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Ethnopharmacological Relevance: Ischemic stroke is an acute central nervous system disease that poses a threat to human health. It induces a series of severe pathological mechanisms, ultimately leading to neuronal cell death in the brain due to local ischemia and hypoxia. Buyang Huanwu decoction (BYHWD), as a representative formula for treating ischemic stroke, has shown good therapeutic effects in stroke patients., Aim of the Study: This study aimed to explore the mechanism of BYHWD in promoting neural remodeling after ischemic stroke from the perspective of neuronal synaptic plasticity, based on the cAMP/PKA/CREB signaling pathway., Materials and Methods: A modified suture technique was employed to establish a rat model of MCAO. The rats were divided into sham, model, and BYHWD (20 g/kg) groups. After the corresponding intervention, rat brains from each group were collected. TMT quantitative proteomics technology was employed for the research. Following proteomics studies, we investigated the mechanism of BYHWD in the intervention of ischemic stroke through animal experiments and cell experiments. The experimental animals were divided into sham, model, and BYHWD (5 g/kg, 10 g/kg, and 20 g/kg) groups. Infarct volume and severity of brain injury were measured by TTC staining. HE staining was utilized to evaluate alterations in tissue morphology. The Golgi staining was used to observe changes in cell body, dendrites, and dendritic spines. Transmission electron microscopy was used to observe the ultrastructure of synapses in the cortex and hippocampus. TUNEL staining was conducted to identify apoptotic neurons. Meanwhile, a stable and reliable (OGD/R) SH-SY5Y cell model was established. The effect of BYHWD-containing serum on SH-SY5Y cell viability was measured by CCK-8 kit. The apoptosis situation of SH-SY5Y cells was determined by Annexin V-FITC/PI. Immunofluorescence was employed to measure the fluorescence intensity of synaptic-related factors Syt1, Psd95, and Syn1. Synaptic plasticity pathways were assessed by using RT-qPCR and Western blot to determine the expression levels of cAMP, Psd95, Prkacb, Creb1/p-Creb1, BDNF, Shank2, Syn1, Syt1, Bcl-2, Bcl-2/Bax mRNA and proteins., Results: After treatment with BYHWD, notable alterations were detected in the signaling pathways linked to synaptic plasticity and the cAMP signaling pathway-related targets among the intervention targets. This trend of change was also reflected in other bioinformatics analyses, indicating the important role of synaptic plasticity changes before and after modeling and drug intervention. The results of vivo and vitro experiments showed that BYHWD improved local pathological changes, and reduced cerebral infarct volume, and neurological function scores in MCAO rats. It increased dendritic spine density, improved synaptic structural plasticity, and had a certain neuroprotective effect. BYHWD increased the postsynaptic membrane thickness, synaptic interface curvature, and synaptic quantity. 10% BYHWD-containing serum was determined as the optimal concentration for treatment. 10% BYHWD-containing serum significantly reduced the overall apoptotic rate of (OGD/R) SH-SY5Y cells. Immunofluorescence experiments demonstrated that 10% BYHWD-containing serum could improve synaptic plasticity and increase the relative expression levels of synaptic-related proteins Syt1, Psd95, and Syn1. BYHWD and decoction-containing serum upregulated the mRNA and protein expression levels in (OGD/R) SH-SY5Y cells and MCAO rats, suggesting its ability to improve damaged neuronal synaptic plasticity and enhance transmission efficiency, which might be achieved through the regulation of the cAMP/PKA/CREB pathway., Conclusions: This study may provide a basis for clinical medication by elucidating the underlying experimental evidence for the promotion of neural plasticity after ischemic stroke by BYHWD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

5-hydroxymethylfurfural (HMF), produced by the Maillard reaction, can indicate thermal processes in food. Previous research has examined the cytotoxic, genotoxic, mutagenic, and carcinogenic characteristics of HMF and its derivatives in different organs. Nevertheless, there is currently no available evidence about the impact of HMF on male reproductive toxicity. In this study, the effects of HMF on testosterone biosynthesis in both mouse testis and TM3 Leydig cells were investigated. HMF was administered to mice at doses of 30 and 300 mg/kg/day for 21 days and to Leydig cells at concentrations of 0.1, 1, and 10 mM for 24 h. The mechanism of action of HMF on testosterone biosynthesis in both mouse testis and Leydig cells was revealed by measuring the amount of testosterone, 3',5'-cyclic adenosine monophosphate (cAMP) levels, and the expression level of some important genes in the steroidogenic pathway. In addition, its effects on general testis were examined through histopathological evaluations. Upon examination of the results, it was observed that HMF had a significant impact on reducing testosterone and cAMP levels. Furthermore, HMF inhibited the expression of steroidogenic genes, including steroidogenic acute regulatory protein, cholesterol side-chain cleavage enzyme, 3β-hydroxy dehydrogenase, and 17β-hydroxy dehydrogenase, as well as transcription factors, such as steroidogenic factor-1, GATA binding protein-4, and nerve growth factor IB. HMF-administrated groups had germinal epithelium degradation, vacuolization, and disorders in the interstitial area. Consequently, it has been proven for the first time that HMF can damage the male reproductive system by detrimentally impacting the production of testosterone., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Tong-Qiao-Huo-Xue Decoction (TQHXD) is a traditional Chinese medicinal formula widely used in the treatment of vascular dementia (VD). Although it has demonstrated good clinical efficacy, the specific molecular mechanisms underlying its therapeutic effects on VD remain unclear., Objective: This study aimed to elucidate the neuroprotective mechanisms of TQHXD to provide a scientific basis for the clinical treatment of VD., Methods: The chemical components of TQHXD were qualitatively analyzed using ultra-performance liquid chromatography (UPLC) and gas chromatography (GC). Network pharmacology predicted the potential protective mechanisms of TQHXD against VD. A rat model of VD was established through bilateral vessel occlusion (2-VO), and an oxygen-glucose deprivation/reperfusion (OGD/R) model was used to induce damage to neuronal cells of the hippocampus. In vivo experiments assessed changes in cerebral blood flow, learning and memory capabilities, hippocampal neuronal morphology, dendritic length, dendritic spine density, and synapse number in rats. We examined the expression of synaptic remodeling-related proteins and pathway proteins in the hippocampal region. In vitro assays evaluated cell viability, apoptosis, reactive oxygen species (ROS) levels, and expression of synaptic remodeling-related proteins and signaling pathway., Results: Multiple active components were identified in TQHXD. KEGG enrichment analysis suggested that the therapeutic effects of TQHXD on VD may be related to the cAMP signaling pathway. Treatment with TQHXD significantly improved learning and memory performance in VD rats, improved hippocampus morphology, and increased dendritic length, dendritic spine density, and number of synapses. Furthermore, TQHXD improved cell viability, reduced apoptosis, and decreased intracellular ROS levels in vitro. Western blotting, immunofluorescence, and enzyme-linked immunosorbent assay results collectively demonstrated that TQHXD upregulated the expression of synaptic remodeling-related proteins and pathway-related proteins both in vivo and in vitro., Conclusions: TQHXD treated VD by promoting synaptic remodeling in hippocampal neurons, likely through activation of the cAMP/PKA/CREB pathway., Competing Interests: Declaration of competing interest All authors of this research paper have directly participated in the planning, execution, or analysis of the study. The contents of this manuscript are not under consideration for publication elsewhere, and will not be copyrighted, submitted, or published elsewhere. There are no directly related manuscripts or abstracts, published or unpublished, by any author(s) of this paper. The authors also claim that there is no competitive financial interest., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Leydig cells are the main testosterone-producing cells in males. During androgen synthesis, cholesterol enters the mitochondria via the STAR protein and is converted into pregnenolone by the CYP11A1 enzyme. This steroid is then exported from the mitochondria to be metabolized to progesterone by the HSD3B1 enzyme in the endoplasmic reticulum. In this study, we used 3'Tag-RNA-Seq to identify progesterone-regulated genes in MA-10 Leydig cells. Our results indicate that high concentrations of progesterone (30 μM) are involved in a negative feedback loop that inhibits cAMP/PKA-dependent activation of Star and Cyp11a1 expression and participate in cAMP/PKA-dependent down-regulation of genes related to the metabolism of steroid hormones. Linked to activation of the MAPK signaling pathway, endoplasmic reticulum stress and apoptosis, most of the genes encoding bZIP transcription factors are upregulated by progesterone in MA-10 Leydig cells. However, only DDIT3 protein levels are increased in response to progesterone in MA-10 Leydig cells. Like normal Leydig cells, MA-10 cells very weakly express the classical nuclear receptor for progesterone, suggesting that gene regulation by progesterone is rather mediated by one of the non-classical membrane receptors for progesterone However, current findings suggest that the inhibitory effect of progesterone on STAR protein increase in response to forskolin is not dependent on PGRMC1/2 or PAQR9. Furthermore, the increase in progesterone synthesis in response to activation of the cAMP/PKA pathway is rather inhibited by siRNA-mediated knockdown of PAQR9. Overall, this study shows that progesterone produced by Leydig cells participates in the regulation of steroidogenesis through autocrine action involving negative feedback upon activation of the cAMP/PKA pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Altered polycystin-mediated endothelial flow mechanosensitivity contributes to the development of hypertension and cardiovascular complications in patients with autosomal dominant polycystic kidney disease (ADPKD). Stimulation of endothelial type 5 dopamine receptors (DR5) can acutely compensate for the endothelial consequences of polycystin deficiency, but the chronic impact of this approach must be evaluated in ADPKD. Nineteen patients with ADPKD on standard of care therapy were randomized to receive a 2-month treatment with the DR agonist rotigotine using transdermal patches, nine at 2 mg/24hours and ten at 4 mg/24hours or while ten were untreated. Rotigotine at the dose of 4 mg/24hours significantly increased nitric oxide release (nitrite levels from 10±30 to 46±34 nmol/L) and radial artery endothelium-dependent flow-mediated dilatation (from 16.4±6.3 to 22.5±7.3%) in response to hand skin heating. Systemic hemodynamics were not significantly modified but aplanation tonometry showed that rotigotine at 4 mg/24hours reduced aortic augmentation index and pulse pressure without affecting carotid-to femoral pulse wave velocity. Plasma creatinine and urea, urinary cyclic AMP, which contributes to cyst growth in ADPKD and copeptin, a surrogate marker of vasopressin, were not affected by rotigotine. In mice with a specific deletion of polycystin-1 in endothelial cells, chronic infusion of the peripheral DR5 agonist fenoldopam also improved mesenteric artery flow-mediated dilatation and reduced blood pressure. Thus, our study demonstrates that in patients with ADPKD, chronic administration of rotigotine improves conduit artery endothelial function through the restoration of flow-induced nitric oxide release as well as hemodynamics suggesting that endothelial DR5 activation may represent a promising pharmacological approach to prevent cardiovascular complications of ADPKD., (Copyright © 2024 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

The presence of cyclic adenosine monophosphate (cAMP) has been considered to be a fundamental factor in ensuring meiotic arrest prior to ovulation. cAMP is regarded as a key molecule in the regulation of oocyte maturation. However, it has been reported that increased levels of intracellular cAMP can result in abnormal cytokinesis, with some MI oocytes leading to symmetrically cleaved 2-cell MII oocytes. Consequently, we aimed to investigate the effects of elevated intracellular cAMP levels on abnormal cytokinesis and oocyte maturation during the meiosis of mouse oocytes. This study found that a high concentration of isobutylmethylxanthine (IBMX) also caused chromatin/chromosomes aggregation (AC) after the first meiosis. The rates of AC increased the greater the concentration of IBMX. In addition, AC formation was found to be reversible, showing that the re-formation of the spindle chromosome complex was possible after the IBMX was removed. In human oocytes, the chromosomes aggregate after the germinal vesicle breakdown and following the first and second polar body extrusions (the AC phase), while mouse oocytes do not have this AC phase. The results of our current study may indicate that the AC phase in human oocytes could be related to elevated levels of intracytoplasmic cAMP.

We often encounter patients with congestive heart failure refractory to conventional diuretics therapy. Kampo goreisan (Tsumura &Co. Tokyo, Japan) is receiving great concern in mediating body water balance, particularly for such a cohort. However, its detailed biological mechanism remains uncertain. Patients who received goreisan to treat congestive heart failure refractory to tolvaptan-incorporated medical therapy were prospectively included and observed for one week during the therapeutic period. The change in urine biomarkers during the first 24 h was assessed as a primary concern. Baseline factors associated with an increase in urine volume during the first 24 h were investigated as a secondary concern. A total of 18 patients were included. Median age was 81 (77, 86) and 12 (67%) were men. During the first 24 h after the initiation of goreisan, urine cyclic AMP tended to decrease, urine aquaporin-2 decreased significantly, urine osmolality decreased significantly, and urine volume tended to increase. Baseline higher common logarithm of plasma B-type natriuretic peptide was associated with any increases in urine volume during the first 24 h with an odds ratio of 73.2 (95% confidence interval 1.04-5149, p = 0.048). Baseline plasma B-type natriuretic peptide level had a positive correlation with a change in urine volume between baseline and day 1 (r = 0.533, p = 0.026). Goreisan may increase urine volume even in patients with congestive heart failure refractory to tolvaptan-incorporated medical therapy by modulating aquaporin-2 systems in the collecting duct, particularly in individuals with advanced heart failure accompanying significant congestion. Goreisan may have a regulatory effect on body fluid, rather than just forcing aquaresis., Competing Interests: Declarations Conflict of interest The authors declare that they have no conflict of interest., (© 2024. Springer Nature Japan KK, part of Springer Nature.)

When hypoxanthine was utilized as the activator for the salvage pathway in cAMP synthesis, xanthine oxidase would generate in quantity leading to low hypoxanthine conversion ratios and cell viability. To enhance cAMP salvage synthesis, fermentations with citrate/luteolin and hypoxanthine coupling added were conducted in a 7 L bioreactor and then multiple physiological indicators of fermentation with luteolin addition were assayed. Due to hypoxanthine feeding, cAMP productivity reached 0.066 g/(L·h) with 43.5% higher than control, however, cAMP synthesis, cell growth and glucose uptake all ceased at 50 h which was shortened by 22 h in comparison to control. The addition of citrate resulted in the cessation of fermentation at 61 h, on the contrary, owing to luteolin addition, cAMP fermentation performance was enhanced significantly during the whole fermentation period (72 h) with higher hypoxanthine conversion ratios and cAMP contents when compared with citrate and only hypoxanthine added batches. Multiple physiological indicators revealed that luteolin inhibited xanthine oxidase activity reducing hypoxanthine decomposition and ROS generation. ATP/AMP, NADH/NAD + and NADPH/NADP + were significantly increased especially at the late phase. Moreover, HPRT, PUP expression contents and corresponding gene transcription levels were also elevated. Luteolin could inhibit xanthine oxidase activity and further decrease hypoxanthine decomposition and ROS generation leading to higher hypoxanthine conversion and less cell damage for cAMP salvage synthesis efficiently., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The biosynthesis of cyclic 3',5'-adenosine monophosphate (cAMP) by mammalian membrane-bound adenylyl cyclases (mACs) is predominantly regulated by G-protein-coupled receptors (GPCRs). Up to now the two hexahelical transmembrane domains of mACs were considered to fix the enzyme to membranes. Here, we show that the transmembrane domains serve in addition as signal receptors and transmitters of lipid signals that control Gsα-stimulated mAC activities. We identify aliphatic fatty acids and anandamide as receptor ligands of mAC isoforms 1-7 and 9. The ligands enhance (mAC isoforms 2, 3, 7, and 9) or attenuate (isoforms 1, 4, 5, and 6) Gsα-stimulated mAC activities in vitro and in vivo. Substitution of the stimulatory membrane receptor of mAC3 by the inhibitory receptor of mAC5 results in a ligand inhibited mAC5-mAC3 chimera. Thus, we discovered a new class of membrane receptors in which two signaling modalities are at a crossing, direct tonic lipid and indirect phasic GPCR-Gsα signaling regulating the biosynthesis of cAMP., Competing Interests: ML, SE, HG, AF, AS, JS No competing interests declared, (© 2024, Landau et al.)

Aquaporin 2 (AQP2) contributes to water reabsorption and urine concentration by migrating to the luminal surface of the collecting ducts in an anti-diuretic hormone-stimulated manner, and the signaling pathway involved in AQP2 subcellular localization is a target for arginine vasopressin receptor antagonists (aquaretics). This study investigated the involvement of AQP2 in the diuretic effect and mechanisms of Goreisan (GRS), a traditional Japanese Kampo medicine used to treat conditions such as edema in patients with decreased urination. GRS exerted diuretic effects on desmopressin (DDAVP)-induced decreases in urine output and the level of AQP2 phosphorylated at Serine269 (pSer269-AQP2) in the renal tissues of mice. Furthermore, GRS inhibited the accumulation of pSer269-AQP2 to the luminal side following forskolin stimulation using a 3D culture model of the kidney collecting duct cell line mIMCD-3. GRS induced a transient increase in the intracellular Ca 2+ concentration via the calcium-sensing receptor (CaSR) and suppressed the forskolin-stimulated increase in cAMP production. These results suggest that GRS regulates urine volume by modulating the subcellular localization of AQP2 via CaSR., Competing Interests: Declarations. Competing interests: All authors are employed by Tsumura & Co. This study received funding from Tsumura & Co. The funder was involved in the following aspects of the study: study design, data collection and analysis, publication decision, and manuscript preparation. The authors declare no other competing interests. Ethics approval: All animal experiments were approved by the Laboratory Animal Committee of Tsumura & Co. (approval nos. 23 − 013, 23–024) and performed in accordance with guidelines for the conduct of animal experiments from the Ministry of Health, Labour and Welfare of Japan. This study is reported in accordance with ARRIVE guidelines., (© 2024. The Author(s).)

In liver transplantation, donor livers are typically stored in a preservation solution at 4°C for up to 12 hours. However, this short preservation duration can lead to various issues, such as suboptimal donor-recipient matching and limited opportunities for organ sharing. Previous studies have developed a long-term preservation method called supercooling liver preservation (SLP) to address these issues. However, in this study using a rat model, we observed that long-term SLP led to more severe liver damage compared with clinically prevalent traditional static cold storage (SCS) for durations less than 8 hours. To understand the potential mechanism of SLP-induced liver injury, we conducted an integrative metabolomic, transcriptomic, and proteomic analysis. We identified the PDE3B-cAMP-autophagy pathway as a key determinant of SLP-induced liver injury. Specifically, we found that PDE3B was elevated during SLP, which promoted a reduction of cAMP metabolites, triggering an AMPK-dependent autophagy process that led to liver injury in rats. We found that blocking the reduction in cAMP using the PDE3B inhibitor cilostamide inhibited autophagy and substantially ameliorated liver injury during 48-hour SLP in rat livers. Furthermore, we validated the effectiveness of cilostamide treatment in preventing liver injury in pig and human liver 48-hour SLP models. In summary, our results reveal that metabolic reprogramming involving the PDE3B-cAMP-autophagy axis is the key determinant of liver injury in long-term SLP and provide an early therapeutic strategy to prevent liver injury in this setting.

Obstruction of bile ducts due to gallstones can lead to biliary acute pancreatitis (BAP). According to Perides et al., G protein-coupled bile acid receptor-1 (GPBAR1) mediates BAP. However, Zi's findings suggest that GPR39, rather than GPBAR1, mediates TLCAS-induced increases in cytosolic calcium and acinar cell necrosis, casting doubt on the role of GPBAR1 in BAP. Numerous G protein-coupled receptors on pancreatic acinar cells utilize Ca 2+ and cyclic adenosine monophosphate (cAMP) as second messengers to manage pancreatic exocrine secretion, with significant cross-talk between these signals. The primary bile acid cholic acid (CA) and its conjugated forms are predominant in the human gallbladder. This study aimed to clarify the role and physiological significance of GPBAR1 by investigating the physiological and pathological effects of CA activation on GPBAR1 in pancreatic acinar cells. Isolated rat pancreatic acinar cells were treated with CA and CCK in vitro to observe the effect of CA-induced cAMP signaling on CCK-induced physiological and pathological calcium signaling. In vivo evaluations involved reverse biliopancreatic duct injections of 5 % sodium taurocholate (STC) or 5 % CA in rats. CA induced intracellular cAMP signaling in a concentration-dependent manner without increasing the intracellular Ca 2+ concentration. CA did not independently cause calcium overload or enzyme activation, nor did it exacerbate calcium overload or enzyme activation from high-dose CCK. Reverse biliopancreatic duct injections of 5 % CA did not cause acute pancreatitis in the rats. Transcriptomic analysis revealed that 50 μM CA induced changes in gene expression related to protein synthesis in the endoplasmic reticulum and ribosomes. Furthermore, 50 μM CA accelerated the calcium waves and increased the enzyme secretion induced by CCK. GPBAR1 was found on the basolateral membrane in rat pancreatic tissue rather than near the apical region of acinar cells. GPBAR1 activation is not crucial for BAP activity but may play a role in bile acid regulation of pancreatic exocrine secretion, suggesting that GPBAR1 is a potential therapeutic target for pancreatic exocrine insufficiency., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jun Li reports financial support was provided by National Natural Science Foundation of China. Jun Li reports financial support was provided by Project of Shaanxi Administration of Traditional Chinese Medicine. Jun Li reports financial support was provided by Xi'an Science and Technology Program. Yudan Fan and Peng An reports financial support was provided by Key Research and Development Program of Shaanxi Province of China. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: Ulcerative colitis (UC) is a chronic, non-specific inflammatory condition of the colon, characterized by recurrent episodes and a notable lack of effective pharmacological treatments. Scutellarin, a natural component, exhibits appreciable pharmacological effects and therapeutic potential for various diseases. However, its effects on UC are not fully understood, and the precise mechanisms remain to be deciphered. This study aimed to assess the therapeutic efficacy of scutellarin and elucidate its underlying mechanisms in treating UC., Methods: This study utilized dextran sulfate sodium (DSS)-induced mice to evaluate the therapeutic potential of scutellarin against UC and to elucidate the mechanisms involving the gut microbiota. An antibiotics cocktail (ABX) and fecal microbiota transplantation (FMT) were also used to determine the mechanistic role of the gut microbiota. An integrative approach combining fecal metabolomics and network pharmacology analysis was used to explore the gut microbiota-directed molecular mechanism., Results: The results showed that scutellarin provided various therapeutic benefits in UC management, including alleviating weight loss, slowing disease progression, and reducing inflammatory damage in colon structures. The improved gut microbiota after scutellarin administration contributed to these effects. Fecal metabolome revealed that scutellarin selectively mitigated DSS-induced dysregulation of gut microbiota-derived metabolites, including glycolic acid, γ-aminobutyric acid, glutamate, tryptophan, xanthine, and β-hydroxypyruvate. Network pharmacology analysis, along with in vivo experimental verification, implicated the cAMP/PKA/NF-κB pathway in the action of these metabolites in treating UC, which may be the mechanism responsible for scutellarin's curative effects on UC., Conclusion: This study demonstrates the potential of scutellarin in alleviating UC by activating the cAMP/PKA/NF-κB pathway through gut microbiota-derived metabolites, highlighting scutellarin as a promising therapeutic agent for UC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Homo- and heteromerization of G protein-coupled receptors (GPCRs) plays an important role in the regulation of receptor functions. Recently, we demonstrated an interaction between the serotonin receptor 7 (5-HT7R), a class A GPCR, and the cell adhesion molecule CD44. However, the functional consequences of this interaction on 5-HT7R-mediated signaling remained enigmatic., Methods: Using a quantitative FRET (Förster resonance energy transfer) approach, we determined the affinities for the formation of homo- and heteromeric complexes of 5-HT7R and CD44. The impact of heteromerization on 5-HT7R-mediated cAMP signaling was assessed using a cAMP responsive luciferase assay and a FRET-based cAMP biosensor under basal conditions as well as upon pharmacological modulation of the 5-HT7R and/or CD44 with specific ligands. We also investigated receptor-mediated G protein activation using BRET (bioluminescence resonance energy transfer)-based biosensors in both, homo- and heteromeric conditions. Finally, we analyzed expression profiles for 5-HT7R and CD44 in the brain during development., Results: We found that homo- and heteromerization of the 5-HT7R and CD44 occur at similar extent. Functionally, heteromerization increased 5-HT7R-mediated cAMP production under basal conditions. In contrast, agonist-mediated cAMP production was decreased in the presence of CD44. Mechanistically, this might be explained by increased Gαs and decreased GαoB activation by 5-HT7R/CD44 heteromers. Unexpectedly, treatment of the heteromeric complex with the CD44 ligand hyaluronic acid boosted constitutive 5-HT7R-mediated cAMP signaling and receptor-mediated transcription, suggesting the existence of a transactivation mechanism., Conclusions: Interaction with the hyaluronan receptor CD44 modulates both the constitutive activity of 5-HT7R as well as its agonist-mediated signaling. Heteromerization also results in the transactivation of 5-HT7R-mediated signaling via CD44 ligand., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

cAMP plays an important role as a second messenger in the stage transition of various protozoan parasites. This signaling pathway relies on multiple effectors, such as protein kinase A (PKA), exchange protein activated by cAMP, and cAMP-response element binding protein transcription factors, to initiate signal transduction in humans. The Giardia genome only contains two adenylate cyclases (ACs), a single phosphodiesterase (PDE) and a single known PKA effector, and the specific functions of these components are not fully understood. In our previous research, we demonstrated the important role of AC2-dependent cAMP signaling in promoting the encystation program. Using the NanoBit technology, we emphasized the significance of AC2-dependent cAMP biosynthesis in regulating the dissociation of the PKA regulatory domain (PKAr) and PKA catalytic domain (PKAc). In this study, our objectives are twofold: first, we used the newly developed Split-Halo to examine subcellular interactions of Gl PKAr and Gl PKAc in Giardia ; and second, we investigated whether PKAc regulates encystation-specific proteins. Our findings revealed distinct subcellular locations where Gl PKAr and Gl PKAc interacted during the trophozoite stage, including the flagella, basal bodies, and cytoplasm. Upon exposure to encystation stimuli, the interaction shifted from the flagella to the cytosol. Knockdown of Gl PKAc resulted in the downregulation of encystation-specific genes, leading to the production of fewer viable and water-resistant cysts indicating a role for PKA in the transcriptional regulation of encystation. These discoveries contribute to a deeper understanding of the cAMP signaling pathway and its important role in governing Giardia 's encystation process., Importance: The precise timing of interactions and subcellular compartmentation play crucial roles in signal transduction. The co-immunoprecipitation assay (CO-IP) has long been utilized to validate protein-protein interactions; however, CO-IPs lack spatial and temporal resolutions. Our recent study used the NanoBit assay, which showcased the reversible protein-protein interaction between PKAr and PKAc in response to cAMP analogs and encystation stimuli. Expanding on this groundwork, this study employs the Split-Halo assay to uncover the subcellular compartments where the PKAr and PKAc protein-protein interactions take place and respond to encystation stimuli. Taken together, these molecular tools provide spatiotemporal information on the protein-protein interaction, which will be useful in the field., Competing Interests: The authors declare no conflict of interest.

Single-fluorescent protein (FP)-based FLIM (fluorescence lifetime imaging microscopy) biosensors can visualize intracellular processes quantitatively. They require a single wavelength for detection, which facilitates multi-color imaging. However, their development has been limited by the absence of a general design framework and complex screening processes. In this study, we engineered FLIM biosensors for ATP (adenosine triphosphate), cAMP (cyclic adenosine monophosphate), citrate, and glucose by inserting each sensing domain into mTurquoise2 (mTQ2) between Tyr-145 and Phe-146 using peptide linkers. Fluorescence intensity-based screening yielded FLIM biosensors with a 0.5 to 1.0 ns dynamic range upon analyte binding, demonstrating that the mTQ2(1-145)-GT-X-EF-mTQ2(146-238) backbone is a versatile platform for FLIM biosensors, allowing for simple intensity-based screening while providing dual-functional biosensors for both FLIM and intensity-based imaging. As a proof of concept, we monitored cAMP and Ca 2+ dynamics simultaneously in living cells by dual-color imaging. Our results complement recent studies, establishing mTQ2 as a valuable framework for developing FLIM biosensors., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

It is widely believed that information storage in neuronal circuits involves nanoscopic structural changes at synapses, resulting in the formation of synaptic engrams. However, direct evidence for this hypothesis is lacking. To test this conjecture, we combined chemical potentiation, functional analysis by paired pre-postsynaptic recordings, and structural analysis by electron microscopy (EM) and freeze-fracture replica labeling (FRL) at the rodent hippocampal mossy fiber synapse, a key synapse in the trisynaptic circuit of the hippocampus. Biophysical analysis of synaptic transmission revealed that forskolin-induced chemical potentiation increased the readily releasable vesicle pool size and vesicular release probability by 146% and 49%, respectively. Structural analysis of mossy fiber synapses by EM and FRL demonstrated an increase in the number of vesicles close to the plasma membrane and the number of clusters of the priming protein Munc13-1, indicating an increase in the number of both docked and primed vesicles. Furthermore, FRL analysis revealed a significant reduction of the distance between Munc13-1 and CaV2.1 Ca2+ channels, suggesting reconfiguration of the channel-vesicle coupling nanotopography. Our results indicate that presynaptic plasticity is associated with structural reorganization of active zones. We propose that changes in potential nanoscopic organization at synaptic vesicle release sites may be correlates of learning and memory at a plastic central synapse., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Brown and beige adipocytes can be activated by β-adrenergic agonist via cAMP-dependent signaling. Performing RNA-sequencing analysis in human cervical area-derived adipocytes, we found that dibutyryl-cAMP, which can mimic in vivo stimulation of browning and thermogenesis, enhanced the expression of browning and batokine genes and upregulated several signaling pathway genes linked to thermogenesis. We observed that the expression of inhibitor of DNA binding and cell differentiation (ID) 1 and particularly ID3 was strongly induced by the adrenergic stimulation. The degradation of ID1 and ID3 elicited by the ID antagonist AGX51 during thermogenic activation prevented the induction of proton leak respiration that reflects thermogenesis and abrogated cAMP analogue-stimulated upregulation of thermogenic genes and mitochondrial complex I, II, and IV subunits, independently of the proximal cAMP-PKA signaling pathway. The presented data suggests that ID proteins contribute to efficient thermogenic response of adipocytes during adrenergic stimulation., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: Phosphodiesterase-4 (PDE4) is responsible for terminating cyclic adenosine monophosphate (cAMP) signalling. PDE4 inhibitors, such as roflumilast (RFM), have anti-inflammatory activity and have been studied in inflammation-induced tissue damage in sepsis. However, the role of RFM on cardiovascular derangements induced by sepsis is still unknown. Thus, we aimed to evaluate the potential effects of RFM on cardiovascular collapse and multiorgan damage caused by sepsis., Methods: Sepsis was induced by cecal ligation and puncture (CLP) in male rats. Six hours after the CLP or sham procedure, animals were randomly assigned to receive either RFM (0.3 mg/kg) or vehicle subcutaneously, and cardiovascular parameters were assessed 24 h after the surgery and organ/plasma samples were collected for further analyses., Results: Sepsis induced hypotension, tachycardia, reduced renal blood flow (RBF) and hyporeactivity to vasoconstrictors both in vivo and ex vivo. RFM treatment increased systemic cAMP levels and RBF. RFM also attenuated hypoperfusion and liver damage induced by CLP. Furthermore, RFM reduced systemic nitric oxide (NO) levels in septic rats, while there were no changes in hepatic NOS-2 expression. Nevertheless, RFM exacerbated sepsis-induced hypotension and tachycardia without ameliorating vascular hyporeactivity., Conclusion: Our data show that PDE-4 inhibition protects septic rats from hepatic injury and improves renal perfusion. However, RFM worsened hemodynamic parameters and showed no protection against sepsis-induced cardiovascular dysfunction and mortality. Thus, despite the anti-inflammatory benefits of RFM, its application in sepsis should be approached cautiously., Competing Interests: Declaration of competing interest The authors have no conflicts of interest or disclosures to report., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Hypersensitivity to odorants like perfumes can induce or promote asthma with non-type 2 inflammation for which therapeutic options are limited. Cell death of primary bronchial epithelial cells (PBECs) and the release of the pro-inflammatory cytokines interleukin-6 (IL-6) and IL-8 are key in the pathogenesis. Extra-nasal olfactory receptors (ORs) can influence cellular processes involved in asthma. This study investigated the utility of ORs in epithelial cells as potential drug targets in this context., Methods: We used the A549 cell line and primary bronchial epithelial cells using air-liquid interface culture system (ALI-PBECs). OR expression was investigated by RT-PCR, Western blot, and Immunofluorescence. Effects of OR activation by specific ligands on intracellular calcium concentration, cAMP, Phospholipase C (PLC), cell viability, and IL-6 and IL-8 secretion were analyzed by calcium imaging, enzyme immunoassays, Annexin V/ propidium iodide -based fluorescence-activated cell staining or by ELISA, respectively., Results: By screening A549 cells, the OR51B5 agonists Farnesol and Isononyl Alcohol and the OR1G1 agonist Nonanal increased intracellular Ca2 + . OR51B5 and OR1G1 mRNAs and proteins were detected. Both receptors showed a preferential intracellular localization. OR51B5- but not OR1G1-induced Ca2 + dependent on both cAMP and PLC signaling. Farnesol, Isononyl Alcohol, and Nonanal, all reduced cell viability and induced IL-8 and IL-6 release. The data were verified in ALI-PBECs., Conclusion: ORs in the lung epithelium might be involved in airway-sensitivity to odorants. Their antagonism could represent a promising strategy in treatment of odorant-induced asthma with non-type 2 inflammation., Competing Interests: Declarations Ethical approval The primary lung epithelial cells were isolated from postmortem lung transplants (ethics of University Duisburg-Essen 18–8024-BO and 19–8717-BO). Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Regeneration of the sciatic nerve is a sophisticated process that involves the interplay of several signaling pathways that orchestrate the cellular responses critical to regeneration. Among the key pathways are the mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/AKT, cyclic adenosine monophosphate (cAMP), and Janus kinase/signal transducers and transcription activators (JAK/STAT) pathways. In particular, the cAMP pathway modulates neuronal survival and axonal regrowth. It influences various cellular behaviors and gene expression that are essential for nerve regeneration. MAPK is indispensable for Schwann cell differentiation and myelination, whereas PI3K/AKT is integral to the transcription, translation, and cell survival processes that are vital for nerve regeneration. Furthermore, GTP-binding proteins, including those of the Ras homolog gene family (Rho), regulate neural cell adhesion, migration, and survival. Notch signaling also appears to be effective in the early stages of nerve regeneration and in preventing skeletal muscle fibrosis after injury. Understanding the intricate mechanisms and interactions of these pathways is vital for the development of effective therapeutic strategies for sciatic nerve injuries. This review underscores the need for further research to fill existing knowledge gaps and improve therapeutic outcomes., Competing Interests: Declarations Ethics Approval We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. Conflict of Interest The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Terpenoids are the largest class of natural products, found across all domains of life. One of the most abundant bacterial terpenoids is the volatile odorant 2-methylisoborneol (2-MIB), partially responsible for the earthy smell of soil and musty taste of contaminated water. Many bacterial 2-MIB biosynthetic gene clusters were thought to encode a conserved transcription factor, named EshA in the model soil bacterium Streptomyces griseus. Here, we revise the function of EshA, now referred to as Sg Enc, and show that it is a Family 2B encapsulin shell protein. Using cryo-electron microscopy, we find that Sg Enc forms an icosahedral protein shell and encapsulates 2-methylisoborneol synthase (2-MIBS) as a cargo protein. Sg Enc contains a cyclic adenosine monophosphate (cAMP) binding domain (CBD)-fold insertion and a unique metal-binding domain, both displayed on the shell exterior. We show that Sg Enc CBDs do not bind cAMP. We find that 2-MIBS cargo loading is mediated by an N-terminal disordered cargo-loading domain and that 2-MIBS activity and Sg Enc shell structure are not modulated by cAMP. Our work redefines the function of EshA and establishes Family 2B encapsulins as cargo-loaded protein nanocompartments involved in secondary metabolite biosynthesis., (© 2024. The Author(s).)

Navigating the duality of opioids' potent analgesia and side effects, including tolerance and hyperalgesia, is a significant challenge in chronic pain management, often prompting hazardous dose escalation to maintain analgesic effects. The peripheral mu-opioid receptor (MOR) is known to mediate these contradictory effects. Here, we show that the fms-like tyrosine kinase receptor 3 (FLT3) in peripheral somatosensory neurons drives morphine tolerance and hyperalgesia in a male rodent model. We found that chronic morphine treatment increases FLT3 and MOR co-expression, and that inhibiting FLT3 represses MOR-induced hyperactivation of the cyclic adenosine monophosphate (cAMP) signaling pathway, mitigating maladaptive excitatory processes engaged after chronic morphine treatment. Furthermore, in postsurgical or inflammatory models of chronic pain, co-administering morphine with a FLT3-specific inhibitor not only prevents or suppresses tolerance and hyperalgesia but also potentiates the analgesic efficacy of morphine, without aggravating other morphine-induced adverse effects. Our findings suggest that pairing morphine with FLT3 inhibitors could become a promising avenue for chronic pain management to safely harness the power of opioids, without the risk of dose escalation. By enhancing morphine analgesic potency through FLT3 inhibition, this approach could minimize opioid dosage, thereby curtailing the risk of addiction and other opioid-related side effects., (© 2024. The Author(s).)

In this issue of Molecular Cell, Luo et al. 1 identify a signaling pathway, OSM1-COLD6, that induces cold tolerance in rice by promoting production of the non-canonical cyclic nucleotide 2',3'-cAMP. The study opens new avenues for enhancing cold tolerance in rice breeding., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

While it is known that temperature sensors trigger calcium (Ca 2+ ) signaling to confer cold tolerance in cells, less is known about sensors that couple with other secondary messengers. Here, we identify a cold sensor complex of CHILLING-TOLERANCE DIVERGENCE 6 (COLD6) and osmotin-like 1 (OSM1), which triggers 2',3'-cyclic adenosine monophosphate (2',3'-cAMP) production to enhance cold tolerance in rice. COLD6, which is encoded by a major quantitative trait locus (QTL) gene, interacts with the rice G protein α subunit (RGA1) at the plasma membrane under normal conditions. Upon exposure to chilling, cold-induced OSM1 binds to COLD6, kicking out RGA1 from interaction. This triggers an elevation of 2',3'-cAMP levels for enhancing chilling tolerance. Genetic data show that COLD6 negatively regulates cold tolerance and functionally depends on OSM1 in chilling stress. COLD6 alleles were selected during rice domestication. Knockout and natural variation of COLD6 in hybrid rice enhanced chilling tolerance, hinting design potential for breeding. This highlighted a module triggering 2',3'-cAMP to improve chilling tolerance in crops., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Phosphodiesterases (PDEs) constitute a family of enzymes that play a pivotal role in the regulation of intracellular levels of cyclic nucleotides, including cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Dysregulation of PDE activity has been implicated in diverse pathological conditions encompassing cardiovascular disorders, pulmonary diseases, and neurological disorders. Small-molecule inhibitors targeting PDEs have emerged as promising therapeutic agents for the treatment of these ailments, some of which have been approved for their clinical use. Despite their success, challenges such as resistance mechanisms and off-target effects persist, urging continuous research for the development of next-generation PDE inhibitors. The objective of this review is to provide an overview of the synthesis and clinical application of representative approved small-molecule PDE inhibitors, with the aim of offering guidance for further advancements in the development of novel PDE inhibitors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Neuroblastoma (NB) is the most common solid extracranial neoplasm found in children and is derived from primitive sympathoadrenal neural precursor. The disease accounts for 15% of all cancer deaths in children. The mortality rate is high in patients presenting with a metastatic tumour even with extensive treatments. This signifies the need for further research towards the development of new additional therapies that can combat not only tumour growth but metastasis, especially amongst the high-risk groups. During metastasis, primary tumour cells become migratory and travel towards a capillary within the tumour. They then degrade the matrix surrounding the pericytes and endothelial cells traversing the endothelial barrier twice to establish a secondary. This led to the hypothesis that modulation of the endothelial cell junctional stability could have an influence on tumour metastasis. To test this hypothesis, agents that modulate endothelial permeability on NB cell line migration and invasion were assessed in vitro in a tissue culture model. The cAMP agonist and its antagonists were found to have no obvious effect on both SK-N-BE2C and SK-N-AS migration, invasion and proliferation. Next, NB cells were cocultured with HDMEC cells and live cell imaging was used to assess the effect of an Epac agonist on trans-endothelial cell migration of NB cells. Epac1 agonist remarkably reduced the trans-endothelial migration of both SK-N-BE2C and SK-N-AS cells. These results demonstrate that an Epac1 agonist may perhaps serve as an adjuvant to currently existing therapies for the high-risk NB patients., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright: © 2024 Inuwa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The phototransduction cascade enables the photoreceptor to detect light over a wide range of intensities without saturation. The main second messenger of the cascade is cGMP and the primary regulatory mechanism is calcium feedback. However, some experimental data suggest that cAMP may also play a role in regulating the phototransduction cascade, but this would require changes in cAMP on a time scale of seconds. Currently, there is a lack of data on the dynamics of changes in intracellular cAMP levels on this timescale. This is largely due to the specificity of the sensory modality of photoreceptors, which makes it practically impossible to use conventional experimental approaches based on fluorescence methods. In this study, we employed the method of rapid cryofixation of retinal samples after light stimulation and subsequent isolation of outer segment preparations. The study employed highly sensitive metabolomics approaches to measure levels of cAMP. Additionally, PKA activity was measured in the samples using a western blot. The results indicate that when exposed to near-saturating but still moderate light, cAMP levels increase transiently within the first second and then return to pre-stimulus levels. The increase in cAMP activates PKA, resulting in the phosphorylation of PKA-specific substrates in frog retinal outer segments., (© 2024 Chernyshkova et al.)

Endometrial disorders, such as infertility and endometriosis, significantly impact reproductive health, thus necessitating better models to study endometrial function. Current in vitro models fail to replicate the complexity of the human endometrium throughout the entire menstrual cycle. This study aimed to assess the physiological response of human endometrial organoids (hEOs) to in vitro hormonal treatments designed to mimic the hormonal fluctuations of the menstrual cycle. Endometrial biopsies from three healthy women were used to develop hEOs, which were treated over 28 days with three hormonal stimulation strategies: (1) estrogen only (E) to mimic the proliferative phase, (2) the addition of progesterone (EP) to simulate the secretory phase, and (3) the further addition of cAMP (EPC) to enhance the secretory functions of hEOs. Gene and protein expression were analyzed using qPCR, IHC, and ELISA. The hEOs exhibited proliferation, gland formation, and appropriate expression of markers such as E-cadherin and Ki67. The hormonal treatments induced significant changes in PR , HSD17B1 , PAEP , SPP1 , and other genes relevant to endometrial function, closely mirroring in vivo physiological responses. The prominent changes were observed in EPC-treated hEOs (week 4) with significantly high expression of uterine milk components such as glycodelin (PAEP) and osteopontin (SPP1), reflecting mid- to late-secretory phase physiology. This model successfully recapitulates human menstrual cycle dynamics and offers a promising platform for studying endometrial disorders and advancing personalized treatments in gynecology.

Aim: Potassium 2-(1-hydroxypentyl)-benzoate (dl-PHPB), puerarin and salvianolic acid B are three natural products or derivatives that can inhibit platelet aggregation. However, the mechanisms of dl-PHPB, puerarin and salvianolic acid B to inhibit platelet aggregation are still not clear., Method: Here, 2-methylthioadenosine diphosphate (2-MeSADP) was used as an inducer to confirm the effects of three drugs on platelet aggregation and illustrate the corresponding mechanisms., Result: The results indicated that dl-PHPB, puerarin and salvianolic acid B significantly inhibited platelet aggregation both in vivo and in vitro. In addition, the content of IP 3 , cAMP and intracellular [Ca 2+ ] i were measured in HEK293 cell lines overexpressing P2Y 1 and P2Y 12 . Dl-PHPB and puerarin could obviously reduce 2-MeSADP-induced IP 3 increase, but salvianolic acid B showed no effects. Unlike dl-PHPB and puerarin, which had no effects on 2-MeSADP-induced cAMP decrease, salvianolic acid B significantly reversed the reduction of cAMP. Both dl-PHPB and puerarin could decrease the enhanced intracellular [Ca 2+ ] i induced by 2-MeSADP; however, salvianolic acid B showed no effect on intracellular [Ca 2+ ] i elevation., Conclusion: These results suggested that dl-PHPB and puerarin inhibited platelet aggregation via targeting at P2Y 1 receptor and P2Y 1 -Gq-IP 3 -Ca 2+ signal pathway. Differently, salvianolic acid B inhibited platelet aggregation via targeting at P2Y 12 receptor and via Gi-AC-cAMP signal pathway., (© 2024 The Author(s). CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

Introduction: PDE2 is a dual-specific enzyme that hydrolyzes two intracellular substrates, cAMP and cGMP. PDE2 is mainly distributed in the brain, which indicates that PDE2 can serve as a potential target for central nervous system diseases without causing other peripheral side effects. Discovery of new mechanisms of PDE2 inhibitors is expected to bring new opportunities for the treatment of central nervous system diseases., Area Covered: This review aims to provide an overview of PDE2 inhibitors reported in patents from 2017 to present., Expert Opinion: In recent years, the development of PDE2 inhibitors and their application in the treatment of brain diseases have received much attention. The main reason is the high expression of PDE2 in the brain, which gives PDE2 a natural advantage as a research target for central nervous system diseases. This review summarizes the scaffolds of PDE2 inhibitors reported in various patents since 2017, as well as the scientific issues that need to be addressed in terms of subtype selectivity and metabolic stability, intending to provide insights for the discovery of highly active and selective PDE2 inhibitors in the future.

Microplastics (MPs) colonized by pathogens pose significant risks to the environment and health of animals and humans, however, the strategies for pathogens colonization in MPs and the effects of its colonization on spread of pathogens have not been fully characterized. Here, we investigated the biofilm formation mechanism regulated by c-di-GMP in Hafnia paralvei Z11, and determined the effect of MPs colonized by H. paralvei Z11 on the spread of strain Z11. Overexpression of yhjH, a c-di-GMP phosphodiesterase gene, attenuated intracellular c-di-GMP level in strain Z11, leading to an increase in biofilm dispersal and a decrease in biofilm formation. Meanwhile, the decline of c-di-GMP inhibited the expression of cAMP phosphodiesterase genes, increasing the cAMP content and promoting bacterial motility, that was responsible for the increase of biofilm dispersal. Furthermore, the formation of biofilms by strain Z11 on MPs promotes its colonization, which contributes to its vertical and horizontal spread in water after colonizing polyvinyl chloride-MPs and polypropylene-MPs, respectively. Therefore, this study reveals, for the first time, MPs colonized by H. paralvei Z11 through biofilms regulated by crosstalk between c-di GMP and cAMP promote the spread of strain Z11 in water, which expands the understanding of colonization strategy of pathogens on MPs and its risk on spread of pathogens., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Phosphodiesterase type 5 (PDE5) is an enzyme primarily found in the smooth muscle of the corpus cavernosum and also highly expressed in the substantia nigra, cerebellum, caudate, hippocampal regions and cerebellar purkinje cells, responsible for selectively breaking down cyclic guanosine monophosphate (cGMP) into 5'-GMP and regulate intracellular cGMP levels. As a second messenger, cyclic GMP enhances signals at postsynaptic receptors and triggers downstream effector molecules, leading to changes in gene expression and neuronal responses. Additionally, cGMP signaling transduction cascade, present in the brain, is also essential for learning and memory processes. Mechanistically, PDE5 inhibitors share structural similarities with cGMP, competitively binding to PDE5 and inhibiting cGMP hydrolysis. This action enhances the effects of nitric oxide, resulting in anti-inflammatory and neuroprotective effects. Neurodegenerative disorders entail the progressive loss of neuron structure, culminating in neuronal cell death, with currently available drugs providing only limited symptomatic relief, rendering neurodegeneration considered incurable. PDE5 inhibitors have recently emerged as a potential therapeutic approach for neurodegeneration, neuroinflammation, and diseases involving cognitive impairment. This review elucidates the principal roles of 3',5'-cyclic adenosine monophosphate (cAMP) and cGMP signaling pathways in neuronal functions, believed to play pivotal roles in the pathogenesis of various neurodegenerative disorders. It provides an updated assessment of PDE5 inhibitors as disease-modifying agents for conditions such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, cerebral ischemia, Huntington's disease, and neuroinflammation. The paper aims to review the current understanding of PDE5 inhibitors, which concurrently regulate both cAMP and cGMP signaling pathways, positing that they may exert complementary and synergistic effects in modifying neurodegeneration, thus presenting a novel direction in therapeutic discovery. Moreover, the review provides critical about biological functions, therapeutic potentials, limitations, challenges, and emerging applications of selective PDE5 inhibitors. This comprehensive overview aims to guide future academic and industrial endeavors in this field., (© 2024 Wiley Periodicals LLC.)

Tumor-associated macrophages (TAMs) are prime therapeutic targets due to their pro-tumorigenic functions, but varying efficacy of macrophage-targeting therapies highlights our incomplete understanding of how macrophages are regulated within the tumor microenvironment (TME). The circadian clock is a key regulator of macrophage function, but how circadian rhythms of macrophages are influenced by the TME remains unknown. Here, we show that conditions associated with the TME such as polarizing stimuli, acidic pH, and lactate can alter circadian rhythms in macrophages. While cyclic AMP (cAMP) has been reported to play a role in macrophage response to acidic pH, our results indicate pH-driven changes in circadian rhythms are not mediated solely by cAMP signaling. Remarkably, circadian disorder of TAMs was revealed by clock correlation distance analysis. Our data suggest that heterogeneity in circadian rhythms within the TAM population level may underlie this circadian disorder. Finally, we report that circadian regulation of macrophages suppresses tumor growth in a murine model of pancreatic cancer. Our work demonstrates a novel mechanism by which the TME influences macrophage biology through modulation of circadian rhythms., (© 2024. The Author(s).)

Pancreatic ductal adenocarcinoma (PDAC) remains a highly malignant cancer with a grim prognosis due to its early metastasis and resistance to current chemotherapies, such as Gemcitabine (GEM). We have previously demonstrated that cAMP exclusion by MRP4 is critical for PDAC cell proliferation, establishing this transporter as a promising prognostic marker and therapeutic target. In search for novel therapeutic options to improve GEM efficacy, we conducted a drug repositioning screening to identify potential inhibitors of cAMP transport by MRP4. Several non-steroidal anti-inflammatory drugs (NSAIDs) can inhibit the transport of certain MRP4 substrates. In this study, we assessed the efficacy of sixteen NSAIDs in inhibiting cAMP transport mediated by MRP4, identifying seven potent inhibitors based on their IC 50 values. The most potent inhibitors were further tested for their effect on cell proliferation and migration. Flurbiprofen emerged as the most potent inhibitor of both MRP4-mediated cAMP transport and cell proliferation. Overexpression of MRP4 in BxPC-3 cells significantly increased GEM resistance, and co-administration of flurbiprofen with GEM markedly enhanced the latter's potency inhibiting PDAC cells proliferation. These findings position flurbiprofen as a potent inhibitor of cAMP transport by MRP4 and a promising adjunctive therapy to enhance GEM effectiveness in PDAC treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Type three secretion system (TTSS)-competent Pseudomonas aeruginosa expressing soluble promiscuous cyclase, exoenzyme Y (ExoY), generates cyclic nucleotides in pulmonary microvascular endothelial cells (PMVECs). Within cells, cyclic nucleotide signals are highly compartmentalized, but these second messengers are also released into the extracellular space. Although agonist stimulation of endogenous adenylyl cyclase (AC) or the presence of ExoY increases cyclic nucleotides, the proportion of the signal that is in the intracellular versus extracellular compartments is unresolved. Furthermore, it is unclear whether P. aeruginosa primary infection or treatment with sterile media supernatants derived from a primary infection alters beta-adrenergic agonist-induced elevations in cAMP in PMVECs. Herein, we determine that PMVECs release cAMP into the extracellular space constitutively, following beta-adrenergic stimulation of endogenous AC, and following infection with P. aeruginosa expressing ExoY. Surprisingly, in PMVECs, only a small proportion of cGMP is detected within the cell at baseline or following P. aeruginosa ExoY infection with a larger proportion of total cGMP being detected extracellularly. Thus, the ability of lung endothelium to generate cyclic nucleotides may be underestimated by examining intracellular cyclic nucleotides alone, since a large portion is delivered into the extracellular compartment. In addition, P. aeruginosa infection or treatment with sterile media supernatants from a primary infection suppresses the beta-adrenergic cAMP response, which is further attenuated by the expression of functional ExoY. These findings reveal an overabundance of extracellular cyclic nucleotides following infection with ExoY expressing TTSS-competent P. aeruginosa . NEW & NOTEWORTHY P. aeruginosa exoenzyme Y (ExoY) infection increases cyclic nucleotides intracellularly, but an overabundance of cAMP and cGMP is also detected in the extracellular space and reveals a greater capacity of pulmonary endothelial cells to generate cAMP and cGMP. P. aeruginosa infection or treatment with sterile media supernatants derived from a primary infection suppresses the β-adrenergic-induced cAMP response in pulmonary endothelial cells, which is exacerbated by the expression of functional ExoY.

Background: Glucagon-like peptide (GLP)-1 receptor (GLP1R) agonists exert a multitude of beneficial cardiovascular effects beyond control of blood glucose levels and obesity reduction. They also have anti-inflammatory actions through both central and peripheral mechanisms. GLP1R is a G protein-coupled receptor (GPCR), coupling to adenylyl cyclase (AC)-stimulatory Gs proteins to raise cyclic 3`-5`-adenosine monophosphate (cAMP) levels in cells. cAMP exerts various anti-apoptotic and anti-inflammatory effects via its effectors protein kinase A (PKA) and Exchange protein directly activated by cAMP (Epac). However, the precise role and importance of cAMP in mediating GLP1R`s anti-inflammatory actions, at least in the heart, remains to be determined. To this end, we tested the effects of the GLP1R agonist liraglutide on lipopolysaccharide (LPS)-induced acute inflammatory injury in H9c2 cardiac cells, either in the absence of cAMP production (AC inhibition) or upon enhancement of cAMP levels via phosphodiesterase (PDE)-4 inhibition with roflumilast., Methods & Results: Liraglutide dose-dependently inhibited LPS-induced apoptosis and increased cAMP levels in H9c2 cells, with roflumilast but also PDE8 inhibition further enhancing cAMP production by liraglutide. GLP1R-stimulated cAMP markedly suppressed the LPS-dependent induction of pro-inflammatory tumor necrosis factor (TNF)-a, interleukin (IL)-1b, and IL-6 cytokine expression, of inducible nitric oxide synthase (iNOS) expression and nuclear factor (NF)-kB activity, of matrix metalloproteinases (MMP)-2 and MMP-9 levels and activities, and of myocardial injury markers in H9c2 cardiac cells. The effects of liraglutide were mediated by the GLP1R since they were abolished by the GLP1R antagonist exendin(9-39). Importantly, AC inhibition completely abrogated liraglutide`s suppression of LPS-dependent inflammatory injury, whereas roflumilast significantly enhanced the protective effects of liraglutide against LPS-induced inflammation. Finally, PKA inhibition or Epac1/2 inhibition alone only partially blocked liraglutide`s suppression of LPS-induced inflammation in H9c2 cardiac cells, but, together, PKA and Epac1/2 inhibition fully prevented liraglutide from reducing LPS-dependent inflammation., Conclusions: cAMP, via activation of both PKA and Epac, is essential for GLP1R`s anti-inflammatory signaling in cardiac cells and that cAMP levels crucially regulate the anti-inflammatory efficacy of GLP1R agonists in the heart. Strategies that elevate cardiac cAMP levels, such as PDE4 inhibition, may potentiate the cardiovascular, including anti-inflammatory, benefits of GLP1R agonist drugs., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The carcinogenic effects of benzidine (BZ) on bladder cancer are well documented, but its potential for promoting upper urinary tract urothelial carcinoma (UTUC) remains unclear. The ability of emodin, a natural pharmaceutical compound, to prevent BZ‑associated UTUC has not been previously explored. To the best of our knowledge, the present study is the first to reveal that BZ significantly enhanced the survival and migration of UTUC cell lines in vitro . Furthermore, in vivo experiments demonstrated that BZ promoted an increase in the size of subcutaneous tumors in nude mice. Further investigation revealed that BZ upregulated the expression of protein kinase A (PKA) and cyclooxygenase 2 (COX2), along with downstream matrix metalloproteinase 9 (MMP9) and vascular endothelial growth factor (VEGF), in UTUC cells. Moreover, BZ increased the levels of cyclic adenosine monophosphate (cAMP) and prostaglandin E2 (PGE2) in cell lysates. By contrast, emodin reduced the PKA and COX2 expression levels compared with the BZ‑treated group. Similarly, the in vivo experiments demonstrated that emodin significantly inhibited tumor growth in BZ‑pretreated nude mice, accompanied by reductions in the cAMP, PGE2, MMP9 and VEGF levels. These findings elucidated the role of BZ in promoting UTUC progression. Additionally, emodin has emerged as a novel inhibitor of BZ‑induced UTUC development through PKA/COX2 inhibition, suggesting its potential as a natural therapeutic agent against BZ‑associated UTUC.

Metabolic syndrome (MetS) increases the risk of coronary artery disease, but effects of this condition on the working myocardium remain to be fully elucidated. In the present study we evaluated the consequences of diet-induced metabolic disorders on cardiac function and myocyte performance using female mice fed with Western diet. Animals maintained on regular chow were used as control (Ctrl). Mice on the Western diet (WesD) had increased body weight, impaired glucose metabolism, preserved diastolic and systolic function, but increased left ventricular (LV) mass, with respect to Ctrl animals. Moreover, WesD mice had reduced heart rate variability (HRV), indicative of altered cardiac sympathovagal balance. Myocytes from WesD mice had increased volume, enhanced cell mechanics, and faster kinetics of contraction and relaxation. Moreover, levels of cAMP and protein kinase A (PKA) activity were enhanced in WesD myocytes, and interventions aimed at stabilizing cAMP/PKA abrogated functional differences between Ctrl and WesD cells. Interestingly, in vivo β-adrenergic receptor (β-AR) blockade normalized the mechanical properties of WesD myocytes and revealed defective cardiac function in WesD mice, with respect to Ctrl. Collectively, these results indicate that metabolic disorders induced by Western diet enhance the cAMP/PKA signaling pathway, a possible adaptation required to maintain cardiac function., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Evidence has been accumulating that elements of the vertebrate pituitary adenylate cyclase-activating polypeptide (PACAP) system are missing in non-chordate genomes, which is at odds with the partial sequence-, immunohistochemical-, and physiological data in the literature. Multilevel experiments were performed on the great pond snail (Lymnaea stagnalis) to explore the role of PACAP in invertebrates. Screening of neuronal transcriptome and genome data did not reveal homologs to the elements of vertebrate PACAP system. Despite this, immunohistochemical investigations with an anti-human PAC 1 receptor antibody yielded a positive signal in the neuronal elements in the heart. Although Western blotting of proteins extracted from the nervous system found a relevant band for PACAP-38, immunoprecipitation and mass spectrometric analyses revealed no corresponding peptide fragments. Similarly to the effects reported in vertebrates, PACAP-38 significantly increased cAMP synthesis in the heart and had a positive ionotropic effect on heart preparations. Moreover, it significantly modulated the effects of serotonin and acetylcholine. Homologs to members of Cluster B receptors, which have shared common evolutionary origin with the vertebrate PACAP receptors, PTHRs, and GCGRs, were identified and shown not to be expressed in the heart, which does not support a potential role in the mediation of PACAP-induced effects. Our findings support the notion that the PACAP system emerged after the protostome-deuterostome divergence. Using antibodies against vertebrate proteins is again highlighted to have little/no value in invertebrate studies. The physiological effects of vertebrate PACAP peptides in protostomes, no matter how similar they are to those in vertebrates, should be considered non-specific., Competing Interests: Declarations Competing interests The authors declare no competing interests. Ethics statement All procedures, methods, and experiments on the specimens were carried out in accordance with the relevant guidelines and regulations approved by the Scientific Committee of Animal Experimentation of the Balaton Limnological Research Institute (VE-I-001/01890-10/2013). Declaration of conflict of interest The authors declare that there is no conflict of interest. Author Statement The authors declare that the recommendations of the ARRIVE 2.0. guideline were considered and followed during the experiments, analysis, and data reporting., (© 2024. The Author(s).)

Agonists at μ opioid receptors relieve acute pain, however, their long-term use is limited by side effects, which may involve β-arrestin2. Agonists biased against β-arrestin2 recruitment may be advantageous. However, the classification of bias may be compromised by assays utilising overexpressed μ receptors which overestimate efficacy for G-protein activation. There is a need for re-evaluation with restricted receptor availability to determine accurate agonist efficacies. We depleted μ receptor availability in PathHunter CHO cells using the irreversible antagonist, β-funaltrexamine (β-FNA), and compared efficacies and apparent potencies of twelve agonists, including several previously reported as biased, in β-arrestin2 recruitment and cAMP assays. With full receptor availability all agonists had partial efficacy for stimulating β-arrestin2 recruitment relative to DAMGO, while only TRV130 and buprenorphine were partial agonists as inhibitors of cAMP accumulation. Limiting receptor availability by prior exposure to β-FNA (100 nM) revealed morphine, oxycodone, PZM21, herkinorin, U47700, tianeptine and U47931e are also partial agonists in the cAMP assay. The efficacies of all agonists, except SR-17018, correlated between β-arrestin2 recruitment and cAMP assays, with depleted receptor availability in the latter. Furthermore, naloxone and cyprodime exhibited non-competitive antagonism of SR-17018 in the β-arrestin2 recruitment assay. Limited antagonism by naloxone was also non-competitive in the cAMP assay, while cyprodime was competitive. Furthermore, SR-17018 only negligibly diminished β-arrestin2 recruitment stimulated by DAMGO (1 μM), whereas fentanyl, morphine and TRV130 all exhibited the anticipated competitive inhibition. The data suggest that SR-17018 achieves bias against β-arrestin2 recruitment through interactions with μ receptors outside the orthosteric agonist site. This article is part of the Special Issue on "Ligand Bias"., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2024. Published by Elsevier Ltd.)

Subarachnoid hemorrhage (SAH) triggers severe neuroinflammation and cognitive impairment, where microglial M1 polarization exacerbates the injury and M2 polarization mitigates damage. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs), carrying microRNA (miR)-140-5p, offer therapeutic promise by targeting the cAMP/PKA/CREB pathway and modulating microglial responses, demonstrating a novel approach for addressing SAH-induced brain injury. This research explored the role of miR-140-5p delivered by MSC-EVs in mitigating brain damage following SAH. Serum from SAH patients and healthy individuals was analyzed for miR-140-5p and cAMP levels. The association between miR-140-5p levels, brain injury severity, and patient survival was examined, along with the target relationship between miR-140-5p and histone deacetylases 7 (HDAC7). MSC-EVs were characterized for their ability to cross the blood-brain barrier and modulate the HDAC7/AKAP12/cAMP/PKA/CREB axis, reducing M1 polarization and inflammation. The therapeutic effect of MSC-EV-miR-140-5p was demonstrated in an SAH mouse model, showing reduced neuronal apoptosis and improved neurological function. This study highlights the potential of MSC-EV-miR-140-5p in mitigating SAH-induced neuroinflammation and brain injury, providing a foundation for developing MSC-EV-based treatments for SAH., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

d-Lactic acid holds significant industrial importance due to its versatility and serves as a crucial component in the synthesis of environmentally friendly and biodegradable thermal-resistant poly-lactic acid. This polymer exhibits promising potential as a substitute for nonbiodegradable, petroleum-based plastics. The production of d-lactic acid from lignocellulosic biomass, a type of biorenewable and nonfood resources, can lower costs and improve product competitiveness. Glucose and xylose are the most abundant sugar monomers in lignocellulosic biomass materials. Despite Escherichia coli possessing native xylose catabolic pathways and transport, their ability to effectively utilize xylose is often hindered in the presence of glucose. Here, the E. coli strain Rec1.0, previously engineered to overcome carbon catabolite repression, was selected as the initial strain for reengineering to produce d-lactic acid. An adaptive evolution approach was employed to achieve highly efficient fermentation of glucose-xylose mixtures. The resulting strain, QJL010, could produce d-lactic acid of 87.5 g/L with a carbon yield of 0.99 mol/mol. Notably, the consumption rates of glucose and xylose reached 0.75 and 0.82 g/gDCW/h, respectively. Further analysis revealed that increased Glk activity, resulting from glk mutations (A142V and R188H), along with their upregulated expression, contributed to an elevated glucose consumption rate. Additionally, a CRP G141D mutation, cAMP-independent, stimulated the expression of the xylR, xylE, and galABC* genes, resulting in an accelerated xylose consumption rate. These findings provide valuable support for the utilization of E. coli platform strains in the production of value-added chemicals from lignocellulosic biomass., (© 2024 Wiley Periodicals LLC.)

Hepatic encephalopathy (HE) is one of the most prevalent and severe hepatic and brain disorders in which escalation of the oxidative, inflammatory and apoptotic trajectories pathologically connects acute liver injury with neurological impairment. Mirabegron (Mira) is a beta3 adrenergic receptor agonist with proven antioxidant and anti-inflammatory activities. The current research pointed to exploring Mira's hepato-and neuroprotective impacts against thioacetamide (TAA)-induced HE in rats. Rats were distributed into three experimental groups: the normal control group, the TAA group, received TAA (200 mg/kg/day for three consecutive days) and the Mira-treated group received Mira (10 mg/kg/day; oral gavage) for 15 consecutive days and intoxicated with TAA from the 13th to the 15th day of the experimental period. Mira counteracted hyperammonemia, enhanced rats' locomotor capability and motor coordination. It attenuated hepatic/neurological injuries by its antioxidant, anti-apoptotic as well as anti-inflammatory potentials. Mira predominantly targeted cyclic adenosine monophosphate (cAMP)/phosphorylated extracellular signal-regulated kinase (p-Erk1/2)/peroxisome proliferator-activated receptor gamma (PPARγ) dependent pathways via downregulation of p S536-nuclear factor kappa B p65 (p S536 NF-κB p 65)/tumor necrosis alpha (TNF-α) axis. Meanwhile, it attenuated nuclear factor erythroid 2-related factor (Nrf2) depletion in parallel with restoring of the neuroprotective defensive pathway by upregulation of cerebral cAMP/PPAR-γ/p-ERK1/2 and p-CREB/BDNF/TrkB besides reduction of GFAP immunoreactivity. Mira showed anti-apoptotic activity through inhibition of Bax immunoreactivity and elevation of Bcl2. To summarize, Mira exhibited a hepato-and neuroprotective effect against TAA-induced HE in rats via shielding antioxidant defense and mitigation of the pathological inflammatory and apoptotic axis besides upregulation of neuroprotective signaling pathways., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The MC1R protein is a receptor found in melanocytes that plays a role in melanin synthesis. Mutations in this protein can impact hair color, skin tone, tanning ability, and increase the risk of skin cancer. The MC1R protein is activated by the alpha-melanocyte-stimulating hormone (α-MSH). Previous studies have shown that mutations affect the interaction between MC1R and α-MSH; however, the mechanism behind this process is poorly understood. Our study aims to shed light on this mechanism using molecular dynamics (MD) simulations to analyze the Asp84Glu and Asp294His variants. We simulated both the wild-type (WT) protein and the mutants with and without ligand. Our results reveal that mutations induce unique conformations during state transitions, hindering the switch between active and inactive states and decreasing cellular levels of cAMP. Interestingly, Asp294His showed increased ligand affinity but decreased protein activity, highlighting that tighter binding does not always lead to increased activation. Our study provides insights into the molecular mechanisms underlying the impact of MC1R mutations on protein activity., (© 2024 Wiley Periodicals LLC.)

The central nervous system (CNS) is constantly surveilled by microglia, highly motile and dynamic cells deputed to act as the first line of immune defense in the brain and spinal cord. Alterations in the homeostasis of the CNS are detected by microglia that respond by extending their processes or - following major injuries - by migrating toward the affected area. Understanding the mechanisms controlling directed cell migration of microglia is crucial to dissect their responses to neuroinflammation and injury. We used a combination of pharmacological and genetic approaches to explore the involvement of calcium (Ca 2+ ) signaling in the directed migration of human induced pluripotent stem cell (iPSC)-derived microglia challenged with a purinergic stimulus. This approach mimics cues originating from injury of the CNS. Unexpectedly, simultaneous imaging of microglia migration and intracellular Ca 2+ changes revealed that this phenomenon does not require Ca 2+ signals generated from the endoplasmic reticulum (ER) and store-operated Ca 2+ entry (SOCE) pathways. Instead, we find evidence that human microglial chemotaxis to purinergic signals is mediated by cyclic AMP in a Ca 2+ -independent manner. These results challenge prevailing notions, with important implications in neurological conditions characterized by perturbation in Ca 2+ homeostasis., Competing Interests: Declaration of competing interest M.B.-J. is a co-founder of NovoGlia Inc. I.F.S. is a consultant for, and holds stock options in NeuroQure, Inc. All other authors declare no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The nanoscale organization of enzymes associated with the dynamics of second messengers is critical for ensuring compartmentation and localization of signaling molecules in cells. Specifically, the spatiotemporal orchestration of cAMP and Ca2+ oscillations is critical for many cellular functions. Previous experimental studies have shown that the formation of nanodomains of A-kinase anchoring protein 79/150 (AKAP150) and adenylyl cyclase 8 (AC8) on the surface of pancreatic MIN6 β cells modulates the phase of Ca2+-cAMP oscillations from out-of-phase to in-phase. In this work, we develop computational models of the Ca2+/cAMP pathway and AKAP/AC nanodomain formation that give rise to the two important predictions: instead of an arbitrary phase difference, the out-of-phase Ca2+/cAMP oscillation reaches Ca2+ trough and cAMP peak simultaneously, which is defined as inversely out-of-phase; the in-phase and inversely out-of-phase oscillations associated with Ca2+-cAMP dynamics on and away from the nanodomains can be explained by an incoherent feedforward loop. Factors such as cellular surface-to-volume ratio, compartment size, and distance between nanodomains do not affect the existence of in-phase or inversely out-of-phase Ca2+/cAMP oscillation, but cellular surface-to-volume ratio and compartment size can affect the time delay for the inversely out-of-phase Ca2+/cAMP oscillation while the distance between two nanodomains does not. Finally, we predict that both the Turing pattern-generated nanodomains and experimentally measured nanodomains demonstrate the existence of in-phase and inversely out-of-phase Ca2+/cAMP oscillation when the AC8 is at a low level, consistent with the behavior of an incoherent feedforward loop. These findings unveil the key circuit motif that governs cAMP and Ca2+ oscillations and advance our understanding of how nanodomains can lead to spatial compartmentation of second messengers., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Qiao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Osteoarthritis (OA) is a degenerative disease of the joints, prevalent worldwide. Polydeoxyribonucleotide (PDRN) is used for treating knee OA. However, the role of PDRN in IL-1β-induced inflammatory responses in human bone marrow-derived mesenchymal stem cells (hBMSCs) remains unknown. Here, we investigated the role of PDRN in IL-1β-induced impairment of chondrogenic differentiation in hBMSCs. hBMSCs treated with PDRN showed a large micromass, enhanced safranin O and alcian blue staining intensity, and increased expression of chondrogenic genes in IL-1β-induced inflammatory responses, in addition to regulation of catabolic and anabolic genes. In addition, PDRN treatment suppressed the expression of inflammatory cytokines and mitigated IL-1β-induced apoptosis in hBMSCs. Mechanistically, PDRN treatment increased the formation of cyclic adenosine monophosphate (cAMP) and upregulated the phosphorylation of cAMP-dependent protein kinase A (PKA)/cAMP response element binding protein (CREB) through the adenosine A2A receptor in hBMSCs and thus blocked the nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) signaling pathway. Thus, IL-1β-induced expression of inflammatory cytokines in hBMSCs was directly reduced by adenosine A2A receptor activation. Based on our results, we suggest that PDRN may be a promising MSC-based therapeutic agent for OA., (© 2024. The Author(s).)

Objective: Invasive lobular breast cancer (ILC) is the most common special type of breast cancer and has unique clinicopathological and molecular hallmarks that differentiate it from the more common invasive carcinoma-no special type (NST). Despite these differences, ILC and NST are treated as a single entity and there is a lack of ILC-targeted therapies. To fill this gap, we sought to identify novel molecular alterations in ILC that could be exploited for targeted therapies., Methods: Differential gene expression and Geneset Enrichment and Variation analyses were performed on RNA-seq data from three large public breast cancer databases-the Sweden Cancerome Analysis Network-Breast (SCAN-B; luminal A ILC N = 263, luminal A NST N = 1162), The Cancer Genome Atlas (TCGA; luminal A ILC N = 157, luminal A NST N = 307) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC; luminal A ILC N = 65, luminal A NST N = 533). Pathways enriched in overlapping differentially expressed genes from these datasets were clustered using Jaccard similarity to identify pathways enriched in ILC. The cAMP/PKA/CREB signaling was studied in ILC, ILC-like and NST cell lines and patient-derived organoids (PDOs) using forskolin, an activator of the pathway., Results: Clinicopathological features of patients with ILC and NST in SCAN-B were similar to prior population-based studies. There was a consistent pattern of up-regulation of cAMP/PKA/CREB related signaling in ILC compared to NST in SCAN-B, TCGA and METABRIC. Treatment with forskolin resulted in a greater increase in phospho-CREB in ILC cell lines and organoids than NST. CRISPR deletion of CDH1 in NST cell lines did not alter response of cells to forskolin as measured by phospho-CREB. Forskolin treatment caused growth inhibition in ILC and NST, with ILC cell lines being more sensitive to forskolin-mediated growth inhibition., Conclusion: In three separate datasets, cAMP/PKA/CREB signaling was identified to be higher in ILC than NST. This in silico finding was validated in cell line and organoid models. Loss of CDH1 was not sufficient to mediate this phenotype. Future studies should investigate the mechanisms for differential cAMP/PKA/CREB signaling and the potential for therapeutic targeting in patients with ILC., (© 2024. The Author(s).)

Bimolecular fluorescence complementation (BiFC) methodology uses split fluorescent proteins to detect interactions between proteins in living cells. To date, BiFC has been used to investigate receptor dimerization by splitting the fluorescent protein between the intracellular portions of different receptor components. We reasoned that attaching these split proteins to the extracellular N-terminus instead may improve the flexibility of this methodology and reduce the likelihood of impaired intracellular signal transduction. As a proof-of-concept, we used receptors for calcitonin gene-related peptide, which comprise heterodimers of either the calcitonin or calcitonin receptor-like receptor in complex with an accessory protein (receptor activity-modifying protein 1). We created fusion constructs in which split mVenus fragments were attached to either the C-termini or N-termini of receptor subunits. The resulting constructs were transfected into Cos7 and HEK293S cells, where we measured cAMP production in response to ligand stimulation, cell surface expression of receptor complexes, and BiFC fluorescence. Additionally, we investigated ligand-dependent internalization in HEK293S cells. We found N-terminal fusions were better tolerated with regards to cAMP signaling and receptor internalization. N-terminal fusions also allowed reconstitution of functional fluorescent mVenus proteins; however, fluorescence yields were lower than with C-terminal fusion. Our results suggest that BiFC methodologies can be applied to the receptor N-terminus, thereby increasing the flexibility of this approach, and enabling further insights into receptor dimerization., (© 2024 The Author(s).)