Project description:Aedes aegypti mosquito ovarian follicles develop synchronously after taking a blood meal. A single layer of follicular epithelial cells surrounding the oocyte is responsible for secreting a majority of eggshell structural components between 18 and 54 hours post blood meal. We previously identified a protein called eggshell organizing factor 1 (EOF1). When we knocked down EOF1 with RNA interference in female adult mosquitoes, they laid eggs that were not properly melanized, and fragile eggs did not contain viable embryos. Here, we aimed to determine putative downstream eggshell proteins in RNAi-EOF1 female mosquitoes. Our eggshell proteomics identified 220 proteins, suggesting that mosquito extracellular eggshells are composed of a complex mixture of proteins. These proteins are involved in the formation of an intact eggshell that protects the embryonic development and larva from the environment for long periods of time.
Project description:Here we show the potential of proteins preserved in Pleistocene eggshell for addressing a longstanding controversy in human and evolution: the identity of the extinct bird that laid the eggs which were exploited by Australia’s first inhabitants. The eggs had been originally attributed to the iconic extinct flightless Genyornis newtoni, and subsequently dated to before 50 ±5 ka by Miller et al. (2016). This was taken to represent the extinction date for this endemic megafaunal species and thus implied a role of humans in its demise. A contrasting hypothesis, according to which the eggshell was laid by a large megapode (mound-builder), would therefore acquit humans of their responsibility in the extinction of Genyornis. Ancient protein sequences were reconstructed and used to assess the evolutionary proximity of the undetermined eggshell to extant birds, rejecting the megapode hypothesis. Ancient DNA could not be retrieved from these highly degraded samples, but morphometric data supported the attribution of the eggshell to Genyornis. When used in triangulation to address well-defined hypotheses, palaeoproteomics is a precious tool for reconstructing the evolutionary history of extinct and extant species. Here we show that the identification of Genyornis eggshell implies a more nuanced understanding of the modes of interactions between humans and their environment.
Project description:Trematodes such as the liver fluke Fasciola hepatica and the rumen fluke Calicophoron daubneyi are endemic throughout Europe where they tyically infect sheep and cattle. Adult fluke shed thousands of eggs which possess a tougher outer eggshell composed of cross-linked eggshell precursor (or vitelline) proteins. Fasciola hepatica usues di-tyrosine crosslinking (typcial of most trematode species) whilst the crosslinks formed in C. daubneyi eggshells appear to be different, based on histochemical data. Here, the composition of the eggshell of both species was investigated by mass spectrometry.
Project description:Claims for exceptional preservation of biomolecules in the fossil record are contested. Here we demonstrate the role of surface stabilisation in significantly prolonging protein sequence survival to ~3.8 million years. The intracrystalline environment of calcite ostrich (Struthionidae) eggshell encapsulates uterine proteins and molecular dynamics simulations of struthiocalcin-1 & -2, the dominant proteins within the eggshell, reveal that they bind to the mineral surface in distinct domains. By ~3.8 million years the struthiocalcin-1 domain with the lowest calculated binding energy is selectively preserved in eggshell samples from equatorial Africa. Sequence survival is explained by entropy loss of the peptide and water, lowering the effective temperature of the local environment at the peptide mineral interface.
Project description:The Del-Mar 14K chip was used to interrogate differential expression of transcripts in the white isthmus (WI) compared with the adjacent magnum (Mg) and uterine (Ut) segments of the hen oviduct. Differential expression of genes common to both comparisons (WI/Mg and WI/Ut) was detected for 204 annotated proteins. Of these, 58 genes were overexpressed in both WI/Mg and WI/Ut, and are therefore considered to be the most interesting candidates for WI - specific functions. Additionally, general analysis revealed 135 clones hybridizing to overexpressed transcripts (WI/Mg + WI/Ut), and corresponding to 102 NCBI annotatated non-redundant Gallus gallus gene ID~s. This combined analysis revealed that structural proteins highly over-expressed in white isthmus were collagen X (COL10A1), Fibrillin (FBN1) and Cysteine Rich Eggshell Membrane Protein (CREMP). In addition, genes encoding collagen-processing enzymes were over-expressed, as were proteins known to regulate disulfide cross-linking, suggesting that coordinated upregulation of gene networks in the white isthmus is associated with eggshell membrane fibre formation. IPA interactome analysis reinforces the key role of the estrogen receptor and SMAD3 in mediating gene regulation during eggshell membrane synthesis. These results will assist with development of selection strategies to improve eggshell quality and food safety of the table egg. Keywords: Laying hen, eggshell, oviduct, Isthmus expression, cDNA microarray, indirect cDNA labelling, Alexa Fluor dyes Keywords: Expression profiling by array
Project description:The Del-Mar 14K chip was used to interrogate differential expression of transcripts in the white isthmus (WI) compared with the adjacent magnum (Mg) and uterine (Ut) segments of the hen oviduct. Differential expression of genes common to both comparisons (WI/Mg and WI/Ut) was detected for 204 annotated proteins. Of these, 58 genes were overexpressed in both WI/Mg and WI/Ut, and are therefore considered to be the most interesting candidates for WI - specific functions. Additionally, general analysis revealed 135 clones hybridizing to overexpressed transcripts (WI/Mg + WI/Ut), and corresponding to 102 NCBI annotatated non-redundant Gallus gallus gene ID~s. This combined analysis revealed that structural proteins highly over-expressed in white isthmus were collagen X (COL10A1), Fibrillin (FBN1) and Cysteine Rich Eggshell Membrane Protein (CREMP). In addition, genes encoding collagen-processing enzymes were over-expressed, as were proteins known to regulate disulfide cross-linking, suggesting that coordinated upregulation of gene networks in the white isthmus is associated with eggshell membrane fibre formation. IPA interactome analysis reinforces the key role of the estrogen receptor and SMAD3 in mediating gene regulation during eggshell membrane synthesis. These results will assist with development of selection strategies to improve eggshell quality and food safety of the table egg. Keywords: Laying hen, eggshell, oviduct, Isthmus expression, cDNA microarray, indirect cDNA labelling, Alexa Fluor dyes Keywords: Expression profiling by array A balanced block hybridization design (Dye switch) was used where half of the samples were labelled with AlexaM-BM-. 555 fluorescent dye and the other half with AlexaM-BM-. 647. A total of 16 microarray slides were used for hybridization to 32 samples that correspond to four tissue contrast (White isthmus versus magnum and uterus versus white isthmus).
Project description:Dry eye is a common ocular inflammatory disorder characterized by tear film instability and reduced tear production. There is increasing evidence that homeostasis of the ocular surface is impacted by the intestinal microbiome. We are interested in investigating the potential role of microbially produced small molecules in mediating the interaction between the intestinal microbiota and the ocular surface. One such molecule is butyrate, a short-chain fatty acid (SCFA) produced by certain members of the gut microbiota through fermentation of dietary fiber. We have shown that oral administration of tributyrin, a prodrug form of butyrate, is protective of the ocular surface in mice undergoing desiccating stress. To gain insight into the mechanism, we analyzed gene expression in conjunctival tissue from mice treated with either tributyrgn or vehicle control.