Phosphodiesterase Activity Research Articles

A Review Article on Synthesis of Different Types of Bioactive Spiropyrazole Derivatives

Pyrazole category with all derivatives have well-known with biological and industrial applications. Pyrazole combined with other cycle (aliphatic or heterocycles) through one carbon atoms afforded spiropyrazoles. This spiropyrazoles characterized with biological importance as antitumor, analgesic, anti-inflammatory, vasodilation, aldosterone antagonistic, antimicrobial, phosphodiesterase, anabolic, androgenic, progestational, and salt-retaining activities. We interested in this review to collect and sort the spiropyrazoles according to their combined rings and illustrated the different methods of synthesis until the middle of 2021.

Structural investigation and gene deletion studies of mycobacterial oligoribonuclease reveal modulation of c-di-GMP-mediated phenotypes

Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger required for normal physiology as well as survival under hypoxic and reductive stress conditions of mycobacterial cells. Complete degradation of c-di-GMP is necessary for signal termination and maintaining its homeostasis inside the cells. Homeostasis of c-di-GMP in mycobacteria is brought about by the bifunctional diguanylate cyclase (DGC) that synthesizes c-di-GMP from two molecules of GTP and also catalyses the asymmetric cleavage of c-di-GMP to linear pGpG through its phosphodiesterase activity. However, the mycobacterial enzyme for the last step of degradation from pGpG to GMP has not been characterized thus far. Here, we present the identification of oligoribonuclease (Orn) as the most likely phosphodiesterase to degrade pGpG to GMP through AlphaFold-empowered structural homology that exhibited in vitro phosphodiesterase activity on pGpG substrates. In order to understand the physiological role of Orn in mycobacteria, we created a deletion mutant of orn in M. smegmatis and analysed the phenotypes that are associated with c-di-GMP signaling. We find that orn plays important roles in vivo and is required not only for proper growth of M. smegmatis in normal and stress conditions but also for biofilm formation.

Twin cyclic mononucleotide cyclase and phosphodiesterase domain architecture as a common feature in complex plant proteins

The majority of proteins in both prokaryote and eukaryote proteomes consist of two or more functional centers, which allows for intramolecular tuning of protein functions. Such architecture, as opposed to animal orthologs, applies to the plant cyclases (CNC) and phosphodiesterases (PDEs), the vast majority of which are part of larger multifunctional proteins. In plants, until recently, only two cases of combinations of CNC-PDE in one protein were reported. Here we propose that in plants, multifunctional proteins in which the PDE motif has been identified, the presence of the additional CNC center is common. Searching the Arabidopsis thaliana proteome with a combined PDE-CNC motif allowed the creation of a database of proteins with both activities. One such example is methylenetetrahydrofolate dehydrogenase, in which we determined the activities of adenylate cyclase (AC) and PDE. Based on biochemical and mutagenesis analyses we assessed the impact of the AC and PDE catalytic centers on the dehydrogenase activity. This allowed us to propose additional regulatory mechanism that govern folate metabolism by cAMP. It is therefore conceivable that the combined CNC-PDE architecture is a common regulatory configuration, where control of the level of cyclic nucleotides (cNMP) influences other catalytic activities of the protein.

The role of the atrium in the development of the Takotsubo Syndrome – investigation of a patient-specific atrial stem cell model

Abstract Background and aims The Takotsubo syndrome (TTS) is characterised by an acute left ventricular dysfunction without exhibiting signs of stenosis. TTS affects mainly the left ventricle, however, its pathophysiology comprises transient impairments in left atrium functions with a prevalence of atrial fibrillation (AF) of around 18% correlating with higher ventricular heart rhythm disorders. In this study, we aimed to prove the hypothesis that molecular and cellular arrhythmic events contribute to the development of TTS under catecholamine stress and to test if the currently clinically used β-blockers (Metoprolol) or the PDE4 activator MR-L2 are suitable for Takotsubo cardiomyopathy in vitro. Methods and results We generated induced pluripotent stem cell-derived atrial cardiomyocytes (TTS-iPSC-aCMs) from TTS patients, confirmed atrial marker expression (MLC2a, PItX2, NR2F2), and treated them with catecholamines (Iso) to mimic TTS-phenotype. Using a cytosolic Förster resonance energy transfer (FRET) based cAMP sensor, we tested the activity of phosphodiesterases (PDEs) in TTS-iPSC-aCMs and observed that after β-AR stimulation, the strong effects of the PDE4 family in the cytosol of atrial control cells were significantly decreased in aCMs of the TTS patients. This effect was rescued after application of PDE4 activator MR-L2 and is in line with the previously described downregulation of PDE4 in human atrial myocardium of AF patients. In functional studies, Iso-induced increase in systolic Ca2+ transient amplitude was more pronounced in TTS iPSC-aCM compared to controls. These effects were rescued by both, the clinically approved β-blocker Metoprolol and by MR-L2. To analyse arrhythmic events in atrial TTS CMs, we performed confocal microscope Ca2+ measurements and demonstrated that the diastolic sarcoplasmic reticulum Ca2+ leak was increased in iPSC aCMs of TTS patients compared to control under basal conditions and after Iso-treatment. In addition, TTS patients displayed faster Ca2+ kinetics compared to control cells, already under basal conditions. These results were underlined on a molecular level by increased phospholamban phosphorylation in TTS iPSC-aCM. Subsequent treatment with Metoprolol rescued the Ca2+ kinetic parameters and the increased calcium sparks in all cell lines. Conclusion In conclusion, we were able to draw a comprehensive picture on the role of the atrium in the development of arrhythmias in TTS. We found TTS-patient-specific differences with reduced PDE4 activity, elevated arrhythmic events and enhanced reactions to catecholamines, which could be rescued by the clinically approved drug Metoprolol and partly by the PDE4 activator MR-L2. Therefore, Metoprolol has proven to be an effective treatment option for TTS and preliminary data of MR-L2 demonstrate promising effects as a new patient-specific therapeutic target for TTS under catecholamine-stress. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): German centre for cardiovascular research

Effects of long-term phosphorus addition on soil ratios of phosphomonoesterase to phosphodiesterase in three tropical forests

AbstractSoil microorganisms in tropical forests can adapt to phosphorus (P)-poor conditions by changing the activity ratios of different types of phosphatases. We tested whether microorganisms in P-poor tropical forest soils increased the phosphomonoesterase (PME) to phosphodiesterase (PDE) activity ratio, because a one-step enzymatic reaction of monoester P degradation might be more adaptive for microbial P acquisition than a two-step reaction of diester P degradation. A continuous 10-year P addition experiment was performed in three tropical forests. The activities of PME and PDE, and their ratio in soil, were determined under the hypothesis that the P-fertilized plots where P shortage is relieved would have lower PME:PDE ratios than the unfertilized controls. We demonstrated that long-term P addition in tropical forest soil did not alter the PME:PDE ratio in primary and secondary forests, whereas P fertilization elevated the PME:PDE ratio in planted forest. These results were in contrast to previous results. The long-term, large-scale P fertilization in our study may have reduced litter- and/or throughfall-derived PDE, which negated the lowered PME:PDE ratio via exogenous P inputs.

Genomic regulation of transcription and RNA processing by the multitasking Integrator complex.

In higher eukaryotes, fine-tuned activation of protein-coding genes and many non-coding RNAs pivots around the regulated activity of RNA polymerase II (Pol II). The Integrator complex is the only Pol II-associated large multiprotein complex that is metazoan specific, and has therefore been understudied for years. Integrator comprises at least 14 subunits, which are grouped into distinct functional modules. The phosphodiesterase activity of the core catalytic module is co-transcriptionally directed against several RNA species, including long non-coding RNAs (lncRNAs), U small nuclear RNAs (U snRNAs), PIWI-interacting RNAs (piRNAs), enhancer RNAs and nascent pre-mRNAs. Processing of non-coding RNAs by Integrator is essential for their biogenesis, and at protein-coding genes, Integrator is a key modulator of Pol II promoter-proximal pausing and transcript elongation. Recent studies have identified an Integrator-specific serine/threonine-protein phosphatase 2A (PP2A) module, which targets Pol II and other components of the basal transcription machinery. In this Review, we discuss how the activity of Integrator regulates transcription, RNA processing, chromatin landscape and DNA repair. We also discuss the diverse roles of Integrator in development and tumorigenesis.

Apremilast ameliorates acute respiratory distress syndrome by inhibiting neutrophil-induced oxidative stress

BackgroundThe pathogenesis of acute respiratory distress syndrome (ARDS) is attributed to the dysregulation of oxidative stress and neutrophil recruitment. We aimed to investigate the anti-inflammatory effects of apremilast on human neutrophils and assess its efficacy for treating ARDS. MethodsWe analysed superoxide anion generation, integrin expression, and adhesion in activated human neutrophils using spectrophotometry, flow cytometry, and immunofluorescence microscopy. Phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) was determined using immunoblotting. A murine lipopolysaccharide (LPS)-induced ARDS model was used to evaluate the therapeutic effects of apremilast. ResultsApremilast significantly decreased superoxide anion production, reactive oxygen species (ROS) generation, cluster of differentiation (CD)11 b expression, and neutrophil adhesion in formyl-l-methionyl-l-leucyl-l-phenylalanine activated human neutrophils. Apremilast elevated cyclic 3′,5′-adenosine monophosphate (cAMP) and protein kinase A (PKA) activity in activated neutrophils. It reduced cellular cAMP-specific phosphodiesterase (PDE) activity and selectively inhibited enzymatic PDE4 activity. The activated cAMP/PKA pathway suppressed the phosphorylation of ERK and JNK as well as Ca2+ mobilization in activated neutrophils. All inhibitory effects of apremilast on activated neutrophils were reversed by a PKA inhibitor. In vivo examinations indicated that apremilast alleviated lung neutrophil infiltration, myeloperoxidase (MPO) activity, pulmonary oedema, and alveolar damage in LPS-induced ARDS. ConclusionApremilast inhibits inflammatory responses after neutrophil activation via cAMP/PKA-dependent inhibition of ERK and JNK activation. Our study revealed apremilast suppresses oxidative stress and chemotaxis by selectively inhibiting PDE4 in neutrophils and thus protects against endotoxin-induced ARDS in mice. Apremilast can be used as an alternative off-label drug in treating acute lung damage.

Polygenic adaptation contributes to the invasive success of the Colorado potato beetle.

How invasive species cope with novel selective pressures with limited genetic variation is a fundamental question in molecular ecology. Several mechanisms have been proposed, but they can lack generality. Here, we addressed an alternative solution, polygenic adaptation, wherein traits that arise from multiple combinations of loci may be less sensitive to loss of variation during invasion. We tested the polygenic signal of environmental adaptation of Colorado potato beetle (CPB) introduced in Eurasia. Population genomic analyses showed declining genetic diversity in the eastward expansion of Eurasian populations, and weak population genetic structure (except for the invasion fronts in Asia). Demographic history showed that all populations shared a strong bottleneck about 100 years ago when CPB was introduced to Europe. Genome scans revealed a suite of genes involved in activity regulation functions that are plausibly related to cold stress, including some well-founded functions (e.g., the activity of phosphodiesterase, the G-protein regulator) and discrete functions. Such polygenic architecture supports the hypothesis that polygenic adaptation and potentially genetic redundancy can fuel the adaptation of CPB despite strong genetic depletion, thus representing a promising general mechanism for resolving the genetic paradox of invasion. More broadly, most complex traits based on polygenes may be less sensitive to invasive bottlenecks, thus ensuring the evolutionary success of invasive species in novel environments.

Abstract P002: Mechanism Of Mesenteric Artery Vasodilation And Blood Pressure Regulation By Statins

Purpose: Statins are the world’s most prescribed drugs with a wide variety of cholesterol-dependent and -independent cardiovascular effects. Pre-clinical and clinical data suggest that statins lower blood pressure (BP) in some patients but have no effect in others. However, the underlying mechanism for such differences remain unclear. By studying the direct vascular effects of therapeutically relevant concentrations of statins in resistance mesenteric arteries from normotensive Sprague Dawley (SD) rats and Spontaneously Hypertensive (SHR) rats, we sought to determine the mechanism of differential BP regulation by statins. Methods: Pressurized arterial myography, phosphodiesterase (PDE) activity assay, ELISA-based cGMP measurement, blood pressure measurement with tail-cuff. Results: Our pressure myography data revealed that statins induce rapid vasodilation in resistance mesenteric arteries. Endothelium denudation, inhibition of endothelial nitric oxide (NO) synthase (eNOS) with L-NNA, smooth muscle cell (SMC) guanylyl cyclase (sGC) inhibition with ODQ, or the inhibition of protein kinase G (PKG) with KT5823 each abolished statin-evoked vasodilation, demonstrating a critical role of endothelium and NO signaling in this process. However, contrary to previous reports, mevalonate application had no effect on vasodilation. Statins selectively inhibited PDE1A in SMCs, elevated [cGMP] i , which could potentiate NO-GC-cGMP-PKG signaling, leading to vasodilation. Statin-evoked vasodilation is attenuated in SHR rat mesenteric arteries that have reduced eNOS expression and impaired NO signaling. Consistent with the vasodilatory action, acute statin treatment lowered systolic and diastolic BP in healthy SD rats. In contrast, acute statin treatment failed to lower systemic BP in SHR rats with reduced eNOS expression. Conclusion: Statins are endothelium-dependent vasodilator, and selective inhibitor of PDE1A in resistance mesenteric arteries. Intact endothelial function and NO production are essential for statin-induced vasodilation and the reduction of BP. Our study could provide explanation to the anti-hypertensive and BP neutral effects of statins.

Control of phosphodiesterase activity in RbdA, a regulator of biofilm dispersal

Control of phosphodiesterase activity in RbdA, a regulator of biofilm dispersal

Abstract P2106: Arrhythmia As Cause For The Development Of The Takotsubo Syndrome In A Patient-specific Atrial Stem Cell Model

Background and Aims: The Takotsubo syndrome (TTS) is characterised by an acute left ventricular dysfunction without exhibiting signs of stenosis. TTS affects mainly the left ventricle, however, its pathophysiology comprises transient impairments in left atrium functions with a prevalence of atrial fibrillation (AF) of around 18% correlating with higher ventricular heart rhythm disorders. In this study, we aimed to prove the hypothesis that molecular and cellular arrhythmic events contribute to the development of TTS under catecholamine stress and to test if the currently clinically used β-blockers (Metoprolol) are suitable for Takotsubo cardiomyopathy in vitro. Methods and Results: We generated induced pluripotent stem cell-derived atrial cardiomyocytes (TTS-iPSC- a CMs) from TTS patients, confirmed atrial marker expression (MLC2a, PItX2, NR2F2), and treated the cells with catecholamines to mimic TTS-phenotype. Using a cytosolic Förster resonance energy transfer (FRET) based cAMP sensor, we tested the activity of phosphodiesterases (PDEs) in TTS-iPSC-aCMs, which are besides β-ARs main players involved in cAMP signaling in CMs. We observed that after β-AR stimulation, the strong effects of the PDE4 family in the cytosol of atrial controls were significantly decreased in aCMs of the TTS patients. This effect was completely rescued after application of PDE4 activator MR-L2 and is in line with the previously described downregulation of PDE4 in human atrial myocardium of AF patients. In functional studies, isoprenaline-induced increase in systolic Ca 2+ transient amplitude was more pronounced in TTS iPSC-aCM compared to controls. These effects were rescued by both, the clinically approved β-blocker Metoprolol as well as the PDE4 activator MR-L2. To analyse arrhythmic events in atrial TTS CMs, we performed confocal Ca 2+ measurements and demonstrated that the diastolic sarcoplasmic reticulum Ca 2+ leak was increased in iPSC aCMs of TTS patients compared to control under basal conditions and after Iso-treatment. In addition, TTS patients displayed faster Ca 2+ kinetics compared to control cells, already under basal conditions. These results were underlined on a molecular level by increased phospholamban phosphorylation in TTS iPSC-aCM .Subsequent treatment with the beta-blocker Metoprolol rescued the Ca 2+ kinetic parameters in all cell lines. Conclusion: In conclusion, we were able to draw a comprehensive picture on the role of the arrhythmia in the development of TTS. We found TTS-specific differences with reduced PDE4 activity, elevated arrhythmic events and enhanced reactions to catecholamines, which could be rescued by the clinically approved drug Metoprolol and partly by the PDE4 activator MR-L2. Therefore, Metoprolol as well as MR-L2 demonstrate promising effects as patient-specific therapeutic target for TTS.

Optogenetic manipulation of cyclic guanosine monophosphate to probe phosphodiesterase activities in megakaryocytes.

Cyclic guanosine monophosphate (cGMP) signalling plays a fundamental role in many cell types, including platelets. cGMP has been implicated in platelet formation, but mechanistic detail about its spatio-temporal regulation in megakaryocytes (MKs) is lacking. Optogenetics is a technique which allows spatio-temporal manipulation of molecular events in living cells or organisms. We took advantage of this method and expressed a photo-activated guanylyl cyclase, Blastocladiella emersonii Cyclase opsin (BeCyclop), after viral-mediated gene transfer in bone marrow (BM)-derived MKs to precisely light-modulate cGMP levels. BeCyclop-MKs showed a significantly increased cGMP concentration after illumination, which was strongly dependent on phosphodiesterase (PDE) 5 activity. This finding was corroborated by real-time imaging of cGMP signals which revealed that pharmacological PDE5 inhibition also potentiated nitric oxide-triggered cGMP generation in BM MKs. In summary, we established for the first-time optogenetics in primary MKs and show that PDE5 is the predominant PDE regulating cGMP levels in MKs. These findings also demonstrate that optogenetics allows for the precise manipulation of MK biology.

Hepatocyte Rap1a contributes to obesity- and statin-associated hyperglycemia.

SUMMARYExcessive hepatic glucose production contributes to the development of hyperglycemia and is a key feature of type 2 diabetes. Here, we report that activation of hepatocyte Rap1a suppresses gluconeogenic gene expression and glucose production, whereas Rap1a silencing stimulates them. Rap1a activation is suppressed in obese mouse liver, and restoring its activity improves glucose intolerance. As Rap1a′s membrane localization and activation depends on its geranylgeranylation, which is inhibited by statins, we show that statin-treated hepatocytes and the human liver have lower active-Rap1a levels. Similar to Rap1a inhibition, statins stimulate hepatic gluconeogenesis and increase fasting blood glucose in obese mice. Geranylgeraniol treatment, which acts as the precursor for geranylgeranyl isoprenoids, restores Rap1a activity and improves statin-mediated glucose intolerance. Mechanistically, Rap1a activation induces actin polymerization, which suppresses gluconeogenesis by Akt-mediated FoxO1 inhibition. Thus, Rap1a regulates hepatic glucose homeostasis, and blocking its activity, via lowering geranylgeranyl isoprenoids, contributes to statin-induced glucose intolerance.

Vibrio cholerae V-cGAP3 Is an HD-GYP Phosphodiesterase with a Metal Tunable Substrate Selectivity.

Cyclic dinucleotides (CDNs) are signaling molecules involved in the immune response and virulence factor production. CDN cellular levels are fine-tuned by metal-dependent phosphodiesterases (PDEs), among which HD-GYPs make up a subclass of the larger HD-domain protein superfamily. The human pathogen Vibrio cholerae (Vc) encodes nine HD-GYPs, one of which is V-cGAP3 (or VCA0931). V-cGAP3 acts on c-di-GMP and 3'3'c-GAMP, and this activity is related to bacterial infectivity. However, the extant chemical makeup of the V-cGAP3 cofactor and steady state parameters have not been established. Employing electron paramagnetic resonance and Mössbauer spectroscopy in tandem with elemental analyses and activity assays, we demonstrate that V-cGAP3 coordinates different dimetal cofactors with variable activities. MnII and FeII afford c-di-GMP hydrolysis with the highest observed rates, while c-GAMP hydrolysis is selectively dependent on Mn. V-cGAP3 has a single functional domain, and this simple architecture allows us to examine the roles of characteristic conserved residues in catalysis. Substitution of the adjacent to the active site GYP residue triad and the specifically conserved in HD-domain PDEs fifth histidine ligand (i.e., H371 in V-cGAP3) with alanines severely compromises CDN hydrolysis but only modestly affects cofactor incorporation. Our data are consistent with V-cGAP3 being the major regulator of 3'3'c-GAMP hydrolysis in Vc and delineate the importance of specific residues in tuning activity in HD-GYPs in general. We propose that HD-GYPs exhibit diversity in their metallocofactors and substrates, which may serve to increase their functional potential in regulatory pathways or allow for PDE activity upon adaptation of the parent organism to diverse environmental niches.

African Swine Fever Virus EP364R and C129R Target Cyclic GMP-AMP To Inhibit the cGAS-STING Signaling Pathway.

ABSTRACTAfrican swine fever virus (ASFV) is a highly pathogenic swine DNA virus with high mortality that causes African swine fever (ASF) in domestic pigs and wild boars. For efficient viral infection, ASFV has developed complex strategies to evade key components of antiviral innate immune responses. However, the immune escape mechanism of ASFV remains unclear. Upon ASFV infection, cyclic GMP-AMP (2′,3′-cGAMP) synthase (cGAS), a cytosolic DNA sensor, recognizes ASFV DNA and synthesizes the second messenger 2′,3′-cGAMP, which triggers interferon (IFN) production to interfere with viral replication. In this study, we demonstrated a novel immune evasion mechanism of ASFV EP364R and C129R, which blocks cellular cyclic 2′,3′-cGAMP-mediated antiviral responses. ASFV EP364R and C129R with nuclease homology inhibit IFN-mediated responses by specifically interacting with 2′,3′-cGAMP and exerting their phosphodiesterase (PDE) activity to cleave 2′,3′-cGAMP. Particularly notable is that ASFV EP364R had a region of homology with the stimulator of interferon genes (STING) protein containing a 2′,3′-cGAMP-binding motif and point mutations in the Y76S and N78A amino acids of EP364R that impaired interaction with 2′,3′-cGAMP and restored subsequent antiviral responses. These results highlight a critical role for ASFV EP364R and C129R in the inhibition of IFN responses and could be used to develop ASFV live attenuated vaccines.IMPORTANCE African swine fever (ASF) is a highly contagious hemorrhagic disease in domestic pigs and wild boars caused by African swine fever virus (ASFV). ASF is a deadly epidemic disease in the global pig industry, but no drugs or vaccines are available. Understanding the pathogenesis of ASFV is essential to developing an effective live attenuated ASFV vaccine, and investigating the immune evasion mechanisms of ASFV is crucial to improve the understanding of its pathogenesis. In this study, for the first time, we identified the EP364R and C129R, uncharacterized proteins that inhibit type I interferon signaling. ASFV EP364R and C129R specifically interacted with 2′,3′-cGAMP, the mammalian second messenger, and exerted phosphodiesterase activity to cleave 2′,3′-cGAMP. In this study, we discovered a novel mechanism by which ASFV inhibits IFN-mediated antiviral responses, and our findings can guide the understanding of ASFV pathogenesis and the development of live attenuated ASFV vaccines.

Atypical cyclic di-AMP signaling is essential for Porphyromonas gingivalis growth and regulation of cell envelope homeostasis and virulence

Microbial pathogens employ signaling systems through cyclic (di-) nucleotide monophosphates serving as second messengers to increase fitness during pathogenesis. However, signaling schemes via second messengers in Porphyromonas gingivalis, a key Gram-negative anaerobic oral pathogen, remain unknown. Here, we report that among various ubiquitous second messengers, P. gingivalis strains predominantly synthesize bis-(3′,5′)-cyclic di-adenosine monophosphate (c-di-AMP), which is essential for their growth and survival. Our findings demonstrate an unusual regulation of c-di-AMP synthesis in P. gingivalis. P. gingivalis c-di-AMP phosphodiesterase (PDE) gene (pdepg) positively regulates c-di-AMP synthesis and impedes a decrease in c-di-AMP concentration despite encoding conserved amino acid motifs for phosphodiesterase activity. Instead, the predicted regulator gene cdaR, unrelated to the c-di-AMP PDE genes, serves as a potent negative regulator of c-di-AMP synthesis in this anaerobe. Further, our findings reveal that pdepg and cdaR are required to regulate the incorporation of ATP into c-di-AMP upon pyruvate utilization, leading to enhanced biofilm formation. We show that shifts in c-di-AMP signaling change the integrity and homeostasis of cell envelope, importantly, the structure and immunoreactivity of the lipopolysaccharide layer. Additionally, microbe–microbe interactions and the virulence potential of P. gingivalis were modulated by c-di-AMP. These studies provide the first glimpse into the scheme of second messenger signaling in P. gingivalis and perhaps other Bacteroidetes. Further, our findings indicate that c-di-AMP signaling promotes the fitness of the residents of the oral cavity and the development of a pathogenic community.

Presynaptic cGMP sets synaptic strength in the striatum and is important for motor learning

The striatum is a subcortical brain region responsible for the initiation and termination of voluntary movements. Striatal spiny projection neurons receive major excitatory synaptic input from neocortex and thalamus, and cyclic nucleotides have long been known to play important roles in striatal function. Yet, the precise mechanism of action is unclear. Here, we combine optogenetic stimulation, 2‐photon imaging, and genetically encoded scavengers to dissect the regulation of striatal synapses in mice. Our data show that excitatory striatal inputs are tonically depressed by phosphodiesterases (PDEs), in particular PDE1. Blocking PDE activity boosts presynaptic calcium entry and glutamate release, leading to strongly increased synaptic transmission. Although PDE1 degrades both cAMP and cGMP, we uncover that the concentration of cGMP, not cAMP, controls the gain of striatal inputs. Disturbing this gain control mechanism in vivo impairs motor skill learning in mice. The tight dependence of striatal excitatory synapses on PDE1 and cGMP offers a new perspective on the molecular mechanisms regulating striatal activity.

Phytochemical Analysis and In Vitro and In Vivo Pharmacological Evaluation of Parthenium hysterophorus Linn.

The main aim of this research was to explore Parthenium hysterophorus Linn phytochemically and pharmacologically. Phytochemical screening is important for the isolation of active compounds before bulk extraction. The crude extracts and their fractions were screened for enzyme (urease, α-glycosidase, and phosphodiesterase) inhibition assays, in vivo analgesic, anti-inflammatory, and sedative effects. Results indicated the presence of steroids, flavonoids, etc. The crude extracts such as methanol, hexane, aqueous, ethyl acetate, chloroform, and butanol exhibited excellent urease inhibitory activities with IC50 = 43.1 ± 1.24, 31.9 ± 2.21, 31.9 ± 2.21, 57.3 ± 1.27, 49.2 ± 2.16, and 35.3 ± 1.12, respectively, as compared to standard acetohydroxamic acid (20.3 ± 0.43). The extracts (methanol, hexane, aqueous, ethyl acetate, chloroform, and butanol) also showed promising α-glycosidase potency with IC50 = 13.1 ± 0.34, 21.2 ± 1.16, 23.1 ± 0.12, 84.2 ± 2.17, 118.6 ± 3.07, and 840 ± 1.73, respectively against acarbose (840 ± 1.73). The phosphodiesterase activity of the mentioned extracts was also excellent with IC50 = 131.1 ± 2.41, 197.2 ± 3.16, 24.2 ± 0.11, 62.4 ± 2.21, 152.4 ± 1.81, and 55.3 ± 2.15, respectively, against the standard (265.5 ± 2.25). Furthermore, butanol (14.96 ± 1.78), ethyl acetate (18.98 ± 1.71), and methanol (16.87 ± 1.00) showed dose-dependent analgesic effects with a maximum inhibition of acetic acid-induced writhes. Whereas, methanolic and butanol extracts exhibited maximum inhibition of inflammation in the carrageenan paw edema test. The aqueous (p < 0.01) and butanol (p < 0.01) extracts exhibited maximum a sedative effect followed by chloroform (p < 0.05), ethyl acetate (p < 0.05), and methanolic (p < 0.05) fractions as compared to the standard drug. The current research concluded that Parthenium hysterophorus Linn has important phytochemical constituents having inhibitory effects on urease, α-glycosidase, and phosphodiesterase enzymes. Furthermore, the plant has analgesic, anti-inflammatory, and sedative effects. The P. hysterophorus needs to further be explored for the candidate molecules responsible for the abovementioned activities.

Abstract 5322: The antitumor activity and immumodulatory effect of a novel phosphodiesterase 10 inhibitor

Abstract Phosphodiesterase 10 (PDE10) is overexpressed in several types of cancer, including lung, breast and colon cancers. It has been proposed that inhibitors targeting PDE10 may exhibit antitumor activities. Previous studies have shown that PDE10 inhibition can selectively activate cGMP-dependent protein kinase to suppress oncogenic β-catenin signaling in colon cancer cells. However, the impact of PDE10 inhibitors on tumor cell immunogenicity has not been examined. Here we report the antitumor activity and immunomodulatory effect of a novel PDE10 inhibitor (ADT030) in preclinical models. We show that administration of ADT030 significantly inhibited the growth of murine colorectal cancer CT26. In a metastatic breast tumor model (4T1), ADT030 administration not only delayed the growth of the primary tumor but also reduced tumor metastasis, resulting in prolonged mouse survival. Immunological analysis revealed profound reduction of MDSCs in mice, and improved T cell tumor infiltration and function after ADT030 treatment. Moreover, the combination of ADT030 and anti-PD-1 antibody therapy led to further improved tumor growth control and mouse survival. These results implicate PDE10 as a promising therapeutic target and its inhibition may enhance the efficacy of cancer immunotherapy. Citation Format: Md Yeashin Gazi, Caitlin Brandle, Ogacheko Okoko, Xi Chen, Adam Keeton, Yulia Maxuitenko, Gary Piazza, Gang Zhou. The antitumor activity and immumodulatory effect of a novel phosphodiesterase 10 inhibitor [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 5322.

Interaction of soil microbial communities and phosphorus fractions under long-term fertilization in paddy soil

Understanding the impact of biological activities on the soil phosphorus (P) distribution under long-term fertilizer application can facilitate better soil P fertility management. Therefore, the primary objectives of this study were to investigate the effect of long-term (since 1981) fertilizer application on the soil P fractions and microbial community and to evaluate correlations between the microbial community structure and P distribution. The following treatments were implemented in a long-term field trial: no fertilization (CK), inorganic N and K (NK), inorganic P and K (PK), inorganic N, P and K (NPK) and manure+NPK (MNPK) fertilization. The study showed that the soil pH, soil organic carbon and total and available N and P concentrations were considerably higher in the MNPK treatment than in the CK treatment. The soil microbial biomass C, N and P concentrations were also significantly higher in the MNPK treatment than in the CK treatment. Among fertilization treatments, the β-1,4-glucosidase, α-1,4-glucosidase, urease, acid phosphatase and phosphodiesterase activities were the highest in the MNPK treatment. Compared to inorganic fertilization, the MNPK treatment increased the labile soil P fractions and decreased the residual soil P concentration. Continuous fertilization significantly affected the soil microbial composition. The total phospholipid fatty acid (PLFA) concentrations in the NK, PK, NPK and MNPK treatments were 23.3, 43.1, 48.7 and 87.7% higher, respectively, than in the CK treatment. A significant correlation was observed between the microbial community and soil P fractions. Moreover, the aggregated boosted tree (ABT) model showed that among the various soil biochemical properties, the total PLFA concentration was the factor that most influenced the active P pool, accounting for 35.4% of the relative influence of all soil biochemical properties examined. These findings reveal that combined manure and inorganic fertilizer application is a better approach than applying inorganic fertilizer alone for sustaining long-term P fertility by mediating soil biological activity.