Project description:Mouse androgenetic haploid embryonic stem cells (mAG-haESCs) can be utilized to uncover gene functions, especially those of genes with recessive effects, and to produce semicloned mice when injected into mature oocytes. However, mouse haploid cells undergo rapid diploidization during long-term culture in vitro and subsequently lose the advantages of haploidy and the factors that drive diploidization are not well understood. In this study, we compared the small RNAs (sRNAs) of mAG-haESCs, normal ESCs and mouse round spermatids by high-throughput sequencing and identified distinct sRNA profiles. Several let-7 family members and miR-290-295 cluster miRNAs were found significantly differentially transcribed. Knockdown and overexpression experiments showed that let-7a and let-7g suppress diploidization while miR-290a facilitates diploidization. Our study revealed the unique sRNA profile of mAG-haESCs and demonstrated that let-7a overexpression can mitigate diploidization in mAG-haESCs. These findings will help us to better understand mAG-haESCs and utilize them as a tool in the future.
Project description:Particle-attached bacterioplankton and eukaryote composition of surface seawater during the spring bloom at Helgoland in the year 2018
Project description:Colorectal cancer (CRC) is one of the most common and mortal types of cancer. There is increasing evidence that some polyunsaturated fatty acids (PUFA) exercise specific inhibitory actions on cancer cells through different mechanisms, as a previous study on the effect on CRC cells of two PUFA free fatty acids (FFA), docosahexaenoic acid (DHA, 22:6n3) and arachidonic acid (ARA, 20:4n6)-FFA, shown. Here we have used the same study design and technology to investigate the actions of DHA and ARA-monoacylglycerols (MAG), and we have compared the results with the previous study of the corresponding FFA. Cell assays revealed that ARA- and DHA-MAG exercised dose- and time-dependent antiproliferative actions, with DHA-MAG acting on cancer cells more efficiently than ARA-MAG. SWATH-MS massive quantitative proteomics, validated by parallel reaction monitoring and followed by pathway analysis, revealed that DHA-MAG had a massive effect in the proteasome complex, while ARA-MAG main effect was related to DNA replication. Prostaglandin synthesis also resulted inhibited by DHA-MAG. Results clearly demonstrated the ability of MAGs to induce cell death in colon cancer cells and suggested a direct relationship between chemical structure and effect.
Project description:Colorectal cancer is one of the most common and widespread disease in the world and the third type of cancer causing a high mortality rate. There is increasing evidence that some polyunsaturated fatty acids (PUFAs) are involved in the reduction of cancer risk and progression. Recent studies showed that sn-2 monoacylglycerols (MAG) exercise specific inhibitory actions on cancer cells through different mechanisms. However, the anticancer effect of PUFA-based MAGs on colorectal cancer has yet to be assessed. In this work we investigated the actions of two PUFAs, docosahexaenoic (DHA, 22: 6n3) and arachidonic acids (ARA, 20:4n6), both as MAG, on colon cancer human (HT-29) cell line. We performed the MTT test, LDH, and caspase-3 assays, while global proteome changes were assessed by SWATH-MS quantitative proteomics followed by pathway analysis in order to find out which molecular mechanisms were being affected. It has been proven that ARA- and DHA-MAG exercises dose- and time-dependent antiproliferative actions. In all cases, DHA-MAG acts on cancer cells more efficiently than ARA-MAG. Results clearly demonstrate the ability of MAGs to induce cell death in colon cancer cells and suggest a direct relationship between their chemical structure and their potency. Therefore, sn-2 MAG are suitable candidate for the production of new functional ingredients.
Project description:In this paper we investigated the mechanism of action of Maganin-2 (Mag-2), a well-known antimicrobial peptide isolated from the African clawed frog Xenopus laevis, by functional proteomic approaches. Several proteins belonging to E. coli macromolecular membrane complexes were identified as Mag-2 putative interactors. Among these, we focused our attention on BamA a membrane protein belonging to the BAM complex responsible for the folding and insertion of nascent β‐barrel Outer Membrane Proteins (OMPs) in the outer membrane. In silico predictions by molecular modelling and in vitro fluorescence binding and Light Scattering experiments carried out using a recombinant form of BamA confirmed the formation of a stable Mag-2/BamA complex and indicated a high affinity of the peptide for BamA. The functional implications of these interactions were investigated by two alternative and complementary approaches. The number of outer membrane proteins OmpA and OmpF produced in E. coli following Mag-2 incubation was evaluated by both western blot analysis and quantitative tandem mass spectrometry in MRM scan mode. In both experiments, a gradual decrease in outer membrane protein production with time was observed because of Mag-2 treatment.
Project description:Bacteria-host interactions are dynamic processes, and understanding transcriptional responses that directly or indirectly regulate the expression of genes involved in initial infection stages would illuminate the molecular events that result in host colonization. We used oligonucleotide microarrays to monitor (in vitro) differential gene expression in group A streptococci during pharyngeal cell adherence, the first overt infection stage. We present neighbor clustering, a new computational method for further analyzing bacterial microarray data that combines two informative characteristics of bacterial genes that share common function or regulation: (1) similar gene expression profiles (i.e., co-expression); and (2) physical proximity of genes on the chromosome. This method identifies statistically significant clusters of co-expressed gene neighbors that potentially share common function or regulation by coupling statistically analyzed gene expression profiles with the chromosomal position of genes. We applied this method to our own data and to those of others, and we show that it identified a greater number of differentially expressed genes, facilitating the reconstruction of more multimeric proteins and complete metabolic pathways than would have been possible without its application. We assessed the biological significance of two identified genes by assaying deletion mutants for adherence in vitro and show that neighbor clustering indeed provides biologically relevant data. Neighbor clustering provides a more comprehensive view of the molecular responses of streptococci during pharyngeal cell adherence.