Human Tongue Fungiform Papilla Transcriptome and Proteome Reveal Sex Differences in Long Intergenic Noncoding RNA, Immune Response and Metabolism Genes [array]
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ABSTRACT: Fungiform papillae (FP) are visible protrusions on the anterior tongue surface that contain taste buds, their nerves, and capillaries, epithelial cells, stromal cells, and immune-surveilling cells. As FP are easily biopsied in a minimally invasive procedure and have been shown to regrow, we compared three different mechanical methods of FP protein extraction and found that mechanical disruption of FP under liquid nitrogen or bead beating were more efficient than mincing in terms of yield and proteomic profile. A successful protocol to extract RNA from individual FP using bead beating was employed for transcriptomic analysis by RNA sequencing and microarray. Relative to female FP, male FP proteomes and transcriptomes showed predominant expression of Y chromosome-encoded Y antigens, such as EIF1AY, DDX3Y, SMCY, NLGN4Y, UTY, and PCDH11Y, validating our findings. We further found significantly higher levels of Human Leucocyte Antigen (HLA)-associated transcripts and HLA-A protein levels in males than in females. Several long intergenic non-coding (LINC) RNAs exhibit sex-specific expression, in particular LIN02888 with unknown function is more highly expressed in female relative to male FP. Transcripts in the interferon response pathway a key element of the innate immune system's defense against pathogens exhibits differential regulation between the sexes. This indicates that FP biopsy is a minimally invasive method to obtain human tissue in order to study, for example, age-related and sex specific changes of interest, while allowing for dynamic investigation of interindividual differences over time in the context of both health and disease.
Project description:Fungiform papillae (FP) are visible protrusions on the anterior tongue surface that contain taste buds, their nerves, and capillaries, epithelial cells, stromal cells, and immune-surveilling cells. As FP are easily biopsied in a minimally invasive procedure and have been shown to regrow, we compared three different mechanical methods of FP protein extraction and found that mechanical disruption of FP under liquid nitrogen or bead beating were more efficient than mincing in terms of yield and proteomic profile. A successful protocol to extract RNA from individual FP using bead beating was employed for transcriptomic analysis by RNA sequencing and microarray. Relative to female FP, male FP proteomes and transcriptomes showed predominant expression of Y chromosome-encoded Y antigens, such as EIF1AY, DDX3Y, SMCY, NLGN4Y, UTY, and PCDH11Y, validating our findings. We further found significantly higher levels of Human Leucocyte Antigen (HLA)-associated transcripts and HLA-A protein levels in males than in females. Several long intergenic non-coding (LINC) RNAs exhibit sex-specific expression, in particular LIN02888 with unknown function is more highly expressed in female relative to male FP. Transcripts in the interferon response pathway a key element of the innate immune system’s defense against pathogens exhibits differential regulation between the sexes. This indicates that FP biopsy is a minimally invasive method to obtain human tissue in order to study, for example, age-related and sex specific changes of interest, while allowing for dynamic investigation of interindividual differences over time in the context of both health and disease
Project description:Taste buds are complex sensory organs that are embedded in the epithelium of fungiform papillae (FP) and circumvallate papillae (CV). The sweet, bitter, and umami taste are sensed by type II taste cells that expressed taste receptors (Tas1rs and Tas2rs) which coupled with taste G-protein α-gustducin. Recent studies revealed that the taste response profiles of α-gustducin-expressing cells are different between FP and CV. We applied the high-throughput single-cell RNA-sequencing combined with fluorescence-activated cell sorting (FACS) to profile the transcriptome of the α-gustducin-expressing taste cells in both fungiform and circumvallatae papillae with transgenic mice expressing green fluorescent protein (GFP).
Project description:The goal of this study was to optimize protein extraction methods to study root-associated bacteria in Arabidopsis. For this we inoculated Arabidopsis seedlings grown in agar plates with a synthetic community (SynSom) composed of four different strains (Variovorax paradoxus, Arthrobacter sp, Agrobacterium sp. and Pseudomonas sp.. Twelve days after inoculation we extracted proteins from the roots using six different protein extraction methods each in triplicates. These methods were a combination of different extraction buffers (SDS or Triton-based) and mechanical disruption methods (bead-beating, N2 grinding, glass homogenizer and freeze-thaw cycles) We found that bead-beating the roots with lysing matrix E in SDT lysis buffer yielded the highest numbers of microbial protein identification and enhanced the detection of proteins derived from gram positive bacteria.
Project description:In this project, the metaproteome of the marine bacterioplankton was analyzed to assess its respone towards an algal bloom in the southern North Sea in spring 2010. Proteins were extracted applying two different methods: (i) applying chemical cell lysis using trifluoroethanol in combination with in-solution digest and (ii) mechanical cell lysis applying bead beating, SDS-PAGE prefractionation and in-gel digest. Both samples were analyzed by nanoLC and ESI-iontrap MS. In case of the TFE lysis samples, also nanoLC-MALDI-TOF MS was applied.
Project description:The present study was to investigate if the incidence, patterns and surgical outcomes of mechanical ileus have changed in the era of minimally invasive surgery (MIS).
Project description:The goal of this study was to optimize protein extraction methods to study root-associated bacteria in maize. For this we inoculated sterile maize plants with a synthetic community composed of seven different bacteria (Ben Niu et al. PNAS 2017, vol 114, n 12). Then, we extracted proteins from maize roots using eight different protein extraction methods in triplicates. These methods were a combination of different extraction buffers (SDS or Triton-based) and mechanical disruption methods (bead-beating, N2 grinding, glass homogenizer and freeze-thaw cycles). We found that vortexing maize roots with glass beads in PBS yielded the highest numbers of microbial protein identification.
Project description:Clinical FFPE tissue proteomic analyses were performed for early lung adenocarcinomas including adenocarcinoma in-situ (AIS), minimally invasive adenocarcinoma (MIA) and lepidic predominant invasive adenocarcinoma (LPA).
2020-01-30 | PXD017334 | JPOST Repository
Project description:Impact of bead-beating intensity on gut microbiome detection by 16S sequencing
Project description:A specific discovery proteomic protocol for intervertebral discs relies on an efficient protein extraction using the bead beating homogenizing method with stainless steel grinding balls coupled with a customized lysis buffer, and followed by the protein detection with specific mass spectrometry setting.
Project description:Background: Human heart failure is characterized by global alterations in the myocardial DNA methylation profile, yet little is known about epigenetic regulation of non- coding transcripts and potential reversibility of DNA methylation with left ventricular assist device (LVAD) support. Method: High-density genome-wide mapping of myocardial DNA methylation was performed in 36 patients with end-stage heart failure at the time of LVAD implant and 8 patients at the time of LVAD explant using bead-based array platform. Transcriptomic and functional studies were performed in human induced pluripotent stem cell derived cardiomyocytes (iPSCs). Results: Etiology-specific analysis revealed 2079 differentially methylated positions (DMPs) in ischemic cardiomyopathy (ICM) and 261 DMPs in non-ischemic cardiomyopathy (NICM). 192 DMPs were common to ICM and NICM. Analysis of paired samples before and after LVAD support demonstrated reverse methylation of only 3.2% of HF-specific DMPs. Methylation-expression correlation analysis yielded several protein-coding genes that are hypomethylated and upregulated (HTRA1, FAM65A, FBXO16, EFCAB13, AKAP13, RPTOR) or hypermethylated and downregulated (TBX3) in ICM and NICM patients. A novel cardiac-specific super-enhancer lncRNA (LINC00881) is hypermethylated and downregulated in the failing human heart. LINC00881 is an upstream regulator of sarcomere and calcium channel gene expression including MYH6, CACNA1C, and RYR2. LINC00881 knockdown significantly reduced peak calcium amplitude in the beating human iPSCs. Conclusions: Failing human heart exhibits etiology-specific changes in DNA methylation including coding and non-coding regions, which are minimally reversible with mechanical unloading. Epigenetic reprogramming may be necessary to achieve transcriptional normalization and sustained clinical recovery from heart failure.