Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is a disease associated with dysbiosis, resulting in compromised intestinal epithelial barrier and ...chronic mucosal inflammation. Patients with IBD present with increased incidence of psychiatric disorders and cognitive impairment. Hippocampus is a brain region where adult neurogenesis occurs with functional implications in mood control and cognition. Using a well-established model of experimental colitis based on the administration of dextran sodium sulfate (DSS) in the drinking water, we sought to characterize the short and long-term effects of colitis on neurogenesis and glia responses in the hippocampus. We show that acute DSS colitis enhanced neurogenesis but with deficits in cell cycle kinetics of proliferating progenitors in the hippocampus. Chronic DSS colitis was characterized by normal levels of neurogenesis but with deficits in the migration and integration of newborn neurons in the functional circuitry of the DG. Notably, we found that acute DSS colitis-induced enhanced infiltration of the hippocampus with macrophages and inflammatory myeloid cells from the periphery, along with elevated frequencies of inflammatory M1-like microglia and increased release of pro-inflammatory cytokines. In contrast, increased percentages of tissue-repairing M2-like microglia, along with elevated levels of the anti-inflammatory cytokine, IL-10 were observed in the hippocampus during chronic DSS colitis. These findings uncover key effects of acute and chronic experimental colitis on adult hippocampal neurogenesis and innate immune cell responses, highlighting the potential mechanisms underlying cognitive and mood dysfunction in patients with IBD.
Chromatin immunoprecipitation (ChIP) protocols have been used to reveal protein-DNA interactions of various cell types and tissues; however, optimization is required for each specific type of sample. ...Here, we present a ChIP protocol from murine inguinal white adipose tissue. We describe steps for tissue harvesting, crosslinking, chromatin extraction, shearing, immunoprecipitation, and purification. We then detail procedures for analysis including library preparation, sequencing, and qRT-PCR validation.
For complete details on the use and execution of this protocol, please refer to Antonia Katsouda et al. (2022).1
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•Illustrates the steps for inguinal fat chromatin IP and analysis•Isolates chromatin from adipose tissue and uses for IP•Analyzes the purified protein-DNA complexes by deep sequencing and qRT-PCR•Provides optimized conditions to ensure high-quality data
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Chromatin immunoprecipitation (ChIP) protocols have been used to reveal protein-DNA interactions of various cell types and tissues; however, optimization is required for each specific type of sample. Here, we present a ChIP protocol from murine inguinal white adipose tissue. We describe steps for tissue harvesting, crosslinking, chromatin extraction, shearing, immunoprecipitation, and purification. We then detail procedures for analysis including library preparation, sequencing, and qRT-PCR validation.
Hydrogen sulfide (H
S), a gasotransmitter with protective effects in the cardiovascular system, is endogenously generated by three main enzymatic pathways: cystathionine gamma lyase (CTH), ...cystathionine beta synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (MPST) enzymes. CTH and MPST are the predominant sources of H
S in the heart and blood vessels, exhibiting distinct effects in the cardiovascular system. To better understand the impact of H
S in cardiovascular homeostasis, we generated a double
knockout (
) mouse and characterized its cardiovascular phenotype. CTH/MPST-deficient mice were viable, fertile and exhibited no gross abnormalities. Lack of both CTH and MPST did not affect the levels of CBS and H
S-degrading enzymes in the heart and the aorta.
mice also exhibited reduced systolic, diastolic and mean arterial blood pressure, and presented normal left ventricular structure and fraction. Aortic ring relaxation in response to exogenously applied H
S was similar between the two genotypes. Interestingly, an enhanced endothelium-dependent relaxation to acetylcholine was observed in mice in which both enzymes were deleted. This paradoxical change was associated with upregulated levels of endothelial nitric oxide synthase (eNOS) and soluble guanylate cyclase (sGC) α1 and β1 subunits and increased NO-donor-induced vasorelaxation. Administration of a NOS-inhibitor, increased mean arterial blood pressure to a similar extent in wild-type and
mice. We conclude that chronic elimination of the two major H
S sources in the cardiovascular system, leads to an adaptive upregulation of eNOS/sGC signaling, revealing novel ways through which H
S affects the NO/cGMP pathway.
Hydrogen sulfide (H2S) has recently been recognized as a novel gaseous transmitter with several anti-inflammatory properties. The role of host- derived H2S in infections by Pseudomonas aeruginosa was ...investigated in clinical and mouse models. H2S concentrations and survival was assessed in septic patients with lung infection. Animal experiments using a model of severe systemic multidrug-resistant P. aeruginosa infection were performed using mice with a constitutive knock-out of cystathionine-γ lyase (Cse) gene (Cse-/-) and wild-type mice with a physiological expression (Cse+/+). Experiments were repeated in mice after a) treatment with cyclophosphamide; b) bone marrow transplantation (BMT) from a Cse+/+ donor; c) treatment with H2S synthesis inhibitor aminooxyacetic acid (ΑΟΑΑ) or propargylglycine (PAG) and d) H2S donor sodium thiosulfate (STS) or GYY3147. Bacterial loads and myeloperoxidase activity were measured in tissue samples. The expression of quorum sensing genes (QS) was determined in vivo and in vitro. Cytokine concentration was measured in serum and incubated splenocytes. Patients survivors at day 28 had significantly higher serum H2S compared to non-survivors. A cut- off point of 5.3 μΜ discriminated survivors with sensitivity 92.3%. Mortality after 28 days was 30.9% and 93.7% in patients with H2S higher and less than 5.3 μΜ (p = 7 x 10-6). In mice expression of Cse and application of STS afforded protection against infection with multidrug-resistant P. aeruginosa. Cyclophosphamide pretreatment eliminated the survival benefit of Cse+/+ mice, whereas BMT increased the survival of Cse-/- mice. Cse-/- mice had increased pathogen loads compared to Cse+/+ mice. Phagocytic activity of leukocytes from Cse-/- mice was reduced but was restored after H2S supplementation. An H2S dependent down- regulation of quorum sensing genes of P.aeruginosa could be demonstrated in vivo and in vitro. Endogenous H2S is a potential independent parameter correlating with the outcome of P. aeruginosa. H2S provides resistance to infection by MDR bacterial pathogens.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
3-mercaptopyruvate sulfurtransferase (3-MST) is an enzyme capable of synthesizing hydrogen sulfide (H
S) and polysulfides. In spite of its ubiquitous presence in mammalian cells, very few studies ...have investigated its contribution to homeostasis and disease development, thus the role of 3-MST remains largely unexplored. Here, we present a clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) induced
mutant zebrafish line, which will allow the study of 3-MST's role in several biological processes. The
zebrafish orthologue was identified using a bioinformatic approach and verified by its ability to produce H
S in the presence of 3-mercaptopyruvate (3-MP). Its expression pattern was analyzed during zebrafish early development, indicating predominantly an expression in the heart and central nervous system. As expected, no detectable levels of 3-Mst protein were observed in homozygous mutant larvae. In line with this, H
S levels were reduced in
zebrafish. Although the mutants showed no obvious morphological deficiencies, they exhibited increased lethality under oxidative stress conditions. The elevated levels of reactive oxygen species, detected following
deletion, are likely to drive this phenotype. In line with the increased ROS, we observed accelerated fin regenerative capacity in
deficient zebrafish. Overall, we provide evidence for the expression of
in zebrafish, confirm its important role in redox homeostasis and indicate the enzyme's possible involvement in the regeneration processes.
Glioblastoma (GBM) is the most common type of adult brain tumor with extremely poor survival. Cystathionine-gamma lyase (CTH) is one of the main Hydrogen Sulfide (H2S) producing enzymes and its ...expression contributes to tumorigenesis and angiogenesis but its role in glioblastoma development remains poorly understood.
and Principal Results: An established allogenic immunocompetent in vivo GBM model was used in C57BL/6J WT and CTH KO mice where the tumor volume and tumor microvessel density were blindly measured by stereological analysis. Tumor macrophage and stemness markers were measured by blinded immunohistochemistry. Mouse and human GBM cell lines were used for cell-based analyses. In human gliomas, the CTH expression was analyzed by bioinformatic analysis on different databases.
In vivo, the genetic ablation of CTH in the host led to a significant reduction of the tumor volume and the protumorigenic and stemness transcription factor sex determining region Y-box 2 (SOX2). The tumor microvessel density (indicative of angiogenesis) and the expression levels of peritumoral macrophages showed no significant changes between the two genotypes. Bioinformatic analysis in human glioma tumors revealed that higher CTH expression is positively correlated to SOX2 expression and associated with worse overall survival in all grades of gliomas. Patients not responding to temozolomide have also higher CTH expression. In mouse or human GBM cells, pharmacological inhibition (PAG) or CTH knockdown (siRNA) attenuates GBM cell proliferation, migration and stem cell formation frequency.
Inhibition of CTH could be a new promising target against glioblastoma formation.
Overview of the proposed mechanisms by which CTH might regulate glioblastoma formation. Pharmacological (PAG) inhibition of CTH on human glioblastoma cells results to lower Glioblastoma Stem Cell (GSC) formation and lower mRNA expression of stem cell markers (i.e, PROM1, NOX4). Bioinformatic analysis reveals also higher CTH expression in human GBM, which is associated with worse overall survival, non-response to temozolomide (TMZ) and higher expression of the GSC marker SOX2. Inhibition of CTH in mouse GBM cells with a brain-permeable pharmacological inhibitor (PAG) or knockdown with siRNA (siCTH) results in lower GBM cell proliferation and migration by an H2S-dependent mechanism, since the effects of CTH inhibition are attenuated by the H2S donor, NaSH. CTH expression in the tumor stroma/microenvironment plays a crucial role for glioblastoma formation and triggers the expression of the GSC marker SOX2 without affecting the tumoral vascular network. CTH KO mice with GBM have significantly lower tumor volume, tumoral SOX2 expression and no alteration in the levels of tumoral vascular density compared to WT mice with GBM. The figure was created by using the Biorender software https://biorender.com/. Display omitted
•CTH inhibition in the tumor host attenuates glioblastoma.•CTH is upregulated in human gliomas and associated with worse overall survival.•CTH inhibition lowers GBM cell proliferation, migration and stem cell formation.
Protein persulfidation is a significant post-translational modification that involves addition of a sulfur atom to the cysteine thiol group and is facilitated by sulfide species. Persulfidation ...targets reactive cysteine residues within proteins, influencing their structure and/or function across various biological systems. This modification is evolutionarily conserved and plays a crucial role in preventing irreversible cysteine overoxidation, a process that becomes prominent with aging. While, persulfidation decreases with age, its levels in the aged heart and the functional implications of such a reduction in cardiac metabolism remain unknown. Here we interrogated the cardiac persulfydome in wild-type adult mice and age-matched mice lacking the two sulfide generating enzymes, namely cystathionine gamma lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST). Our findings revealed that cardiac persulfidated proteins in wild type hearts are less abundant compared to those in other organs, with a primary involvement in mitochondrial metabolic processes. We further focused on one specific target, NDUFB7, which undergoes persulfidation by both CSE and 3MST derived sulfide species. In particular, persulfidation of cysteines C80 and C90 in NDUFB7 protects the protein from overoxidation and maintains the complex I activity in cardiomyocytes. As the heart ages, the levels of CSE and 3MST in cardiomyocytes decline, leading to reduced NDUFB7 persulfidation and increased cardiac NADH/NAD+ ratio. Collectively, our data provide compelling evidence for a direct link between cardiac persulfidation and mitochondrial complex I activity, which is compromised in aging.
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•Cardiac proteins are differentially persulfidated by CSE and 3MST.•Endogenous cardiac sulfides modify mainly mitochondrial proteins.•Cardiac NDUFB7 persulfidation maintains complex I activity and preserves NAD + levels.•Reduced CSE and 3MST expression in aging results in impaired sulfide sensing and impacts on cardiac NAD + bioavailability.
Reduced adipose tissue H2S in obesity Katsouda, Antonia; Szabo, Csaba; Papapetropoulos, Andreas
Pharmacological research,
February 2018, 20180201, Letnik:
128
Journal Article
Recenzirano
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Hydrogen sulfide (H2S) is an endogenously produced signaling molecule synthesized by cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS) and 3-mercaptopyruvate ...sulfurtransferase (3-MST). Given that H2S exerts significant effects on bioenergetics and metabolism, the goal of the current study was to determine the expression of H2S-producing enzymes in adipose tissues in models of obesity and metabolic disruption. Mice fed a western diet expressed lower mRNA levels of all three enzymes in epididymal fat (EWAT), while only CSE and 3-MST were reduced in brown adipose tissue (BAT). At the protein level 3-MST was reduced in all fat depots studied. Using db/db mice, a genetic model of obesity, we found that CSE, CBS and 3-MST mRNA were reduced in white fat, while only CSE was reduced in BAT. CBS and CSE protein levels were suppressed in all three fat depots. In a model of age-related weight gain, no reduction in the mRNA of any of the enzymes was noted. Smaller amounts of 3-MST protein were found in EWAT, while both CSE and 3-MST were reduced in BAT. Tissue levels of H2S were lower in WAT in HFD mice; both WAT and BAT contained lower H2S amounts in db/db animals. Taken together, our data suggest that obesity is associated with a decreased expression of H2S-synthesizing enzymes and reduced H2S levels in adipose tissues of mice. We propose that the reduction in H2S may contribute to the metabolic response associated with obesity. Further work is needed to determine whether restoring H2S levels may have a beneficial effect on obesity-associated metabolic alterations.
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Recent studies have implicated endogenously produced H2S in the angiogenic process. On one hand, pharmacological inhibition and silencing of the enzymes involved in H2S synthesis ...attenuate the angiogenic properties of endothelial cells, including proliferation, migration and tube-like structure network formation. On the other hand, enhanced production of H2S by substrate supplementation or over-expression of H2S-producing enzymes leads to enhanced angiogenic responses in cultured endothelial cells. Importantly, H2S up-regulates expression of the key angiogenic factor vascular endothelial growth factor (VEGF) and contributes to the angiogenic signaling in response to VEGF. The signaling pathways mediating H2S-induced angiogenesis include mitogen-activated protein kinases, phosphoinositide-3 kinase, nitric oxide/cGMP-regulated cascades and ATP-sensitive potassium channels. Endogenously produced H2S has also been shown to facilitate neovascularization in prototypical model systems in vivo, and to contribute to wound healing, post-ischemic angiogenesis in the heart and other tissues, as well as in tumor angiogenesis. Targeting of H2S synthesizing enzymes might offer novel therapeutic opportunities for angiogenesis-related diseases.
Reduced adipose tissue H 2 S in obesity Katsouda, Antonia; Szabo, Csaba; Papapetropoulos, Andreas
Pharmacological research,
02/2018, Letnik:
128
Journal Article
Recenzirano
Hydrogen sulfide (H
S) is an endogenously produced signaling molecule synthesized by cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST). ...Given that H
S exerts significant effects on bioenergetics and metabolism, the goal of the current study was to determine the expression of H
S-producing enzymes in adipose tissues in models of obesity and metabolic disruption. Mice fed a western diet expressed lower mRNA levels of all three enzymes in epididymal fat (EWAT), while only CSE and 3-MST were reduced in brown adipose tissue (BAT). At the protein level 3-MST was reduced in all fat depots studied. Using db/db mice, a genetic model of obesity, we found that CSE, CBS and 3-MST mRNA were reduced in white fat, while only CSE was reduced in BAT. CBS and CSE protein levels were suppressed in all three fat depots. In a model of age-related weight gain, no reduction in the mRNA of any of the enzymes was noted. Smaller amounts of 3-MST protein were found in EWAT, while both CSE and 3-MST were reduced in BAT. Tissue levels of H
S were lower in WAT in HFD mice; both WAT and BAT contained lower H
S amounts in db/db animals. Taken together, our data suggest that obesity is associated with a decreased expression of H
S-synthesizing enzymes and reduced H
S levels in adipose tissues of mice. We propose that the reduction in H
S may contribute to the metabolic response associated with obesity. Further work is needed to determine whether restoring H
S levels may have a beneficial effect on obesity-associated metabolic alterations.
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