Project description:Identification of differentially expressed genes from RNA-seq data of non-embryogenic and embryogenic ortets. Selected ortets were previously cloned, thereby somatic embryogenesis rates are known for these ortets. Ortets fitting the study criteria were supplied by two agencies, namely L1 and L2. Principal Component Analysis indicated that variance between agencies were higher than the variance between embryogenesis groups within the agency. Therefore, differential analysis was conducted separately for each agency. Differential expression analysis using DESeq2 package suggested the L2 transcriptomes of zero and low embryogenesis groups were more similar compared to the high embryogenesis group. The L1 transcriptomes consisting of zero and low embryogenesis groups similarly showed overlapping clusters. Differential expression analysis was conducted on the L1 samples (low vs. zero embryogenesis) using DESeq2 R package and the identified differentially expressed genes (DEGs) was used for clustering analysis of the L2 samples. The clustering profiles suggested that expression of these DEGs in L2 samples were able to differentiate high embryogenesis from zero-low embryogenesis L2 groups.
Project description:Samples from fruit juice vesicle tissue from three lemon genotypes (Frost Lisbon, Faris "sour" and Faris "sweet") differing in fruit acidity were compared at two developmental timepoints (immature, mature). Faris lemon appears to be a graft chimera with the L2 layer derived from normal acid lemon and layer L1 from Millsweet limetta or a closely related genotype. Fruit of Faris sour and Faris sweet grew on different branches of the same tree, with sour fruit developing on branches with L1 and L2 from acid lemon.
Project description:Lysosomal acid lipase (LAL) is the key enzyme of lysosomal lipid hydrolysis, which degrades cholesteryl esters (CE), triacylglycerols (TG), diacylglycerols (DG), and retinyl esters. The role of LAL in various cellular processes has mostly been studied in LAL-deficient (Lal-/-) mice, which share phenotypical characteristics with humans suffering from LAL deficiency. In vitro, the cell-specific functions of LAL have been commonly investigated by using the LAL inhibitors Lalistat-1 (L1) and Lalistat-2 (L2). Here, we show that pharmacological LAL inhibition but not genetic loss of LAL impairs isoproterenol-stimulated lipolysis and neutral TG hydrolase (TGH) and CE hydrolase (CEH) activities in mature adipocytes, indicating that L1 and L2 inhibit other lipid hydrolases apart from LAL. Since adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are the major enzymes that degrade cytosolic TG and CE, respectively, at neutral pH, we hypothesized that L1 and L2 also inhibit ATGL and/or HSL through off-target effects. In fact, both inhibitors drastically reduced neutral CEH activity in cells overexpressing mouse and human HSL and neutral TGH activity in cells overexpressing mouse and human ATGL, albeit to a lesser extent. By performing serine hydrolase-specific activity-based labeling in combination with quantitative proteomics, we confirmed that L2 inhibits HSL and other lipid hydrolases, whereas L1 treatment results in less pronounced inhibition of neutral lipid hydrolases. These results demonstrate that commonly used concentrations of L2 (and L1) are not suitable for investigating the role of LAL-specific lipolysis in lysosomal function, signaling pathways, and autophagy.
Project description:All above ground organs of higher plants are ultimately derived from specialized organogenic structures termed shoot apical meristems (SAMs). The SAM exhibits distinctive structural organization, marked by cell layering. Maize SAMs are comprised of two cell layers, L1 (single cell layered tunica) and L2 (corpus). To identify genes required for layer-specific functions intact maize SAMs were fixed, embedded in paraffin and sectioned. L1 and L2 cells were isolated from these sections via laser capture microdissection (LCM). RNA was isolated from six biological replications of L1 and L2, amplified and hybridized to microarrays spotted with ~37,000 maize cDNA clones. This experiment identified ~700 ESTs that are preferentially expressed in the L1 or the L2 (P <0.001). The L1-up-regulated ESTs included ZmOCL1 and ZmOCL4, which are known to exhibit L1-specific expression in the maize SAM. The L2-up-regulated ESTs included KNOTTED1, whose transcripts are known to accumulate in the L2 but not in the L1 of the maize SAM. Differentially expressed ESTs included genes involved in transcription, signal transduction, transport and metabolism, many of which are novel candidates that are required for layer-specific functions in the maize SAM. Several L1-up-regulated ESTs were annotated as yabby family genes or basic helix-loop-helix transcription factor-like genes, which have not previously been reported as having layer-specific expression in the SAM. Novel WW domain-containing genes (WW genes) were identified in this study. The WW domain mediates protein-protein interactions, often with signal transduction components. These WW genes were substantially up-regulated in the L1 relative to the L2. Keywords: Cell Type Comparison
Project description:All above ground organs of higher plants are ultimately derived from specialized organogenic structures termed shoot apical meristems (SAMs). The SAM exhibits distinctive structural organization, marked by cell layering. Maize SAMs are comprised of two cell layers, L1 (single cell layered tunica) and L2 (corpus). To identify genes required for layer-specific functions intact maize SAMs were fixed, embedded in paraffin and sectioned. L1 and L2 cells were isolated from these sections via laser capture microdissection (LCM). RNA was isolated from six biological replications of L1 and L2, amplified and hybridized to microarrays spotted with ~37,000 maize cDNA clones. This experiment identified ~700 ESTs that are preferentially expressed in the L1 or the L2 (P <0.001). The L1-up-regulated ESTs included ZmOCL1 and ZmOCL4, which are known to exhibit L1-specific expression in the maize SAM. The L2-up-regulated ESTs included KNOTTED1, whose transcripts are known to accumulate in the L2 but not in the L1 of the maize SAM. Differentially expressed ESTs included genes involved in transcription, signal transduction, transport and metabolism, many of which are novel candidates that are required for layer-specific functions in the maize SAM. Several L1-up-regulated ESTs were annotated as yabby family genes or basic helix-loop-helix transcription factor-like genes, which have not previously been reported as having layer-specific expression in the SAM. Novel WW domain-containing genes (WW genes) were identified in this study. The WW domain mediates protein-protein interactions, often with signal transduction components. These WW genes were substantially up-regulated in the L1 relative to the L2. Keywords: Cell Type Comparison An experimental aim is to identify genes that are differentially expressed in distinct histological cell layers of maize SAM by comparing the transcript accumulation between L1 (single cell layered tunica) and L2 (corpus) using cDNA microarrays that have over 37,000 informative spots from maize.
Project description:Disrupting PD-1/PD-L1 interaction rejuvenates antitumor immunity. Clinical successes by blocking PD-1/PD-L1 binding have grown across wide-ranging cancer histologies, but innate therapy resistance is evident in the majority of treated patients1. Cancer cells can express robust surface levels of PD-L1 to tolerize tumor-specific T cells, but regulation of PD-L1 protein levels in the cancer cell is poorly understood. Quasi-mesenchymal tumor cells up-regulate PD-L1/L2 and induce an immune-suppressive microenvironment, including expansion of M2-like macrophages and regulatory T cells and exclusion of CD8+ T-cell infiltration2. Targeted therapy, including MAPK inhibitor therapy in melanoma, leads to quasi-mesenchymal transitions and resistance3, and both MAPK inhibitor treatment and mesenchymal signatures are associated with innate anti-PD-1 resistance4,5. Here we identify ITCH as an E3 ligase that downregulates tumor cell-surface PD-L1/L2 in PD-L1/L2-high cancer cells, including MAPK inhibitor-resistant melanoma, and suppresses acquired MAPK inhibitor resistance in and only in immune-competent mice. ITCH interacts with and poly-ubiquitinates PD-L1/L2, and ITCH deficiency increases cell-surface PD-L1/L2 expression and reduces T cell activation. Mouse melanoma tumors grow faster with Itch knockdown only in syngeneic hosts but not in immune-deficient mice. MAPK inhibitor therapy induces tumor cell-surface PD-L1 expression in murine melanoma, recapitulating the responses of clinical melanoma3, and this induction is more robust with Itch knockdown. Notably, suppression of ITCH expression first elicits a shift toward an immune-suppressive microenvironment and then accelerates resistance development. These findings collectively identify ITCH as a critical negative regulator of PD-L1 tumor cell-surface expression and provide insights into previously unexplained role of PD-L1 in adaptive resistance to therapy.
Project description:Purpose: the technology of Solexa/Illumina (RNA-seq) is an attractive alternative to the transcriptome sequencing. The goals of this study are to investigate transcriptional changes in replanted R. glutinosa leaves and identify genes responding to the disease. Methods:Totally 6.01and 6.15 million raw tags were measured from leaves of the first year planted (L1) and the second year replanted R. glutinosa (L2) at the tuberous root expansion stage, respectively. mRNA profiles of leaves of the first year planted (L1) and the second year replanted R. glutinosa (L2) at the tuberous root expansion stage, were generated by deep sequencing (Illumina HiSeq™ 2000 system). qRT–PCR validation was performed using SYBR Green assays. Results: we obtained a total of 6.01 and 6.15 million total tags including 377,200 and 361,659 distinct tags from the L1 and L2 samples, respectively. Removal of low quality tags, 161,298 and 149,290 distinct clean tags were remained.With reference to the 94,544 non-redundant consensus sequences defined by the RNA-seq transcriptomic procedure, 60,574 genes had the diagnostic CATG site, which were taken as reference distinct sequences for DGE analysis. 47,458 of the L1 tag library and 42,247 of the L2 tag library were perfectly matched the reference sequences. we screened differentially expressed genes in the L1 and L2 libraries by digital gene expression (DGE) technique. Finally, a set of 1,954 genes may be in differential expression of L2. By bioinformatics and qRT-PCR, the 117 most strongly differentially expressed ones were considered to be prime candidates for responsible for replanting disease. Conclusions: The study provides an important resource for further investigating the cause of replanting disease and developing the methods to control or subtract its injury.
Project description:Purpose: the technology of Solexa/Illumina (RNA-seq) is an attractive alternative to the transcriptome sequencing. The goals of this study are to investigate transcriptional changes in replanted R. glutinosa leaves and identify genes responding to the disease. Methods:Totally 6.01and 6.15 million raw tags were measured from leaves of the first year planted (L1) and the second year replanted R. glutinosa (L2) at the tuberous root expansion stage, respectively. mRNA profiles of leaves of the first year planted (L1) and the second year replanted R. glutinosa (L2) at the tuberous root expansion stage, were generated by deep sequencing (Illumina HiSeq⢠2000 system). qRTâPCR validation was performed using SYBR Green assays. Results: we obtained a total of 6.01 and 6.15 million total tags including 377,200 and 361,659 distinct tags from the L1 and L2 samples, respectively. Removal of low quality tags, 161,298 and 149,290 distinct clean tags were remained.With reference to the 94,544 non-redundant consensus sequences defined by the RNA-seq transcriptomic procedure, 60,574 genes had the diagnostic CATG site, which were taken as reference distinct sequences for DGE analysis. 47,458 of the L1 tag library and 42,247 of the L2 tag library were perfectly matched the reference sequences. we screened differentially expressed genes in the L1 and L2 libraries by digital gene expression (DGE) technique. Finally, a set of 1,954 genes may be in differential expression of L2. By bioinformatics and qRT-PCR, the 117 most strongly differentially expressed ones were considered to be prime candidates for responsible for replanting disease. Conclusions: The study provides an important resource for further investigating the cause of replanting disease and developing the methods to control or subtract its injury. Leaf mRNA profiles of L1 (The first year planted R. glutinosa leaves ) and L2 (the second year replanted R. glutinosa leaves) root expansion stage were generated by deep sequencing (using Illumina HiSeq⢠2000 system)
Project description:All above ground organs of higher plants are ultimately derived from shoot apical meristems (SAMs). The SAM exhibits distinctive structural organization, and monocot SAMs such as maize are comprised of two cell layers, a single cell layered tunica (L1) and a corpus (L2). Although recent research has revealed roles of these cell layers in the SAM, intra- and inter-cell-layer signaling networks involved in organ development remain largely unknown except for a few differentially expressed genes. Here, we used Illumnia technology to conduct RNA-seq of L1 and L2 cell layers in maize B73 maize shoot apical meristem. Single sequencing library was constructed for L1 and L2 cell layer. Each library was sequenced using 2 lanes on a Solexa flow cell. Processed data file 'ZmB73_4a.53_filtered_genes.fasta' and its README file are linked below as supplementary files. The fasta file contains the gene model ID and corresponding sequence generated from maize genome project. This fasta file was used for the following samples: GSM418173, GSM418174, GSM420173, GSM420174, GSM422828, GSM422829.
Project description:The study goal is to identify the gene expression profile of iBAT-related ganglia (SG/T1 & T3) and iWAT-related ganglia (T13/L1 & L2). RNA sequencing demonstrated clear separation of gene expression between brown adipose tissue related ganglia (SG and T3) and white adipose tissue related ganglia (T13/L1 and L2). Each sample type also showed clear separation from each other. No significant separation was observed in different genders.