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Zheng, Yue, Jie Zhou, Stefan M. Cooper, Clement Opoku-Temeng, Amanda Moreira De Brito, and Herman O. Sintim. "Structure–activity relationship studies of c-di-AMP synthase inhibitor, bromophenol-thiohydantoin." Tetrahedron 72, no. 25 (June 2016): 3554–58. http://dx.doi.org/10.1016/j.tet.2015.10.073.
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Opoku-Temeng, Clement, and Herman O. Sintim. "Potent inhibition of cyclic diadenylate monophosphate cyclase by the antiparasitic drug, suramin." Chemical Communications 52, no. 19 (2016): 3754–57. http://dx.doi.org/10.1039/c5cc10446g.
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Zheng, Yue, Jie Zhou, David A. Sayre, and Herman O. Sintim. "Identification of bromophenol thiohydantoin as an inhibitor of DisA, a c-di-AMP synthase, from a 1000 compound library, using the coralyne assay." Chem. Commun. 50, no. 76 (2014): 11234–37. http://dx.doi.org/10.1039/c4cc02916j.
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Voronkov, Andrey, and Dmitry Pozdnyakov. "Endothelotropic Activity of 4-Hydroxy-3,5-Di-Tret-Butylcinnamic Acid in the Conditions of Experimental Cerebral Ischemia." Research Results in Pharmacology 4, no. 2 (July 19, 2018): 1–10. http://dx.doi.org/10.3897/rrpharmacology.4.26519.
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Introduction: The aim of the study was to evaluate the endothelioprotective activity of 4-hydroxy-3,5-di-tret-butylcinnamic acid in conditions of experimental cerebral ischemia. Materials and Methods: The brain ischemia was reproduced by the method of irreversible right-sided thermocoagulation of the middle cerebral artery. As comparative drugs, mexidol (30 mg/kg) and sulodexide (30 U/kg) were used. The vasodilating function of the vascular endothelium was assessed by the change in the rate of cerebral blood flow when the synthesis of nitric oxide was modified. Antithrombotic function was assessed by changes in the concentration of thromboxane A2, fibrinogen, von Willebrand factor activity and platelet aggregation activity. Serum concentration of C-reactive protein served as a marker of the state of anti-inflammatory endothelial function. To determine the potential mechanism of endothelioprotective activity of 4-4-hydroxy-3,5-di-tret-butylcinnamic acid, the anti-radical activity of this compound toward superoxide and nitrosy-radicals was assessed; and the effect of the compound on the mitochondrial function was studied, by evaluating the functional activity of mitochondrial ATP synthetase and cytochrome-c-oxidase by ELISA. Results and Discussion: In the course of the study, a positive effect of 4-hydroxy-3,5-di-tret-butylcinnamic acid on the state of endothelial function in cerebral ischemia was established, which was expressed in the preservation of vasodilating (restoring the vascular reaction to acetylcholine, nitro-L-arginine methyl ether, L-arginine), antithrombotic (a decrease in the concentration of thromboxane A2, fibrinogen and von Willebrand factor activity by 241.9% (p <0.05), 73.5% (p <0.05), 20.4% (p <0.05), respectively, a decrease in the degree of aggregation and platelet aggregation rate by 56.7 % (p <0.05) and 52.8% (p <0.05), respectively, and anti-inflammatory vascular endothelial function (99.1% C-reactive protein reduction (p <0.05)). The 4-hydroxy-3,5-di-tret-butylcinnamic acid compound in vitro tests suppressed generation of superoxide (IC50 = 1.99 mg/ml) and nitrosyl radical (IC50 = 1.92 mg/ml), eliminated NO-synthase uncoupling, and restored the mitochondrial function (increase in mitochondrial ATP synthase and cytochrome-c-oxidase activity by 23.5% (p <0.05) and 110.8% (p <0.05), respectively). Conclusion: The study demonstrated the presence of endotheliotropic activity of 4-hydroxy-3,5-di-tret-butylcinnamic acid, which is expressed in the preservation of vasodilating, antithrombotic and anti-inflammatory functions of the vascular endothelium in conditions of cerebral ischemia. At the same time, the anti-radical properties of this compound, as well as the direct effect on the functional activity of the NO-synthase system and the improvement of the mitochondrial function, may underlie the endotheliotropic effects of 4-hydroxy-3,5-di-tret-butylcinnamic acid.
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Opoku-Temeng, Clement, Neetu Dayal, Jacob Miller, and Herman O. Sintim. "Hydroxybenzylidene-indolinones, c-di-AMP synthase inhibitors, have antibacterial and anti-biofilm activities and also re-sensitize resistant bacteria to methicillin and vancomycin." RSC Advances 7, no. 14 (2017): 8288–94. http://dx.doi.org/10.1039/c6ra28443d.
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Hydroxybenzylidene-indolinones, newly identified inhibitors of c-di-AMP synthases, inhibit biofilm formation, Gram-positive bacterial growth and sensitize resistant bacteria to methicillin and vancomycin.
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Schmidt, Andrew J., Dmitri A. Ryjenkov, and Mark Gomelsky. "The Ubiquitous Protein Domain EAL Is a Cyclic Diguanylate-Specific Phosphodiesterase: Enzymatically Active and Inactive EAL Domains." Journal of Bacteriology 187, no. 14 (July 2005): 4774–81. http://dx.doi.org/10.1128/jb.187.14.4774-4781.2005.
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ABSTRACT The EAL domain (also known as domain of unknown function 2 or DUF2) is a ubiquitous signal transduction protein domain in the Bacteria. Its involvement in hydrolysis of the novel second messenger cyclic dimeric GMP (c-di-GMP) was demonstrated in vivo but not in vitro. The EAL domain-containing protein Dos from Escherichia coli was reported to hydrolyze cyclic AMP (cAMP), implying that EAL domains have different substrate specificities. To investigate the biochemical activity of EAL, the E. coli EAL domain-containing protein YahA and its individual EAL domain were overexpressed, purified, and characterized in vitro. Both full-length YahA and the EAL domain hydrolyzed c-di-GMP into linear dimeric GMP, providing the first biochemical evidence that the EAL domain is sufficient for phosphodiesterase activity. This activity was c-di-GMP specific, optimal at alkaline pH, dependent on Mg2+ or Mn2+, strongly inhibited by Ca2+, and independent of protein oligomerization. Linear dimeric GMP was shown to be 5′pGpG. The EAL domain from Dos was overexpressed, purified, and found to function as a c-di-GMP-specific phosphodiesterase, not as a cAMP-specific phosphodiesterase, in contrast to previous reports. The EAL domains can hydrolyze 5′pGpG into GMP, however, very slowly, thus implying that this activity is irrelevant in vivo. Therefore, c-di-GMP is the exclusive substrate of EAL. Multiple-sequence alignment revealed two groups of EAL domains hypothesized to correspond to enzymatically active and inactive domains. The domains in the latter group have mutations in residues conserved in the active domains. The enzymatic inactivity of EAL domains may explain their coexistence with GGDEF domains in proteins possessing c-di-GMP synthase (diguanulate cyclase) activity.
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Li, Haotian, Tingting Li, Wenjin Zou, Minghui Ni, Qiao Hu, Xiuxiu Qiu, Zhiming Yao, et al. "IPA-3: An Inhibitor of Diadenylate Cyclase of Streptococcus suis with Potent Antimicrobial Activity." Antibiotics 11, no. 3 (March 21, 2022): 418. http://dx.doi.org/10.3390/antibiotics11030418.
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Antimicrobial resistance (AMR) poses a huge threat to public health. The development of novel antibiotics is an effective strategy to tackle AMR. Cyclic diadenylate monophosphate (c-di-AMP) has recently been identified as an essential signal molecule for some important bacterial pathogens involved in various bacterial physiological processes, leading to its synthase diadenylate cyclase becoming an attractive antimicrobial drug target. In this study, based on the enzymatic activity of diadenylate cyclase of Streptococcus suis (ssDacA), we established a high-throughput method of screening for ssDacA inhibitors. Primary screening with a compound library containing 1133 compounds identified IPA-3 (2,2′-dihydroxy-1,1′-dinapthyldisulfide) as an ssDacA inhibitor. High-performance liquid chromatography (HPLC) analysis further indicated that IPA-3 could inhibit the production of c-di-AMP by ssDacA in vitro in a dose-dependent manner. Notably, it was demonstrated that IPA-3 could significantly inhibit the growth of several Gram-positive bacteria which harbor an essential diadenylate cyclase but not E. coli, which is devoid of the enzyme, or Streptococcus mutans, in which the diadenylate cyclase is not essential. Additionally, the binding site in ssDacA for IPA-3 was predicted by molecular docking, and contains residues that are relatively conserved in diadenylate cyclase of Gram-positive bacteria. Collectively, our results illustrate the feasibility of ssDacA as an antimicrobial target and consider IPA-3 as a promising starting point for the development of a novel antibacterial.
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Bhat, Aaqib M., Bhopal C. Mohapatra, Insha Mushtaq, Sukanya Chakraborty, Samikshan Dutta, Sameer Mirza, Matthew D. Storck, et al. "Abstract 2411: Di-ganglioside GD2 expression and role in promoting tumorigenicity in prostate cancer." Cancer Research 82, no. 12_Supplement (June 15, 2022): 2411. http://dx.doi.org/10.1158/1538-7445.am2022-2411.
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Abstract Background & Significance: Prostate cancer (PCa) is the second leading cause of cancer deaths (~34,000 in 2021 (ACS)) in American men. Castration resistance and resistance to the next-gen androgen receptor (AR) targeted drugs are major challenges. Castration resistance involves multiple mechanisms, including androgen-independent signaling by androgen receptor (AR)- or its variants, and lineage plasticity (LP) with AR-indifferent neuroendocrine (NE) differentiation. Identifying new vulnerabilities across these multitude mechanisms could provide new therapeutic avenues against castration-resistant PCa (CRPC). The cell-surface di-ganglioside GD2 is overexpressed in neural crest cell tumors such as neuroblastoma & melanoma and chimeric (Dinutuximab) or humanized (Naxitamab) anti-GD2 antibodies are now FDA-approved for high-risk neuroblastoma therapy. GD2 expression is reported in other cancers such as breast cancer and glioma and is linked to cancer stem cell behavior. While limited prior studies have detected GD2 expression in PCa cell lines or tumor tissues, nothing is known about the functional role of GD2 in PCa. Objectives: We hypothesized that GD2 overexpression in PCa could play a pro-tumorigenic role and that linkage of GD2 overexpression with CRPC progression may reveal the potential of targeting GD2 for CRPC therapy. Study Design & Results: Immunohistochemical analysis of PCa patient and patient-derived xenograft tissue microarrays (TMAs) revealed GD2 expression in a subset of tumor cells. Fluorescence-activated cell sorter analysis of PCa cell lines showed strong constitutive GD2 expression on murine CRPC cell line RM-1 (derived from mutant Ras and c-Myc overexpressing prostatic epithelial cells) and human PCa line 22Rv1 (overexpresses wild-type AR and ARv7 splice variant). GD2 expression was induced de novo upon induction of lineage plasticity in GD2-negative LNCaP C4-2 prostate adenocarcinoma cell line by shRNA knockdown (KD) of RB1 or TP53. High GD2 expression was also induced when C4-2B cells were made enzalutamide resistant (C4-2BER). Induction of GD2 expression correlated with increased expression of rate-limiting GD2 biosynthetic pathway enzyme GD3 synthase (GD3S). CRISPR-Cas9 mediated stable GD3S knockout (KO) in the RM1 cell line led to the loss of GD2 expression. The GD3S-KO RM1 cells exhibited reduced proliferation, migration, invasion, and tumor sphere forming ability compared to the control cells. Intratibial injections in castrated male C57BL/6 mice showed a significant reduction in tumor development by GD3S KO RM1 cells compared to control cells. Conclusions: Our studies demonstrate that GD2 is expressed in a subset of prostate cancers. Cell line-based studies show that GD2 expression promotes pro-tumorigenic traits. Future studies will assess the biological roles of GD2 in PCa and the potential of targeting GD2+ CRPC with antibody-based approved therapeutic agents. Citation Format: Aaqib M. Bhat, Bhopal C. Mohapatra, Insha Mushtaq, Sukanya Chakraborty, Samikshan Dutta, Sameer Mirza, Matthew D. Storck, Subodh M. Lele, Ming-Fong Lin, Bruce J. Trock, Karen S. Sfanos, Colm Morrissey, Eva Corey, Jonathan Melamed, Leah Cook, Kaustubh Datta, Jane Meza, Jawed Siddiqui, Surinder K. Batra, Vimla Band, Hamid Band. Di-ganglioside GD2 expression and role in promoting tumorigenicity in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2411.
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Wu, Yongxia, Chih-Hang Anthony Tang, Corey Mealer, David Bastian, Mohammed Hanief Sofi, Linlu Tian, Steven Douglas Schutt, et al. "Sting Negatively Regulates Allogeneic T-Cell Responses By Constraining Antigen-Presenting Cell Function." Blood 136, Supplement 1 (November 5, 2020): 37–38. http://dx.doi.org/10.1182/blood-2020-139860.
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The endoplasmic-reticulum-resident protein STING (Stimulator of IFN genes) is a downstream signaling effector of cytosolic DNA sensor cGAS (cyclic GMP-AMP synthase). STING-mediated innate immune activation plays a key role in tumor- and self-DNA elicited anti-tumor immunity and autoimmunity, respectively, yet the mechanism remains largely unclear. We utilized murine models of allogeneic hematopoietic cell transplantation (allo-HCT) to study the biology of STING in antigen-presetting cells (APCs) and T cells. STING expression in donor T cells was dispensable for their ability to induce graft-versus-host disease (GVHD), a major complication of allo-HCT in the clinic. However, when STING-deficient mice were used as recipients, more severe disease was induced after allo-HCT. Using bone marrow (BM) chimeras where STING was absent in different compartments, we found that STING-deficiency on host hematopoietic cells (Fig. A), but not on non-hematopoietic cells, was primarily responsible for exacerbating the disease. Furthermore, STING expression on host CD11c+ cells played a dominant role in the regulation of allogeneic T-cell responses (Fig. B). Mechanistically, STING deficiency resulted in increased survival, activation and function of irradiated APCs, including macrophages and dendritic cells (DCs, fig. C-D). To further determine the role of STING in APCs, we generated a STING V154M knock-in mouse model, in which V154M mutation in TMEM173 causes constitutive activation of STING. Consistently, constitutive activation of STING attenuated the survival, activation and function of APCs isolated from STING V154M knock-in mice. In addition, STING-deficient APCs augmented donor T-cell expansion, chemokine receptor expression and migration into intestinal tissues (Fig. E), resulting in accelerated/exacerbated disease. Using pharmacologic approaches, we demonstrate that systemic administration of a STING agonist (c-di-GMP) to recipient mice before transplantation significantly reduced GVHD mortality (Fig. F). In conclusion, we report an inhibitory role of STING in regulating survival and T-cell priming function of hematopoietic APCs, especially CD11c+ cells, after allo-HCT. We validate that pharmacological activation of STING may serve as a potential therapeutic strategy to constrain APCs and induce immune tolerance. Figure Disclosures No relevant conflicts of interest to declare.
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Raineri, Alice, Rachele Campagnari, Roberto Dal Toso, Stefano Copetti, Macarena Gomez-Lira, and Marta Menegazzi. "3,5-Dicaffeoylquinic Acid Lowers 3T3-L1 Mitotic Clonal Expansion and Adipocyte Differentiation by Enhancing Heme Oxygenase-1 Expression." Molecules 26, no. 16 (August 19, 2021): 5027. http://dx.doi.org/10.3390/molecules26165027.
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Adipogenesis is a complex process in which cell commitment and mitotic clonal expansion (MCE) are in-sequence crucial events leading to terminal adipocyte differentiation. The molecules able to block some key signals in this cascade can hamper adipogenesis becoming promising agents to counteract hyperplasia and hypertrophy of adipose tissue. Mono- and di-caffeoylquinic acid isomers are biologically active polyphenols, displaying in vitro and in vivo antioxidant, hepatoprotective, anti-diabetic and anti-obesity properties. Among these isomers, 3,5-dicaffeoylquinic acid (DCQA) has been reported to inhibit lipid accumulation in adipose cells more successfully than others. Thus, we investigated DCQA effects and molecular mechanisms on 3T3-L1 pre-adipocytes induced to differentiate with a hormonal cocktail (MDI). Oil Red O incorporation assessed that DCQA pre-treatment inhibited lipid accumulation in 3T3-L1 cells induced to differentiate for 10 days. At this time, an increased phosphorylation of both AMP-activated kinase and acetyl-CoA carboxylase, as well as a strong decrease in fatty acid synthase protein level, were registered by immunoblotting, thereby suggesting that DCQA treatment can reduce fatty acid anabolism in 3T3-L1 adipocytes. Furthermore, BrdU incorporation assay, performed 48 h after hormonal stimulation, revealed that DCQA treatment was also able to hinder the 3T3-L1 cell proliferation during the MCE, which is an essential step in the adipogenic process. Thus, we focused our attention on early signals triggered by the differentiation stimuli. In the first hours after hormonal cocktail administration, the activation of ERK1/2 and Akt kinases, or CREB and STAT3 transcription factors, was not affected by DCQA pre-treatment. Whereas 24 h after MDI induction, DCQA pre-treated cells showed increased level of the transcription factor Nrf2, that induced the expression of the antioxidant enzyme heme oxygenase 1 (HO-1). In control samples, the expression level of HO-1 was reduced 24 h after MDI induction in comparison with the higher amount of HO-1 protein found at 2 h. The HO-1 decrease was functional by allowing reactive oxygen species to boost and allowing cell proliferation induction at the beginning of MCE phase. Instead, in DCQA-treated cells the HO-1 expression was maintained at high levels for a further 24 h; in fact, its expression decreased only 48 h after MDI stimulation. The longer period in which HO-1 expression remained high led to a delay of the MCE phase, with a subsequent inhibition of both C/EBP-α expression and adipocyte terminal differentiation. In conclusion, DCQA counteracting an excessive adipose tissue expansion may become an attractive option in obesity treatment.
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Ünlü, Ayşe, Kerem Teralı, Zübeyde Uğurlu Aydın, Ali A. Dönmez, Hasan Soliman Yusufoğlu, and İhsan Çalış. "Isolation, Characterization and In Silico Studies of Secondary Metabolites from the Whole Plant of Polygala inexpectata Peşmen & Erik." Molecules 27, no. 3 (January 21, 2022): 684. http://dx.doi.org/10.3390/molecules27030684.
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Polygala species are frequently used worldwide in the treatment of various diseases, such as inflammatory and autoimmune disorders as well as metabolic and neurodegenerative diseases, due to the large number of secondary metabolites they contain. The present study was performed on Polygala inexpectata, which is a narrow endemic species for the flora of Turkey, and resulted in the isolation of nine known compounds, 6,3′-disinapoyl-sucrose (1), 6-O-sinapoyl,3′-O-trimethoxy-cinnamoyl-sucrose (tenuifoliside C) (2), 3′-O-(O-methyl-feruloyl)-sucrose (3), 3′-O-(sinapoyl)-sucrose (4), 3′-O-trimethoxy-cinnamoyl-sucrose (glomeratose) (5), 3′-O-feruloyl-sucrose (sibiricose A5) (6), sinapyl alcohol 4-O-glucoside (syringin or eleutheroside B) (7), liriodendrin (8), and 7,4′-di-O-methylquercetin-3-O-β-rutinoside (ombuin 3-O-rutinoside or ombuoside) (9). The structures of the compounds were determined by the spectroscopic methods including 1D-NMR (1H NMR, 13C NMR, DEPT-135), 2D-NMR (COSY, NOESY, HSQC, HMBC), and HRMS. The isolated compounds were shown in an in silico setting to be accommodated well within the inhibitor-binding pockets of myeloperoxidase and inducible nitric oxide synthase and anchored mainly through hydrogen-bonding interactions and π-effects. It is therefore plausible to suggest that the previously established anti-inflammatory properties of some Polygala-derived phytochemicals may be due, in part, to the modulation of pro-inflammatory enzyme activities.
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Faozia, Sabrina, Tazin Fahmi, Gary C. Port, and Kyu Hong Cho. "c-di-AMP-Regulated K + Importer KtrAB Affects Biofilm Formation, Stress Response, and SpeB Expression in Streptococcus pyogenes." Infection and Immunity 89, no. 4 (March 17, 2021). http://dx.doi.org/10.1128/iai.00317-20.
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The second messenger cyclic di-AMP (c-di-AMP) controls biofilm formation, stress response, and virulence in Streptococcus pyogenes . The deletion of the c-di-AMP synthase gene, dacA , results in pleiotropic effects including reduced expression of the secreted protease SpeB.
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Krüger, Larissa, Christina Herzberg, Dennis Wicke, Heike Bähre, Jana L. Heidemann, Achim Dickmanns, Kerstin Schmitt, Ralf Ficner, and Jörg Stülke. "A meet-up of two second messengers: the c-di-AMP receptor DarB controls (p)ppGpp synthesis in Bacillus subtilis." Nature Communications 12, no. 1 (February 22, 2021). http://dx.doi.org/10.1038/s41467-021-21306-0.
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AbstractMany bacteria use cyclic di-AMP as a second messenger to control potassium and osmotic homeostasis. In Bacillus subtilis, several c-di-AMP binding proteins and RNA molecules have been identified. Most of these targets play a role in controlling potassium uptake and export. In addition, c-di-AMP binds to two conserved target proteins of unknown function, DarA and DarB, that exclusively consist of the c-di-AMP binding domain. Here, we investigate the function of the c-di-AMP-binding protein DarB in B. subtilis, which consists of two cystathionine-beta synthase (CBS) domains. We use an unbiased search for DarB interaction partners and identify the (p)ppGpp synthetase/hydrolase Rel as a major interaction partner of DarB. (p)ppGpp is another second messenger that is formed upon amino acid starvation and under other stress conditions to stop translation and active metabolism. The interaction between DarB and Rel only takes place if the bacteria grow at very low potassium concentrations and intracellular levels of c-di-AMP are low. We show that c-di-AMP inhibits the binding of DarB to Rel and the DarB–Rel interaction results in the Rel-dependent accumulation of pppGpp. These results link potassium and c-di-AMP signaling to the stringent response and thus to the global control of cellular physiology.
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Chatterji, Dipankar, Sudhanshu Gautam, Avisek Mahapa, Lahari Yeramala, Apoorv Gandhi, Sushma Krishnan, and Kutti R. Vinothkumar. "Structure-Function Relationship in C-Di-Amp Synthase (Msdisa) from Mycobacterium Smegmatis." SSRN Electronic Journal, 2022. http://dx.doi.org/10.2139/ssrn.4164354.
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Fahmi, Tazin, Sabrina Faozia, Gary C. Port, and Kyu Hong Cho. "The Second Messenger c-di-AMP Regulates Diverse Cellular Pathways Involved in Stress Response, Biofilm Formation, Cell Wall Homeostasis, SpeB Expression, and Virulence inStreptococcus pyogenes." Infection and Immunity 87, no. 6 (April 1, 2019). http://dx.doi.org/10.1128/iai.00147-19.
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ABSTRACTCyclic di-AMP (c-di-AMP) is a recently discovered second messenger in bacteria. The cellular level of c-di-AMP inStreptococcus pyogenesis predicted to be controlled by the synthase DacA and two putative phosphodiesterases, GdpP and Pde2. To investigate the role of c-di-AMP inS. pyogenes, we generated null mutants in each of these proteins by gene deletion. Unlike those in other Gram-positive pathogens such asStaphylococcus aureusandListeria monocytogenes, DacA inS. pyogeneswas not essential for growth in rich media. The DacA null mutant presented a growth defect that manifested through an increased lag time, produced no detectable biofilm, and displayed increased susceptibility toward environmental stressors such as high salt, low pH, reactive oxygen radicals, and cell wall-targeting antibiotics, suggesting that c-di-AMP plays significant roles in crucial cellular processes involved in stress management. The Pde2 null mutant exhibited a lower growth rate and increased biofilm formation, and interestingly, these phenotypes were distinct from those of the null mutant of GdpP, suggesting that Pde2 and GdpP play distinctive roles in c-di-AMP signaling. DacA and Pde2 were critical to the production of the virulence factor SpeB and to the overall virulence ofS. pyogenes, as both DacA and Pde2 null mutants were highly attenuated in a mouse model of subcutaneous infection. Collectively, these results show that c-di-AMP is an important global regulator and is required for a proper response to stress and for virulence inS. pyogenes, suggesting that its signaling pathway could be an attractive antivirulence drug target againstS. pyogenesinfections.
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Annibal, Andrea, Roberto Ripa, Eugen Ballhysa, Christian Latza, Nadine Hochhard, and Adam Antebi. "Mass spectrometric characterization of cyclic dinucleotides (CDNs) in vivo." Analytical and Bioanalytical Chemistry, September 2, 2021. http://dx.doi.org/10.1007/s00216-021-03628-6.
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AbstractCyclic dinucleotides (CDNs) are key secondary messenger molecules produced by cyclic dinucleotide synthases that trigger various cellular signaling cascades from bacteria to vertebrates. In mammals, cyclic GMP-AMP synthase (cGAS) has been shown to bind to intracellular DNA and catalyze the production of the dinucleotide 2′3′ cGAMP, which signals downstream effectors to regulate immune function, interferon signaling, and the antiviral response. Despite the importance of CDNs, sensitive and accurate methods to measure their levels in vivo are lacking. Here, we report a novel LC-MS/MS method to quantify CDNs in vivo. We characterized the mass spectrometric behavior of four different biologically relevant CDNs (c-di-AMP, c-di-GMP, 3′3′ cGAMP, 2′3′ cGAMP) and provided a means of visually representing fragmentation resulting from collision-induced dissociation at different energies using collision energy breakdown graphs. We then validated the method and quantified CDNs in two in vivo systems, the bacteria Escherichia coli OP50 and the killifish Nothobranchius furzeri. We found that optimization of LC-MS/MS parameters is crucial to sensitivity and accuracy. These technical advances should help illuminate physiological and pathological roles of these CDNs in in vivo settings.
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Lu, Zengzeng, Yuqian Fu, Xueyuan Zhou, Hekang Du, and Qi Chen. "Cyclic dinucleotides mediate bacterial immunity by dinucleotide cyclase in Vibrio." Frontiers in Microbiology 13 (December 22, 2022). http://dx.doi.org/10.3389/fmicb.2022.1065945.
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The cyclic GMP-AMP (cGAMP) synthase (cGAS) recognizes cytosolic DNA and synthesizes the second messenger, cGAMP, thus activating the adaptor protein stimulator of interferon genes (STING) and initiating the innate immune responses against microbial infections. cGAS-STING pathway has been crucially implicated in autoimmune diseases, cellular senescence, and cancer immunotherapy, while the cGAS-like receptors in bacteria can protect it against viral infections. Dinucleotide cyclase in Vibrio (DncV) is a dinucleotide cyclase originally identified in Vibrio cholerae. The synthesis of cyclic nucleotides by DncV, including c-di-GMP, c-di-AMP, and cGAMP mediates bacterial colonization, cell membrane formation, and virulence. DncV is a structural and functional homolog of the mammalian cytoplasmic DNA sensor, cGAS, implicating cGAS-STING signaling cascades may have originated in the bacterial immune system. Herein, we summarize the roles of DncV in bacterial immunity, which are expected to provide insights into the evolution of cGAS-STING signaling.
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Gautam, Sudhanshu, Avisek Mahapa, Lahari Yeramala, Apoorv Gandhi, Sushma Krishnan, Kutti R. Vinothkumar, and Dipankar Chatterji. "Regulatory mechanisms of c‐di‐AMP synthase ( MsDisA ) protein from Mycobacterium smegmatis revealed by a structure – function analysis." Protein Science, January 20, 2023. http://dx.doi.org/10.1002/pro.4568.
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McFarland, Adelle P., Thomas P. Burke, Alexie A. Carletti, Rochelle C. Glover, Hannah Tabakh, Matthew D. Welch, and Joshua J. Woodward. "RECON-Dependent Inflammation in Hepatocytes EnhancesListeria monocytogenesCell-to-Cell Spread." mBio 9, no. 3 (May 15, 2018). http://dx.doi.org/10.1128/mbio.00526-18.
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ABSTRACTThe oxidoreductase RECON is a high-affinity cytosolic sensor of bacterium-derived cyclic dinucleotides (CDNs). CDN binding inhibits RECON’s enzymatic activity and subsequently promotes inflammation. In this study, we sought to characterize the effects of RECON on the infection cycle of the intracellular bacteriumListeria monocytogenes, which secretes cyclic di-AMP (c-di-AMP) into the cytosol of infected host cells. Here, we report that during infection of RECON-deficient hepatocytes, which exhibit hyperinflammatory responses,L. monocytogenesexhibits significantly enhanced cell-to-cell spread. Enhanced bacterial spread could not be attributed to alterations in PrfA or ActA, two virulence factors critical for intracellular motility and intercellular spread. Detailed microscopic analyses revealed that in the absence of RECON,L. monocytogenesactin tail lengths were significantly longer and there was a larger number of faster-moving bacteria. Complementation experiments demonstrated that the effects of RECON onL. monocytogenesspread and actin tail lengths were linked to its enzymatic activity. RECON enzyme activity suppresses NF-κB activation and is inhibited by c-di-AMP. Consistent with these previous findings, we found that augmented NF-κB activation in the absence of RECON caused enhancedL. monocytogenescell-to-cell spread and thatL. monocytogenesspread correlated with c-di-AMP secretion. Finally, we discovered that, remarkably, increased NF-κB-dependent inducible nitric oxide synthase expression and nitric oxide production were responsible for promotingL. monocytogenescell-to-cell spread. The work presented here supports a model wherebyL. monocytogenessecretion of c-di-AMP inhibits RECON’s enzymatic activity, drives augmented NF-κB activation and nitric oxide production, and ultimately enhances intercellular spread.IMPORTANCETo date, bacterial CDNs in eukaryotes are solely appreciated for their capacity to activate cytosolic sensing pathways in innate immunity. However, it remains unclear whether pathogens that actively secrete CDNs benefit from this process. Here, we provide evidence that secretion of CDNs leads to enhancement ofL. monocytogenescell-to-cell spread. This is a heretofore-unknown role of these molecules and suggestsL. monocytogenesmay benefit from their secretion in certain contexts. Molecular characterization revealed that, surprisingly, nitric oxide was responsible for the enhanced spread. Pathogens act to prevent nitric oxide production or, likeL. monocytogenes, they have evolved to resist its direct antimicrobial effects. This study provides evidence that intracellular bacterial pathogens not only tolerate nitric oxide, which is inevitably encountered during infection, but can also capitalize on the changes this pleiotropic molecule enacts on the host cell.
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Modena, M., C. Vecoli, C. Caselli, G. Todiere, R. Poddighe, S. Valente, F. Bandini, et al. "Association of eNOS Glu298Asp polymorphism with cardiometabolic risk and inducible myocardial ischemia in patients with stable coronary artery disease." European Heart Journal 43, Supplement_2 (October 1, 2022). http://dx.doi.org/10.1093/eurheartj/ehac544.1112.
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Abstract Background The endothelial nitric oxide synthase (eNOS) gene deficiency is known to cause insulin resistance, hypertension, hypertriglyceridemia and impaired coronary vasodilating capability in animal models. In the general clinical population, the eNOS gene polymorphism (Glu298Asp, G894T), able to reduce eNOS activity, was associated either with features of the metabolic syndrome or prevalence of coronary artery disease (CAD). Purpose To investigate the possible association of Glu298Asp polymorphism with cardiometabolic risk [insulin resistance, increased triglycerides (TG) and low HDL-cholesterol (HDL-C)], obstructive CAD and inducible myocardial ischemia in stable patients with suspected coronary disease. Methods Six cardiology units enrolled a total of 506 consecutive patients (314 males; mean age 62±9 years) referred for suspected CAD within the BIOGEN-CARE Tuscan Region Italian Study. Among these, 325 patients underwent stress ECG or cardiac imaging to assess the presence of inducible ischemia and 436 patients underwent non invasive computerized tomography or invasive coronary angiography to assess the presence of obstructive CAD (>50% stenosis in at least one major coronary vessel). Blood samples were collected from each patient for genotyping and measurements of lipid and glucose parameters. The TG/HDL-C ratio and the TyG-index [ln(TG × Fasting plasma glucose/2)] were used as synthetic markers of atherogenic dyslipidemia and insulin resistance, main components of the cardiometabolic risk. Results In the whole population, 49.6% of patients were homozygous for the G894allele, 40.9% heterozygotes, and 9.5% homozygous for T894. Myocardial ischemia was documented in 160/325 (49.2%) patients undergoing stress testing and obstructive CAD in 178/436 (40.8%) patients undergoing coronary angiography. Patients carrying the T allele (dominant model TT+GT vs GG) had higher TG/HDL ratio (2.7±1.8 vs 2.5±1.9, P=0.03) (Figure) without differences in other lipid and glucose markers. Independent predictors of obstructive CAD were age, gender, obesity, diabetes and TG/HDL-C ratio but not the the T allele (OR 0.80; CI 0.51–1.25; ns). Independent predictors of inducible ischemia were age, gender, obesity and the T allele (OR 1.91; CI 01.19–3.08; P=0.007). Stratifying the population for both obstructive CAD and ischemia, the T allele was associated with increased risk of ischemia (OR 1.96; CI 1.11–3.44; P=0.02) even after adjustment for the presence of obstructive CAD (OR 3.09; CI 1.85–5.78; P<0.001) (Figure 1). Conclusions In stable patients with suspected CAD, the eNOS Glu298Asp gene polymorphism is an independent risk factor for inducible myocardial ischemia and is significantly associated with the specific cardiometabolic risk expressed by high TG and low HDL-C which independently predicts obstructive CAD. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): “BIOhumoral and GENetic predictors of CARdiac Evolving phenotype in Ischemic Heart Disease (BIOGENCARE-IHD)”; funded by Toscan Region-Programma per la ricerca regionale in materia di Salute 2009
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Peterson, Bret N., Megan K. M. Young, Shukun Luo, Jeffrey Wang, Aaron T. Whiteley, Joshua J. Woodward, Liang Tong, Jue D. Wang, and Daniel A. Portnoy. "(p)ppGpp and c-di-AMP Homeostasis Is Controlled by CbpB in Listeria monocytogenes." mBio 11, no. 4 (August 11, 2020). http://dx.doi.org/10.1128/mbio.01625-20.
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ABSTRACT The facultative intracellular pathogen Listeria monocytogenes, like many related Firmicutes, uses the nucleotide second messenger cyclic di-AMP (c-di-AMP) to adapt to changes in nutrient availability, osmotic stress, and the presence of cell wall-acting antibiotics. In rich medium, c-di-AMP is essential; however, mutations in cbpB, the gene encoding c-di-AMP binding protein B, suppress essentiality. In this study, we identified that the reason for cbpB-dependent essentiality is through induction of the stringent response by RelA. RelA is a bifunctional RelA/SpoT homolog (RSH) that modulates levels of (p)ppGpp, a secondary messenger that orchestrates the stringent response through multiple allosteric interactions. We performed a forward genetic suppressor screen on bacteria lacking c-di-AMP to identify genomic mutations that rescued growth while cbpB was constitutively expressed and identified mutations in the synthetase domain of RelA. The synthetase domain of RelA was also identified as an interacting partner of CbpB in a yeast-2-hybrid screen. Biochemical analyses confirmed that free CbpB activates RelA while c-di-AMP inhibits its activation. We solved the crystal structure of CbpB bound and unbound to c-di-AMP and provide insight into the region important for c-di-AMP binding and RelA activation. The results of this study show that CbpB completes a homeostatic regulatory circuit between c-di-AMP and (p)ppGpp in Listeria monocytogenes. IMPORTANCE Bacteria must efficiently maintain homeostasis of essential molecules to survive in the environment. We found that the levels of c-di-AMP and (p)ppGpp, two nucleotide second messengers that are highly conserved throughout the microbial world, coexist in a homeostatic loop in the facultative intracellular pathogen Listeria monocytogenes. Here, we found that cyclic di-AMP binding protein B (CbpB) acts as a c-di-AMP sensor that promotes the synthesis of (p)ppGpp by binding to RelA when c-di-AMP levels are low. Addition of c-di-AMP prevented RelA activation by binding and sequestering CbpB. Previous studies showed that (p)ppGpp binds and inhibits c-di-AMP phosphodiesterases, resulting in an increase in c-di-AMP. This pathway is controlled via direct enzymatic regulation and indicates an additional mechanism of ribosome-independent stringent activation.
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Makieva, Sofia, Marco Reschini, Stefania Ferrari, Francesca Bonesi, Elisa Polledri, Silvia Fustinoni, Liliana Restelli, Veronica Sarais, Edgardo Somigliana, and Paola Viganò. "Oral Vitamin D supplementation impacts gene expression in granulosa cells in women undergoing IVF." Human Reproduction, December 11, 2020. http://dx.doi.org/10.1093/humrep/deaa262.
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Abstract STUDY QUESTION Does oral Vitamin D supplementation alter the hormonal milieu of follicular fluid (FF) and the transcriptomic profile of luteinised granulosa cells (GCs) in women with Vitamin D deficiency undergoing IVF? SUMMARY ANSWER A transcriptomic signature relevant to oral Vitamin D supplementation in luteinised GCs was demonstrated, although Vitamin D supplementation did not alter hormone levels in FF. WHAT IS KNOWN ALREADY Vitamin D deficiency is linked to lower live birth rates among women undergoing IVF. It is unclear whether Vitamin D elicits a targeted action in reproductive physiology or is a surrogate marker of overall well-being. Several in-vitro studies, but none in vivo, have examined the impact of Vitamin D on the periovulatory follicle, focusing on GCs as a proxy marker of oocyte competence. STUDY DESIGN, SIZE, DURATION We present a report of secondary outcomes from the SUNDRO clinical trial, which was launched in 2016 to determine whether Vitamin D supplementation can improve the IVF outcomes of women who are deficient in Vitamin D (<30 ng/ml). FF samples of 145 women who were randomised to receive Vitamin D or placebo from March 2017 to January 2019 were collected. All follicles that were aspirated in our study measured ≥11 mm on the day of hCG trigger. The first cohort of samples was collected from the dominant follicle of each participant and utilised for hormone profiling (n = 50 Vitamin D, n = 45 Placebo). For the second cohort, the follicle aspirates of each participant were pooled to create a single FF sample, which was used for the isolation of GCs for gene expression studies (n = 20 Vitamin D, n = 30 placebo). Six of the samples from the second cohort were used for RNA-sequencing analysis (n = 3 Vitamin D, n = 3 placebo). PARTICIPANTS/MATERIALS, SETTING, METHODS Two academic infertility units were involved in the recruitment of the participants, who received a single dose of oral 25-hydroxyvitamin D (600 000 IU) or placebo, 2–12 weeks before oocyte retrieval. Women in both groups were deficient in Vitamin D, aged 18–39 years with a normal BMI (18–25 kg/m2) and <3 previous IVF cycles. The FF was aspirated at the time of oocyte retrieval and stored. Liquid chromatography tandem mass spectrometry was used to measure FF abundance of 25-hydroxyvitamin D, aldosterone, androstenedione, cortisol, cortisone, corticosterone, 11-deoxycorticosterone, 11-deoxycortisol, 21-deoxycortisol, dehydroepiandrosterone, dehydroepiandrosterone sulfate, dihydrotestosterone, oestradiol (E2), 17-OH-hydroxyprogesterone, progesterone (P4) and testosterone. GCs were isolated from pooled FFs and the transcriptome was evaluated by RNA-sequencing and RT-PCR. Ingenuity pathway analysis (IPA) was used to assess the top canonical pathways and upstream regulators mediating the action of Vitamin D. MAIN RESULTS AND THE ROLE OF CHANCE At oocyte retrieval, FF concentration of 25-hydroxyvitamin D was 2.8-fold higher (P < 0.001) in the Vitamin D group (39.5 ng/ml; n = 50) compared to placebo (13.8 ng/ml; n = 45) but no other hormonal differences were detected. In the placebo group, but not the Vitamin D group, weak correlations of 25-hydroxyvitamin D concentration with P4 (r = 0.31, P = 0.03) and E2 (r = 0.45, P = 0.002) were observed. RNA-sequencing identified 44 differentially expressed genes in the GCs of patients who received Vitamin D (n = 3) compared to placebo (n = 3). RT-PCR demonstrated upregulation of VDR (vitamin D receptor), GSTA3 (glutathione S-transferase A3) and IL21R (interleukin 21 receptor), and downregulation of P T GS2 (prostaglandin-endoperoxide synthase 2), KLF4 (kruppel-like factor 4), T RP C4 (transient receptor potential cation channel subfamily C member 4), VEGF (vascular endothelial growth factor), RXRB (retinoid X receptor beta) and AGER (advanced glycosylation end-product specific receptor) genes in the Vitamin D (n = 17) versus placebo (n = 27) group. IPA suggested roles of Vitamin D in antioxidant defence. LIMITATIONS, REASONS FOR CAUTION Interpretation of the data is influenced by our intervention strategy (2–12 weeks prior to retrieval). As folliculogenesis may last 5–6 months, our protocol can only examine with confidence the impact of Vitamin D on the final stages of follicular growth. Furthermore, we examined the hormonal profile of the dominant follicle only, while the GC data reflect the transcriptome of all (pooled) follicles large enough to be used for IVF. Luteinised GCs from controlled ovarian stimulation were used in this study, which may be functionally distinct from the GCs of developing follicles. Moreover, the sample size for RNA-sequencing analysis was low (n = 3 per group), regardless of validation by RT-PCR that was performed on a larger cohort, introducing complexity to the IPA analysis, which required an input of data with P-adjusted <0.08 instead of <0.05 to be informative. WIDER IMPLICATIONS OF THE FINDINGS This is the first in-vivo study to show that Vitamin D supplementation alters gene expression in luteinised GCs. In contrast to some in-vitro evidence, no effect of the intervention on expression of genes encoding steroidogenic enzymes was observed. Unlike other studies, our results suggest that supplementation with Vitamin D is unlikely to directly influence hormone availability in FF. Our findings instead reinforce the hypothesis that Vitamin D could be considered one of the gatekeepers in protecting against an exaggerated response to ovarian stimulation. STUDY FUNDING/COMPETING INTEREST(S) The study has been funded by the Italian Ministry of Health (RF-2013-02358757) following peer review in the competitive ‘Bando di Ricerca Finalizzata e Giovani Ricercatori 2013’ for the clinical trial SUNDRO (EudraCT registration number 2015-004233-27). There are no competing interests. TRIAL REGISTRATION NUMBER EudraCT registration number 2015-004233-27.
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Colomer-Winter, C., A. L. Flores-Mireles, S. Kundra, S. J. Hultgren, and J. A. Lemos. "(p)ppGpp and CodY Promote Enterococcus faecalis Virulence in a Murine Model of Catheter-Associated Urinary Tract Infection." mSphere 4, no. 4 (July 24, 2019). http://dx.doi.org/10.1128/msphere.00392-19.
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ABSTRACT In Firmicutes, the nutrient-sensing regulators (p)ppGpp, the effector molecule of the stringent response, and CodY work in tandem to maintain bacterial fitness during infection. Here, we tested (p)ppGpp and codY mutant strains of Enterococcus faecalis in a catheter-associated urinary tract infection (CAUTI) mouse model and used global transcriptional analysis to investigate the relationship of (p)ppGpp and CodY. The absence of (p)ppGpp or single inactivation of codY led to lower bacterial loads in catheterized bladders and diminished biofilm formation on fibrinogen-coated surfaces under in vitro and in vivo conditions. Single inactivation of the bifunctional (p)ppGpp synthetase/hydrolase rel did not affect virulence, supporting previous evidence that the association of (p)ppGpp with enterococcal virulence is not dependent on the activation of the stringent response. Inactivation of codY in the (p)ppGpp0 strain restored E. faecalis virulence in the CAUTI model as well as the ability to form biofilms in vitro. Transcriptome analysis revealed that inactivation of codY restores, for the most part, the dysregulated metabolism of (p)ppGpp0 cells. While a clear linkage between (p)ppGpp and CodY with expression of virulence factors could not be established, targeted transcriptional analysis indicates that a possible association between (p)ppGpp and c-di-AMP signaling pathways in response to the conditions found in the bladder may play a role in enterococcal CAUTI. Collectively, data from this study identify the (p)ppGpp-CodY network as an important contributor to enterococcal virulence in catheterized mouse bladder and support that basal (p)ppGpp pools and CodY promote virulence through maintenance of a balanced metabolism under adverse conditions. IMPORTANCE Catheter-associated urinary tract infections (CAUTIs) are one of the most frequent types of infection found in the hospital setting that can develop into serious and potentially fatal bloodstream infections. One of the infectious agents that frequently causes complicated CAUTI is the bacterium Enterococcus faecalis, a leading cause of hospital-acquired infections that are often difficult to treat due to the exceptional multidrug resistance of some isolates. Understanding the mechanisms by which E. faecalis causes CAUTI will aid in the discovery of new druggable targets to treat these infections. In this study, we report the importance of two nutrient-sensing bacterial regulators, named (p)ppGpp and CodY, for the ability of E. faecalis to infect the catheterized bladder of mice.