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Luo HM, Ye JR, Pu FQ, Luo HL, Zhang WJ. Role and therapeutic target of P2X2/3 receptors in visceral pain. Neuropeptides 2023; 101:102355. [PMID: 37390743 DOI: 10.1016/j.npep.2023.102355] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/12/2023] [Revised: 06/19/2023] [Accepted: 06/21/2023] [Indexed: 07/02/2023]
Abstract
Visceral pain (VP) is caused by internal organ disease. VP is involved in nerve conduction and related signaling molecules, but its specific pathogenesis has not yet been fully elucidated. Currently, there are no effective methods for treating VP. The role of P2X2/3 in VP has progressed. After visceral organs are subjected to noxious stimulation, cells release ATP, activate P2X2/3, enhance the sensitivity of peripheral receptors and the plasticity of neurons, enhance sensory information transmission, sensitize the central nervous system, and play an important role in the development of VP. However, antagonists possess the pharmacological effect of relieving pain. Therefore, in this review, we summarize the biological functions of P2X2/3 and discuss the intrinsic link between P2X2/3 and VP. Moreover, we focus on the pharmacological effects of P2X2/3 antagonists on VP therapy and provide a theoretical basis for its targeted therapy.
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Affiliation(s)
- Hong-Mei Luo
- Department of Rheumatology, The Affiliated Hospital of Jiangxi University of Chinese Medicine, Nanchang City, Jiangxi province 343000, China
| | - Jia-Rong Ye
- Department of Gastrointestinal surgery, The Second Affiliated Hospital, Nanchang University, Nanchang City, Jiangxi province 343000, China
| | - Fan-Qin Pu
- Department of Rheumatology, The Affiliated Hospital of Jiangxi University of Chinese Medicine, Nanchang City, Jiangxi province 343000, China
| | - Hong-Liang Luo
- Department of Rehabilitation Medicine, The Second Affiliated Hospital, Nanchang University, Nanchang City, Jiangxi province 343000, China
| | - Wen-Jun Zhang
- Department of Rehabilitation Medicine, The Second Affiliated Hospital, Nanchang University, Nanchang City, Jiangxi province 343000, China.
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Zhang Y, Chen Q, Chen D, Zhao W, Wang H, Yang M, Xiang Z, Yuan H. SerpinA3N attenuates ischemic stroke injury by reducing apoptosis and neuroinflammation. CNS Neurosci Ther 2021; 28:566-579. [PMID: 34897996 PMCID: PMC8928918 DOI: 10.1111/cns.13776] [Citation(s) in RCA: 34] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2021] [Revised: 11/14/2021] [Accepted: 11/27/2021] [Indexed: 11/30/2022] Open
Abstract
Objective To assess the effect of serine protein inhibitor A3N (serpinA3N) in ischemic stroke and to explore its mechanism of action. Methods Mouse ischemic stroke model was induced by transient middle cerebral artery occlusion followed by reperfusion. The expression pattern of serpinA3N was assessed using immunofluorescence, Western blot analysis, and real‐time quantitative PCR. An adeno‐associated virus (AAV) and recombinant serpinA3N were administered. Additionally, co‐immunoprecipitation‐mass spectrometry and immunofluorescence co‐staining were used to identify protein interactions. Results SerpinA3N was upregulated in astrocytes and neurons within the ischemic penumbra after stroke in the acute phase. The expression of serpinA3N gradually increased 6 h after reperfusion, peaked on the day 2–3, and then decreased by day 7. Overexpression of serpinA3N by AAV significantly reduced the infarct size and improved motor function, associated with alleviated inflammation and oxidative stress. SerpinA3N treatment also reduced apoptosis both in vivo and in vitro. Co‐immunoprecipitation‐mass spectrometry and Western blotting revealed that clusterin interacts with serpinA3N, and Akt‐mTOR pathway members were upregulated by serpinA3N both in vivo and in vitro. Conclusions SerpinA3N is expressed in astrocytes and penumbra neurons after stroke in mice. It reduces brain damage possibly via interacting with clusterin and inhibiting neuronal apoptosis and neuroinflammation.
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Affiliation(s)
- Yu Zhang
- Department of Anesthesiology, Second Affiliated Hospital of Naval Medical University, Shanghai, China
| | - Qianbo Chen
- Department of Anesthesiology, Third Affiliated Hospital of Naval Medical University, Shanghai, China
| | - Dashuang Chen
- Department of Anesthesiology, Second Affiliated Hospital of Naval Medical University, Shanghai, China
| | - Wenqi Zhao
- Department of Anesthesiology, Second Affiliated Hospital of Naval Medical University, Shanghai, China
| | - Haowei Wang
- Department of Anesthesiology, Second Affiliated Hospital of Naval Medical University, Shanghai, China
| | - Mei Yang
- Department of Anesthesiology, Second Affiliated Hospital of Naval Medical University, Shanghai, China
| | - Zhenghua Xiang
- Department of Neurobiology, Key Laboratory of Molecular Neurobiology, Ministry of Education, Naval Medical University, Shanghai, China
| | - Hongbin Yuan
- Department of Anesthesiology, Second Affiliated Hospital of Naval Medical University, Shanghai, China
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Sivcev S, Slavikova B, Ivetic M, Knezu M, Kudova E, Zemkova H. Lithocholic acid inhibits P2X2 and potentiates P2X4 receptor channel gating. J Steroid Biochem Mol Biol 2020; 202:105725. [PMID: 32652201 DOI: 10.1016/j.jsbmb.2020.105725] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/27/2020] [Revised: 06/21/2020] [Accepted: 07/05/2020] [Indexed: 02/02/2023]
Abstract
The family of ATP-gated purinergic P2X receptors comprises seven bunits (P2X1-7) that are unevenly distributed in the central and peripheral nervous systems as well as other organs. Endogenous modulators of P2X receptors are phospholipids, steroids and neurosteroids. Here, we analyzed whether bile acids, which are natural products derived from cholesterol, affect P2X receptor activity. We examined the effects of primary and secondary bile acids and newly synthesized derivatives of lithocholic acid on agonist-induced responses in HEK293T cells expressing rat P2X2, P2X4 and P2X7 receptors. Electrophysiology revealed that low micromolar concentrations of lithocholic acid and its structural analog 4-dafachronic acid strongly inhibit ATP-stimulated P2X2 but potentiate P2X4 responses, whereas primary bile acids and other secondary bile acids exhibit no or reduced effects only at higher concentrations. Agonist-stimulated P2X7 responses are significantly potentiated by lithocholic acid at moderate concentrations. Structural modifications of lithocholic acid at positions C-3, C-5 or C-17 abolish both inhibitory and potentiation effects to varying degrees, and the 3α-hydroxy group contributes to the ability of the molecule to switch between potentiation and inhibition. Lithocholic acid allosterically modulates P2X2 and P2X4 receptor sensitivity to ATP, reduces the rate of P2X4 receptor desensitization and antagonizes the effect of ivermectin on P2X4 receptor deactivation. Alanine-scanning mutagenesis of the upper halve of P2X4 transmembrane domain-1 revealed that residues Phe48, Val43 and Tyr42 are important for potentiating effect of lithocholic acid, indicating that modulatory sites for lithocholic acid and ivermectin partly overlap. Lithocholic acid also inhibits ATP-evoked currents in pituitary gonadotrophs expressing native P2X2, and potentiates ATP currents in nonidentified pituitary cells expressing P2X4 receptors. These results indicate that lithocholic acid is a bioactive steroid that may help to further unveil the importance of the P2X2, and P2X4 receptors in many physiological processes.
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Affiliation(s)
- Sonja Sivcev
- Institute of Physiology, Czech Academy of Sciences, Prague, Czech Republic; Faculty of Science, Charles University, Prague, Czech Republic
| | - Barbora Slavikova
- Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic
| | - Milorad Ivetic
- Institute of Physiology, Czech Academy of Sciences, Prague, Czech Republic; Faculty of Science, Charles University, Prague, Czech Republic
| | - Michal Knezu
- Institute of Physiology, Czech Academy of Sciences, Prague, Czech Republic; Faculty of Science, Charles University, Prague, Czech Republic
| | - Eva Kudova
- Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic
| | - Hana Zemkova
- Institute of Physiology, Czech Academy of Sciences, Prague, Czech Republic.
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Sisco NJ, Luu DD, Kim M, Van Horn WD. PIRT the TRP Channel Regulating Protein Binds Calmodulin and Cholesterol-Like Ligands. Biomolecules 2020; 10:E478. [PMID: 32245175 PMCID: PMC7175203 DOI: 10.3390/biom10030478] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2020] [Revised: 03/13/2020] [Accepted: 03/17/2020] [Indexed: 01/04/2023] Open
Abstract
Transient receptor potential (TRP) ion channels are polymodal receptors that have been implicated in a variety of pathophysiologies, including pain, obesity, and cancer. The capsaicin and heat sensor TRPV1, and the menthol and cold sensor TRPM8, have been shown to be modulated by the membrane protein PIRT (Phosphoinositide-interacting regulator of TRP). The emerging mechanism of PIRT-dependent TRPM8 regulation involves a competitive interaction between PIRT and TRPM8 for the activating phosphatidylinositol 4,5-bisphosphate (PIP2) lipid. As many PIP2 modulated ion channels also interact with calmodulin, we investigated the possible interaction between PIRT and calmodulin. Using microscale thermophoresis (MST), we show that calmodulin binds to the PIRT C-terminal α-helix, which we corroborate with a pull-down experiment, nuclear magnetic resonance-detected binding study, and Rosetta-based computational studies. Furthermore, we identify a cholesterol-recognition amino acid consensus (CRAC) domain in the outer leaflet of the first transmembrane helix of PIRT, and with MST, show that PIRT specifically binds to a number of cholesterol-derivatives. Additional studies identified that PIRT binds to cholecalciferol and oxytocin, which has mechanistic implications for the role of PIRT regulation of additional ion channels. This is the first study to show that PIRT specifically binds to a variety of ligands beyond TRP channels and PIP2.
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Affiliation(s)
- Nicholas J. Sisco
- The School of Molecular Sciences, Arizona State University, Tempe, AZ 85287, USA
- The Virginia G. Piper Biodesign Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, AZ 85281, USA
| | - Dustin D. Luu
- The School of Molecular Sciences, Arizona State University, Tempe, AZ 85287, USA
- The Virginia G. Piper Biodesign Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, AZ 85281, USA
| | - Minjoo Kim
- The School of Molecular Sciences, Arizona State University, Tempe, AZ 85287, USA
- The Virginia G. Piper Biodesign Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, AZ 85281, USA
| | - Wade D. Van Horn
- The School of Molecular Sciences, Arizona State University, Tempe, AZ 85287, USA
- The Virginia G. Piper Biodesign Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, AZ 85281, USA
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Sisco NJ, Helsell CVM, Van Horn WD. Competitive Interactions between PIRT, the Cold Sensing Ion Channel TRPM8, and PIP 2 Suggest a Mechanism for Regulation. Sci Rep 2019; 9:14128. [PMID: 31575973 PMCID: PMC6773951 DOI: 10.1038/s41598-019-49912-5] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2018] [Accepted: 09/02/2019] [Indexed: 01/18/2023] Open
Abstract
TRPM8 is a member of the transient receptor potential ion channel family where it functions as a cold and pain sensor in humans and other higher organisms. Previous studies show that TRPM8 requires the signaling phosphoinositide lipid PIP2 to function. TRPM8 function is further regulated by other diverse mechanisms, including the small modulatory membrane protein PIRT (phosphoinositide regulator of TRP). Like TRPM8, PIRT also binds PIP2 and behavioral studies have shown that PIRT is required for normal TRPM8-mediated cold-sensing. To better understand the molecular mechanism of PIRT regulation of TRPM8, solution nuclear magnetic resonance (NMR) spectroscopy was used to assign the backbone resonances of full-length human PIRT and investigate the direct binding of PIRT to PIP2 and the human TRPM8 S1-S4 transmembrane domain. Microscale thermophoresis (MST) binding studies validate the NMR results and identify a competitive PIRT interaction between PIP2 and the TRPM8 S1-S4 domain. Computational PIP2 docking to a human TRPM8 comparative model was performed to help localize where PIRT may bind TRPM8. Taken together, our data suggest a mechanism where TRPM8, PIRT, and PIP2 form a regulatory complex and PIRT modulation of TRPM8 arises, at least in part, by regulating local concentrations of PIP2 accessible to TRPM8.
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Affiliation(s)
- Nicholas J Sisco
- School of Molecular Sciences, Arizona State University, Tempe, AZ, 85287, USA
- The Biodesign Institute, Arizona State University, Tempe, AZ, 85281, USA
- The Virginia G. Piper Center for Personalized Diagnostics, Arizona State University, Tempe, AZ, 85281, USA
- The Magnetic Resonance Research Center, Arizona State University, Tempe, AZ, 85287, USA
| | - Cole V M Helsell
- School of Molecular Sciences, Arizona State University, Tempe, AZ, 85287, USA
- The Biodesign Institute, Arizona State University, Tempe, AZ, 85281, USA
- The Virginia G. Piper Center for Personalized Diagnostics, Arizona State University, Tempe, AZ, 85281, USA
- The Magnetic Resonance Research Center, Arizona State University, Tempe, AZ, 85287, USA
| | - Wade D Van Horn
- School of Molecular Sciences, Arizona State University, Tempe, AZ, 85287, USA.
- The Biodesign Institute, Arizona State University, Tempe, AZ, 85281, USA.
- The Virginia G. Piper Center for Personalized Diagnostics, Arizona State University, Tempe, AZ, 85281, USA.
- The Magnetic Resonance Research Center, Arizona State University, Tempe, AZ, 85287, USA.
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Portrait of Tissue-Specific Coexpression Networks of Noncoding RNAs (miRNA and lncRNA) and mRNAs in Normal Tissues. COMPUTATIONAL AND MATHEMATICAL METHODS IN MEDICINE 2019; 2019:9029351. [PMID: 31565069 PMCID: PMC6745163 DOI: 10.1155/2019/9029351] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/10/2019] [Revised: 08/01/2019] [Accepted: 08/10/2019] [Indexed: 02/01/2023]
Abstract
Genes that encode proteins playing a role in more than one biological process are frequently dependent on their tissue context, and human diseases result from the altered interplay of tissue- and cell-specific processes. In this work, we performed a computational approach that identifies tissue-specific co-expression networks by integrating miRNAs, long-non-coding RNAs, and mRNAs in more than eight thousands of human samples from thirty normal tissue types. Our analysis (1) shows that long-non coding RNAs and miRNAs have a high specificity, (2) confirms several known tissue-specific RNAs, and (3) identifies new tissue-specific co-expressed RNAs that are currently still not described in the literature. Some of these RNAs interact with known tissue-specific RNAs or are crucial in key cancer functions, suggesting that they are implicated in tissue specification or cell differentiation.
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Hilton JK, Salehpour T, Sisco NJ, Rath P, Van Horn WD. Phosphoinositide-interacting regulator of TRP (PIRT) has opposing effects on human and mouse TRPM8 ion channels. J Biol Chem 2018; 293:9423-9434. [PMID: 29724821 DOI: 10.1074/jbc.ra118.003563] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2018] [Revised: 04/25/2018] [Indexed: 12/24/2022] Open
Abstract
Transient receptor potential melastatin 8 (TRPM8) is a cold-sensitive ion channel with diverse physiological roles. TRPM8 activity is modulated by many mechanisms, including an interaction with the small membrane protein phosphoinositide-interacting regulator of TRP (PIRT). Here, using comparative electrophysiology experiments, we identified species-dependent differences between the human and mouse TRPM8-PIRT complexes. We found that human PIRT attenuated human TPRM8 conductance, unlike mouse PIRT, which enhanced mouse TRPM8 conductance. Quantitative Western blot analysis demonstrates that this effect does not arise from decreased trafficking of TRPM8 to the plasma membrane. Chimeric human/mouse TRPM8 channels were generated to probe the molecular basis of the PIRT modulation, and the effect was recapitulated in a pore domain chimera, demonstrating the importance of this region for PIRT-mediated regulation of TRPM8. Moreover, recombinantly expressed and purified human TRPM8 S1-S4 domain (comprising transmembrane helices S1-S4, also known as the sensing domain, ligand-sensing domain, or voltage sensing-like domain) and full-length human PIRT were used to investigate binding between the proteins. NMR experiments, supported by a pulldown assay, indicated that PIRT binds directly and specifically to the TRPM8 S1-S4 domain. Binding became saturated as the S1-S4:PIRT mole ratio approached 1. Our results have uncovered species-specific TRPM8 modulation by PIRT. They provide evidence for a direct interaction between PIRT and the TRPM8 S1-S4 domain with a 1:1 binding stoichiometry, suggesting that a functional tetrameric TRPM8 channel has four PIRT-binding sites.
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Affiliation(s)
- Jacob K Hilton
- From the School of Molecular Sciences, Arizona State University, Tempe, Arizona 85287.,the Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, Arizona 85281, and.,The Magnetic Resonance Research Center, Arizona State University, Tempe, Arizona 85287
| | - Taraneh Salehpour
- From the School of Molecular Sciences, Arizona State University, Tempe, Arizona 85287.,the Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, Arizona 85281, and.,The Magnetic Resonance Research Center, Arizona State University, Tempe, Arizona 85287
| | - Nicholas J Sisco
- From the School of Molecular Sciences, Arizona State University, Tempe, Arizona 85287.,the Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, Arizona 85281, and.,The Magnetic Resonance Research Center, Arizona State University, Tempe, Arizona 85287
| | - Parthasarathi Rath
- From the School of Molecular Sciences, Arizona State University, Tempe, Arizona 85287.,the Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, Arizona 85281, and.,The Magnetic Resonance Research Center, Arizona State University, Tempe, Arizona 85287
| | - Wade D Van Horn
- From the School of Molecular Sciences, Arizona State University, Tempe, Arizona 85287, .,the Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, Arizona 85281, and.,The Magnetic Resonance Research Center, Arizona State University, Tempe, Arizona 85287
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Dal Ben D, Antonioli L, Lambertucci C, Fornai M, Blandizzi C, Volpini R. Purinergic Ligands as Potential Therapeutic Tools for the Treatment of Inflammation-Related Intestinal Diseases. Front Pharmacol 2018; 9:212. [PMID: 29593540 PMCID: PMC5861216 DOI: 10.3389/fphar.2018.00212] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2017] [Accepted: 02/26/2018] [Indexed: 12/13/2022] Open
Abstract
Inflammation-related intestinal diseases are a set of various conditions presenting an overactive enteric immune system. A continuous overproduction of pro-inflammatory cytokines and a decreased production of anti-inflammatory modulators are generally observed, while morpho-functional alterations of the enteric nervous system lead to intestinal secretory and motor dysfunctions. The factors at the basis of these conditions are still to be totally identified and current therapeutic strategies are aimed only at achieving and maintaining remission states, by using therapeutic tools like aminosalicylates, corticosteroids, immunomodulators, biological drugs (i.e., monoclonal antibodies), and eventually surgery. Recent reports described a key role of purinergic mediators (i.e., adenosine and its nucleotides ATP and ADP) in the regulation of the activity of immune cells and enteric nervous system, showing also that alterations of the purinergic signaling are linked to pathological conditions of the intestinal tract. These data prompted to a series of investigations to test the therapeutic potential for inflammation-related intestinal conditions of compounds able to restore or modulate an altered purinergic signaling within the gut. This review provides an overview on these investigations, describing the results of preclinical and/or clinical evaluation of compounds able to stimulate or inhibit specific P2 (i.e., P2X7) or P1 (i.e., A2A or A3) receptor signaling and to modify the adenosine levels through the modulation of enzymes activity (i.e., Adenosine Deaminase) or nucleoside transporters. Recent developments in the field are also reported and the most promising purine-based therapeutic strategies for the treatment of inflammation-related gastrointestinal disorders are schematically summarized.
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Affiliation(s)
- Diego Dal Ben
- Medicinal Chemistry Unit, School of Pharmacy, University of Camerino, Camerino, Italy
| | - Luca Antonioli
- Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy
| | - Catia Lambertucci
- Medicinal Chemistry Unit, School of Pharmacy, University of Camerino, Camerino, Italy
| | - Matteo Fornai
- Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy
| | - Corrado Blandizzi
- Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy
| | - Rosaria Volpini
- Medicinal Chemistry Unit, School of Pharmacy, University of Camerino, Camerino, Italy
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