ABSTRACT: 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.
Project description:Hypertension is a pathological condition of persistent high blood pressure (BP) of which the underlying mechanisms remain largely obscure. Here, we show that the afferent nerves in perirenal adipose tissue are a regulatory site in triggering and maintaining pathological high BP, without affecting physiological BP. Bilateral perirenal adipose tissue (PRAT) ablation leads to a long-term and effective reduction of BP in spontaneous hypertensive rats (SHR) and high fat and high salt diet fed rats, but has no effect on normal BP in control Wistar-Kyoto or control SD rats. Moreover, gain- and loss-of-function studies show that augmented activities of L1-L2 dorsal root ganglia (DRG) neurons are responsible for hypertension in SHR. Further, L1-L2 DRG neuron transcriptomics uncovers significant changes in neuron development, remodeling, and plasticity. Importantly, calcitonin gene-related peptide (CGRP), a systemic vasodilator, is induced after PRAT ablation. We went on to show that CGRP antagonist blocks the BP-lowering effect of PRAT ablation. CGRP is therefore a key endogenous suppressor of hypertension that is sequestered by anti-hypertensive PRAT in SHRs. Taken together, we identify PRAT afferent nerves as a pathological node of hypertension that sustains high BP via suppressing CGRP, thereby providing a therapeutic target to tackle primary hypertension.
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:In our study, MS-based high-resolution proteomic analysis of CSF have been employed in order to gain deeper insights into changes occurring during IVDH in dogs, monitoring the pathophysiological processes at the protein and metabolite levels. The proteomics study involved canine patients classified within two groups. Healthy dogs (control group) (N=6) were mix breeds (20 kg average body weight), age 2-6 years, while the intervertebral disc herniation group (N=9) were mixed breeds (body weight 6-15 kg), age 7-12 years. In IVDH dogs, disc herniation was detected in T13/L1/L2 discs by CT. The dogs in IVDH group were paraplegic with preserved deep pain reflex and underwent a surgery procedure for decompression of the spinal cord.
Project description:Stress granules (SG) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SG act as a protective mechanism to cope with acute stress, but persistent SG have cytotoxic effects that are associated with several age-related diseases. Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP. MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Importantly, knockout of the MAGE-B2 mouse ortholog confers hypersensitivity of the male germline to heat stress in vivo. Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermo-sensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress through fine-tuning phase separation and could aid in the development of male fertility therapies
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: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: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:Piwi-related Argonaute proteins play important roles in maintaining germline integrity and fertility and have been linked to a class of germline-enriched small RNAs termed piRNAs. Caenorhabditis elegans encodes two Piwi family proteins called PRG-1 and PRG-2, and PRG-1 interacts with the C. elegans piRNAs (21U-RNAs). Previous studies found that the prg-1 mutation causes a marked reduction in the expression of 21U-RNAs, temperature-sensitive defects in fertility and other phenotypic defects.To systematically demonstrate the function of PRG-1 on regulating small RNAs and their targets. We use recent advances in high-throughput sequencing technology to show that expression of non-coding small RNAs in six stages(embryo,L1,L2,L3,L4,young audlt) and mRNAs in four stages (L1,L2,L3,L4) after prg-1 mutation. prg-1 mutation can not only lead to a decrease in the expression of 21U-RNAs, but also cause 35~40% of miRNAs to be significantly down-regulated; approximately 3% (6.00% in L4) of protein-coding genes are differentially expressed after mutating prg-1, and 60~70% of these substantially changed protein-coding genes are up-regulated. Examination of small RNA expression in six different developmental stages (embryo, L1, L2, L3, L4, young adult) and mRNA expression in four stages (L1,L2,L3,L4) of C. elegans prg-1 mutant (wm161) .
Project description:Piwi-related Argonaute proteins play important roles in maintaining germline integrity and fertility and have been linked to a class of germline-enriched small RNAs termed piRNAs. Caenorhabditis elegans encodes two Piwi family proteins called PRG-1 and PRG-2, and PRG-1 interacts with the C. elegans piRNAs (21U-RNAs). Previous studies found that the prg-1 mutation causes a marked reduction in the expression of 21U-RNAs, temperature-sensitive defects in fertility and other phenotypic defects.To systematically demonstrate the function of PRG-1 on regulating small RNAs and their targets. We use recent advances in high-throughput sequencing technology to show that expression of non-coding small RNAs in six stages(embryo,L1,L2,L3,L4,young audlt) and mRNAs in four stages (L1,L2,L3,L4) after prg-1 mutation. prg-1 mutation can not only lead to a decrease in the expression of 21U-RNAs, but also cause 35~40% of miRNAs to be significantly down-regulated; approximately 3% (6.00% in L4) of protein-coding genes are differentially expressed after mutating prg-1, and 60~70% of these substantially changed protein-coding genes are up-regulated. Examination of small RNA expression in six different developmental stages (embryo, L1, L2, L3, L4, young adult) and mRNA expression in four stages (L1,L2,L3,L4) of C. elegans prg-1 mutant (wm161) .
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.