ABSTRACT: In this study, a metaproteomic approach was used for a detailed analysis of the structure of the microbial community, temporal changes and the functional role of the microbiota during pozol fermentation.
Project description:In this study, a metaproteomic approach was used for a detailed analysis of the structure of the microbial community, temporal changes and the functional role of the microbiota during pozol fermentation.
Project description:In this study, a metaproteomic approach was used for a detailed analysis of the structure of the microbial community, temporal changes and the functional role of the microbiota during pozol fermentation.
Project description:The essential myosin light chain (ELC) is part of the cardiac myosin motor regulating the contractility of the heart. Recently, we have demonstrated ELC phosphorylation to be responsible not only for basal maintenance of cardiac contractility, but also for the adaptation of sarcomere function to increased demands in adult zebrafish (Scheid et al. Cardiovascular Research, 2016). However, the direct interacting kinases, phosphatases as well as the signaling pathway mediating the ELC phosphorylation are mostly unknown. Here, we aim to identify ELC interacting proteins in zebrafish heart tissue using an ELC-specific precipitation assay followed by mass spectrometry. In brief, ELC specific co-immunoprecipitation was performed by using the Dynabeads Co-immunoprecipitation Kit (Invitrogen, #14321D). Custom-specific polyclonal ELC antibodies (Eurogentec, peptide for immunization: NH2-CAPVPETPKEPEVDLK-CONH2) against zebrafish ELC were covalently coupled to DynabeadsTM M-270 Epoxy (Invitrogen, #14302D) and whole zebrafish heart protein was used as input. All steps were carried out according to the manufacturer's instructions.
Project description:NIMA-related kinase 9 (NEK9) was identified to interact with the essential myosin light chain (ELC). To validate these findings ascorbate peroxidase (APEX) catalyzed proximity labeling (Hung et al. Nat Protoc. 2016) was performed. To do so, human ELC fused to APEX was overexpressed in HEK293 cells. Empty pcDNA3-APEX2-NES vector serves as negative control. Half of each sample was treated with H2O2, which catalyzes protein labeling in close proximity of ELC. After streptavidin pull down of labeled proteins, the samples were analyzed by mass spectrometry.
Project description:The myosin light chain protein family consists of two classes, the regulatory myosin light chains (RLC) and the essential myosin light chains (ELC). Functionally, the MLC proteins are directly involved in sarcomeric activity and force transmission contributing to cardiac contractility. Besides regulating contractility by protein-protein interactions alone, MLCs modulate force transmission through posttranslational phosphorylation. The MLC phosphorylation status in human cardiac disease is under investigation. In contrast to RLC, the phosphorylation pattern of ELC in human heart disease is not well understood. Here, 2-dimensional (2D) gel electrophoresis followed by ELC specific immunoblot (IB) was performed to detect the ELC phosphorylation pattern of human left ventricular tissue. Cardiac proteins were separated by isoelectric point (pI) and molecular weight (MW) (vELC: MW 21,932 kDa, pI 5.03; vRLC: MW 19 kDa, pI 4.89). Next, we performed mass-spectrometry analysis after cutting the spot-regions in question from silver-stained 2D gels of proteins from the human tissue. The aim was 1) to validate the detection of the MLC migration pattern seen in 2D IB by mass spectrometry, 2) to distinguish between atrial and ventricular MLC isoforms as these two forms might converge on 2D gel (vELC: MW 21.932 kDa, pI 5.03; aELC: 21.550 kDa, pI 4.98) and 3) to detect phosphorylated amio acid residues of ELC and RLC in human ventricular tissue.
Project description:The cuticles of arthropods, including aquatic crustaceans like Daphnia, provide an interface between the organism and its environment. Thus, the cuticle’s structure influences how the organism responds to and interacts with its surroundings. Here, we used label-free quantification proteomics to provide a proteome of the molted cuticle of Daphnia magna, which has long been a prominent subject of studies on ecology, evolution, and developmental biology, anddetected 278 high confidence proteins. Using protein sequence domain and functional enrichment analyses, we identified chitin-binding structural proteins and chitin modifying enzymes as most abundant protein groups in the cuticle proteome.Structural cuticular protein families showed a similar distribution to those found in other arthropods and indicated proteins responsible for the soft and flexible structure of the Daphnia cuticle . Finally, cuticle protein genes were clustered as tandem gene arrays in the Daphnia genome, indicating their importance for adaptation to environmental change. The cuticle proteome presented here will be a valuable resource to the Daphnia research community, informing investigations on diverse topics such as the genetic basis of interactions with predators and parasites.
Project description:A large body of work has demonstrated that leptospires regulate and modify gene expression in response to environmental cues, as encountered during disease transmission, including changes in temperature, osmolarity, concentration of iron, the presence of serum, and interaction with macrophages. However, since leptospires are not readily amenable to genetic manipulation, the functional and biological significance of these differentially expressed genes often remains unclear. More recently, it is been shown that proteins, in response to changing environmental conditions, can be modified further by specific post-translational modifications. Indeed, both saprophytic and pathogenic leptospires have comprehensive systems to modify proteins. The paucibacillary nature of spirochetal infections, combined with the challenges associated with acquiring pathogens free from contaminating host proteins makes the study of these bacteria in a mammalian host-adapted state inherently difficult. As an alternative approach, we developed a model in which leptospires are cultivated in a dialysis membrane chamber (DMC) implanted within the peritoneal cavity of rats, where they are exposed to the environmental cues encountered during host infection. This strategy has been successfully applied to compare the transcriptome of L. interrogans cultivated within DMC with that of leptospires grown under standard in vitro conditions. In addition to determining the relative expression levels of ‘‘core’’ housekeeping genes under both growth conditions, we identified 166 genes that were differentially-expressed by L. interrogans in response to mammalian host signals. The proteome of DMC (dialysis membrane chamber) cultivated leptospires was compared to that of in vivo derived leptospires and with leptospires cultivated in vitro at 30°C or 37°C by 2-dimensional difference in gel electrophoresis (2-D DIGE). Our analysis indicates that the abundance of leptospire-proteins is modulated the expression of a range of proteins which are differentially expressed in response to mammalian host signals, and not temperature alone . In addition, we confirm that in several proteins there is a change in the presence of modify the expression of the post-translational modifications trimethyllysine and acetyllysine in response to environmental cues encountered during persistent renal colonization in a reservoir host of infection. These results provide novel insights in the proteome level changes, including to differential protein and post-translational modifications, expressed in response to mammalian host signals which can be used to further define the unique equilibrium that exists between pathogenic leptospires and their reservoir host of infection.
Project description:Chlamydomonas wild type CC 4533 and psb28 mutant were grown in TAP medium at 25°C and ~30 µmol photons m^-2 s^-1 on a rotary shaker. Protein complexes of three wild type (wt) and three psb28 (mut) samples were separated by BN-PAGE and cut into 36 slices. Subsequent mass spectrometry enables the analysis of protein complex migration patterns of wild type and mutant complexes.
Project description:Purpose: we want to see gene expression changes during in vitro expansion of VM-derived NSCs (VM-NSCs) with cell passges in the absence or presence of Lin28a overexpression. changes upon Lin28 overexpression in P1 and P3 stages of Neural stem cells. RNA-seq, sRNA-seq, and Polysome-seq with/without Lin28 overexpression in P1 and P3 stages of Neural stem cells.
Project description:Phenotypic plasticity, the ability of one genotype to express different phenotypes in response to changing environmental conditions, is one of the most common phenomena characterising the living world and is not only relevant for the ecology but also for the evolution of species. Daphnia, the waterflea, is a textbook example for predator induced phenotypic plastic defences including changes in life-history, behaviour and morphology. However, the analysis of molecular mechanisms underlying these inducible defences is still in its early stages.<br><br>We exposed Daphnia magna to chemical cues of the predator Triops cancriformis to identify key processes underlying plastic defensive trait formation. D. magna is known to develop an array of morphological changes in the presence of T. cancriformis including changes of carapace morphology and cuticle hardening. To get a more comprehensive idea of this phenomenon, we studied four different genotypes originating from habitats with different predation history, reaching from predator-free to temporary habitats containing T. cancriformis.<br><br>We analysed the morphologies as well as proteomes of predator-exposed and control animals. Three genotypes showed morphological changes when the predator was present. Using a high-throughput proteomics approach, we found 294 proteins which were significantly altered in their abundance after predator exposure in a general or genotype dependant manner. Proteins connected to genotype dependant responses were related to the cuticle, protein synthesis and calcium binding whereas the yolk protein vitellogenin increased in abundance in all genotypes, indicating their involvement in a more general response. Furthermore, genotype dependant responses at the proteome level correlated well with local adaptation to Triops predation.<br><br>Altogether, our study provides new insights concerning genotype dependant and general molecular processes involved in predator-induced phenotypic plasticity in D. magna.