complex (MTBC) greatly impact human and animal health worldwide. The mycobacterial life cycle is complex, and the mechanisms resulting in pathogen infection and survival in host cells are not fully understood. Eurasian wild boar (Sus scrofa) are natural reservoir hosts for MTBC and a model for mycobacterial infection and tuberculosis (TB). In the wild boar TB model, mycobacterial infection affects the expression of innate and adaptive immune response genes in mandibular lymph nodes and oropharyngeal tonsils, and biomarkers have been proposed as correlates with resistance to natural infection. However, the mechanisms used by mycobacteria to manipulate host immune response are not fully characterized. Our hypothesis is that the immune system proteins under-represented in infected animals, when compared to uninfected controls, are used by mycobacteria to guarantee pathogen infection and transmission. To address this hypothesis, a comparative proteomics approach was used to compare host response between uninfected (TB-) and M. bovis-infected young (TB+) and adult animals with different infection status [TB lesions localized in the head (TB+) or affecting multiple organs (TB++)]. The results identified host immune system proteins that play an important role in host response to mycobacteria. Calcium binding protein A9, Heme peroxidase, Lactotransferrin, Cathelicidin and Peptidoglycan-recognition protein were under-represented in TB+ animals when compared to uninfected TB- controls, but protein levels were higher as infection progressed in TB++ animals when compared to TB- and/or TB+ adult wild boar. MHCI was the only protein over-represented in TB+ adult wild boar when compared to uninfected TB- controls. The results reported here suggest that M. bovis manipulates host immune response by reducing the production of immune system proteins. However, as infection progresses, wild boar immune response recovers to limit pathogen

  10. A Phenotypic High-Content Screening Assay to Identify Regulators of Membrane Protein Localization.

    PubMed

    Smith, Lorey K; Thomas, Daniel W; Simpson, Kaylene J; Humbert, Patrick O

    2016-10-01

    Correct subcellular localization of proteins is a requirement for appropriate function. This is especially true in epithelial cells, which rely on the precise localization of a diverse array of epithelial polarity and cellular adhesion proteins. Loss of cell polarity and adhesion is a hallmark of cancer, and mislocalization of core polarity proteins, such as Scribble, is observed in a range of human epithelial tumors and is prognostic of poor survival. Despite this, little is known about how Scribble membrane localization is regulated. Here, we describe the development and application of a phenotypic high-content screening assay that is designed to specifically quantify membrane levels of Scribble to identify regulators of its membrane localization. A screening platform that is capable of resolving individual cells and quantifying membrane protein localization in confluent epithelial monolayers was developed by using the cytoplasm-to-cell-membrane bioapplication integrated with the Cellomics ArrayScan high-content imaging platform. Application of this method to a boutique human epithelial polarity and signaling small interfering RNA (siRNA) library resulted in highly robust coefficient-of-variance and Z' factor values. As proof of concept, we present two candidate genes whose depletion specifically reduces Scribble protein levels at the membrane. Data mining revealed that these proteins interact with components of the Scribble polarity complex, providing support for the utility of the screening approach. This method is broadly applicable to genome-wide and large-scale compound screening of membrane-bound proteins, and when coupled with pathway analysis the dataset becomes even more valuable and can provide predictive mechanistic insight.

  11. UV, VISIBLE AND NIR SPECTRAL ANALYSIS OF EGGSHELLS IN THE CHARADRIIDAE FAMILY OF BIRDS

    EPA Science Inventory

    We employed reflectance spectrophotometry to quantify color and mineral composition of eggshells from several species of the bird family Charadriidae to characterize species physiology and to distinguish nesting habitat preferences. We used a Shimadzu spectrophotometer to measur...

  12. UV, VISIBLE AND NIR SPECTRAL ANALYSIS OF EGGSHELLS IN THE CHARADRIIDAE FAMILY OF BIRDS

    EPA Science Inventory

    We employed reflectance spectrophotometry to quantify color and mineral composition of eggshells from several species of the bird family Charadriidae to characterize species physiology and to distinguish nesting habitat preferences. We used a Shimadzu spectrophotometer to measur...

  13. Filling the gaps of dinosaur eggshell phylogeny: Late Jurassic Theropod clutch with embryos from Portugal

    PubMed Central

    Araújo, Ricardo; Castanhinha, Rui; Martins, Rui M. S.; Mateus, Octávio; Hendrickx, Christophe; Beckmann, F.; Schell, N.; Alves, L. C.

    2013-01-01

    The non-avian saurischians that have associated eggshells and embryos are represented only by the sauropodomorph Massospondylus and Coelurosauria (derived theropods), thus missing the basal theropod representatives. We report a dinosaur clutch containing several crushed eggs and embryonic material ascribed to the megalosaurid theropod Torvosaurus. It represents the first associated eggshells and embryos of megalosauroids, thus filling an important phylogenetic gap between two distantly related groups of saurischians. These fossils represent the only unequivocal basal theropod embryos found to date. The assemblage was found in early Tithonian fluvial overbank deposits of the Lourinhã Formation in West Portugal. The morphological, microstructural and chemical characterization results of the eggshell fragments indicate very mild diagenesis. Furthermore, these fossils allow unambiguous association of basal theropod osteology with a specific and unique new eggshell morphology. PMID:23722524

  14. Ultrastructural features and elemental distribution in eggshell during pre and post hatching periods in the green turtle, Chelonia mydas at Ras Al-Hadd, Oman.

    PubMed

    Al-Bahry, S N; Mahmoud, I Y; Al-Amri, I S; Ba-Omar, T A; Melgheit, K O; Al-Kindi, A Y

    2009-06-01

    Eggshells were randomly collected from turtle nests immediately after oviposition and at the end of incubation to examine the ultrastructural features using scanning JSM-5600LV microscopy. Three layers were recognized; an outer calcareous, a middle multistrata and an inner membrane. The calcareous layer had loose nodular units varying in shape and size without interlocking attachments. In freshly laid eggs, each nodular unit had spicules arranged in folded stacks. The spicules became unfolded during incubation, to form radiating configurations. Elemental composition and mapping of the layers were analyzed using energy dispersive spectroscopy (EDS). The elements were unevenly distributed throughout the eggshell and Ca(2+) decreased significantly after hatching. X-ray diffraction was used to identify the crystals of the eggshells. It revealed that nodular units of the calcareous were made up of CaCO(3), as aragonite (91%), calcite (6%) and vaterite (3%). The middle layer consisted of organic amorphous material with aragonite (89%) and calcite (11%). The shell membrane consisted of reticular fibers with crystals predominantly of NaCl halite. Thermogravimetry analysis of the calcareous layer indicated a complete evaporation of bonded H(2)O at 480 degrees C and CO(2) at 830 degrees C. Using the differential thermal analysis (DTA), aragonite was transformed to stable calcite at 425 degrees C.

  15. Identifying biological concepts from a protein-related corpus with a probabilistic topic model.

    PubMed

    Zheng, Bin; McLean, David C; Lu, Xinghua

    2006-02-08

    Biomedical literature, e.g., MEDLINE, contains a wealth of knowledge regarding functions of proteins. Major recurring biological concepts within such text corpora represent the domains of this body of knowledge. The goal of this research is to identify the major biological topics/concepts from a corpus of protein-related MEDLINE titles and abstracts by applying a probabilistic topic model. The latent Dirichlet allocation (LDA) model was applied to the corpus. Based on the Bayesian model selection, 300 major topics were extracted from the corpus. The majority of identified topics/concepts was found to be semantically coherent and most represented biological objects or concepts. The identified topics/concepts were further mapped to the controlled vocabulary of the Gene Ontology (GO) terms based on mutual information. The major and recurring biological concepts within a collection of MEDLINE documents can be extracted by the LDA model. The identified topics/concepts provide parsimonious and semantically-enriched representation of the texts in a semantic space with reduced dimensionality and can be used to index text.

  16. Identifying biological concepts from a protein-related corpus with a probabilistic topic model

    PubMed Central

    Zheng, Bin; McLean, David C; Lu, Xinghua

    2006-01-01

    Background Biomedical literature, e.g., MEDLINE, contains a wealth of knowledge regarding functions of proteins. Major recurring biological concepts within such text corpora represent the domains of this body of knowledge. The goal of this research is to identify the major biological topics/concepts from a corpus of protein-related MEDLINE© titles and abstracts by applying a probabilistic topic model. Results The latent Dirichlet allocation (LDA) model was applied to the corpus. Based on the Bayesian model selection, 300 major topics were extracted from the corpus. The majority of identified topics/concepts was found to be semantically coherent and most represented biological objects or concepts. The identified topics/concepts were further mapped to the controlled vocabulary of the Gene Ontology (GO) terms based on mutual information. Conclusion The major and recurring biological concepts within a collection of MEDLINE documents can be extracted by the LDA model. The identified topics/concepts provide parsimonious and semantically-enriched representation of the texts in a semantic space with reduced dimensionality and can be used to index text. PMID:16466569

  17. The yeast signal sequence trap identifies secreted proteins of the hemibiotrophic corn pathogen Colletotrichum graminicola.

    PubMed

    Krijger, Jorrit-Jan; Horbach, Ralf; Behr, Michael; Schweizer, Patrick; Deising, Holger B; Wirsel, Stefan G R

    2008-10-01

    The hemibiotroph Colletotrichum graminicola is the causal agent of stem rot and leaf anthracnose on Zea mays. Following penetration of epidermal cells, the fungus enters a short biotrophic phase, followed by a destructive necrotrophic phase of pathogenesis. During both phases, secreted fungal proteins are supposed to determine progress and success of the infection. To identify genes encoding such proteins, we constructed a yeast signal sequence trap (YSST) cDNA-library from RNA extracted from mycelium grown in vitro on corn cell walls and leaf extract. Of the 103 identified unigenes, 50 showed significant similarities to genes with a reported function, 25 sequences were similar to genes without a known function, and 28 sequences showed no similarity to entries in the databases. Macroarray hybridization and quantitative reverse-transcriptase polymerase chain reaction confirmed that most genes identified by the YSST screen are expressed in planta. Other than some genes that were constantly expressed, a larger set showed peaks of transcript abundances at specific phases of pathogenesis. Another set exhibited biphasic expression with peaks at the biotrophic and necrotrophic phase. Transcript analyses of in vitro-grown cultures revealed that several of the genes identified by the YSST screen were induced by the addition of corn leaf components, indicating that host-derived factors may have mimicked the host milieu.

  18. Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

    PubMed Central

    Laventie, Benoît-Joseph; Nesper, Jutta; Ahrné, Erik; Glatter, Timo; Schmidt, Alexander; Jenal, Urs

    2015-01-01

    Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail. We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling. PMID:25867682

  19. A Genome-Wide Association Study Identifies Protein Quantitative Trait Loci (pQTLs)

    PubMed Central

    Corsi, Anna-Maria; Stevens, Kara; Rafferty, Ian; Lauretani, Fulvio; Murray, Anna; Gibbs, J. Raphael; Paolisso, Giuseppe; Rafiq, Sajjad; Simon-Sanchez, Javier; Lango, Hana; Scholz, Sonja; Weedon, Michael N.; Arepalli, Sampath; Rice, Neil; Washecka, Nicole; Hurst, Alison; Britton, Angela; Henley, William; van de Leemput, Joyce; Li, Rongling; Newman, Anne B.; Tranah, Greg; Harris, Tamara; Panicker, Vijay; Dayan, Colin; Bennett, Amanda; McCarthy, Mark I.; Ruokonen, Aimo; Jarvelin, Marjo-Riitta; Guralnik, Jack; Bandinelli, Stefania; Frayling, Timothy M.; Singleton, Andrew; Ferrucci, Luigi

    2008-01-01

    There is considerable evidence that human genetic variation influences gene expression. Genome-wide studies have revealed that mRNA levels are associated with genetic variation in or close to the gene coding for those mRNA transcripts – cis effects, and elsewhere in the genome – trans effects. The role of genetic variation in determining protein levels has not been systematically assessed. Using a genome-wide association approach we show that common genetic variation influences levels of clinically relevant proteins in human serum and plasma. We evaluated the role of 496,032 polymorphisms on levels of 42 proteins measured in 1200 fasting individuals from the population based InCHIANTI study. Proteins included insulin, several interleukins, adipokines, chemokines, and liver function markers that are implicated in many common diseases including metabolic, inflammatory, and infectious conditions. We identified eight Cis effects, including variants in or near the IL6R (p = 1.8×10−57), CCL4L1 (p = 3.9×10−21), IL18 (p = 6.8×10−13), LPA (p = 4.4×10−10), GGT1 (p = 1.5×10−7), SHBG (p = 3.1×10−7), CRP (p = 6.4×10−6) and IL1RN (p = 7.3×10−6) genes, all associated with their respective protein products with effect sizes ranging from 0.19 to 0.69 standard deviations per allele. Mechanisms implicated include altered rates of cleavage of bound to unbound soluble receptor (IL6R), altered secretion rates of different sized proteins (LPA), variation in gene copy number (CCL4L1) and altered transcription (GGT1). We identified one novel trans effect that was an association between ABO blood group and tumour necrosis factor alpha (TNF-alpha) levels (p = 6.8×10−40), but this finding was not present when TNF-alpha was measured using a different assay , or in a second study, suggesting an assay-specific association. Our results show that protein levels share some of the features of the genetics of gene expression. These

  20. Estimating the purebred-crossbred genetic correlation for uniformity of eggshell color in laying hens.

    PubMed

    Mulder, Han A; Visscher, Jeroen; Fablet, Julien

    2016-05-05

    Uniformity of eggs is an important aspect for retailers because consumers prefer homogeneous products. One of these characteristics is the color of the eggshell, especially for brown eggs. Existence of a genetic component in environmental variance would enable selection for uniformity of eggshell color. Therefore, the objective of this study was to quantify the genetic variance in environmental variance of eggshell color in purebred and crossbred laying hens, to estimate the genetic correlation between environmental variance of eggshell color in purebred and crossbred laying hens and to estimate genetic correlations between environmental variance at different times of the laying period. We analyzed 167,651 and 79,345 eggshell color records of purebred and crossbred laying hens, respectively. The purebred and crossbred laying hens originated mostly from the same sires. Since eggshell color records of crossbred laying hens were collected per cage, these records could be related only to cage and sire family. A double hierarchical generalized linear sire model was used to estimate the genetic variance of the mean of eggshell color and its environmental variance. Approximate standard errors for heritability and the genetic coefficient of variation for environmental variance were derived. The genetic variance in environmental variance at the log scale was equal to 0.077 and 0.067, for purebred and crossbred laying hens, respectively. The genetic coefficient of variation for environmental variance was equal to 0.28 and 0.26, for purebred and crossbred laying hens, respectively. A genetic correlation of 0.70 was found between purebred and crossbred environmental variance of eggshell color, which indicates that there is some reranking of sires for environmental variance of eggshell color in purebred and crossbred laying hens. Genetic correlations between environmental variance of eggshell color in different laying periods were generally higher than 0.85, except between

  1. Development of egg-shell nano catalysts with porous hollow silica supports for hydrogenation.

    PubMed

    Xia, Zeng-Min; Chen, Jian-Feng; Li, Jian-Feng; Song, Ji-Rui; Wen, Li-Xiong

    2009-02-01

    Self-synthesized novel porous hollow silica nanoparticles (PHSNs) were applied as supports to prepare egg-shell nano catalysts for hydrogenation. By an impregnation method, different catalytic actives, such as Pd, Ag or Pt, and some promoters could be evenly loaded on the external surface, the pore channels and the internal surface of PHSNs. The prepared egg-shell catalysts were tested for CO hydrogenation and showed both improved activity and selectivity over those catalysts prepared with conventional support materials.

  2. Combined Use of Electron and Light Microscopy Techniques Reveals False Secondary Shell Units in Megaloolithidae Eggshells

    PubMed Central

    Ba