Cyclic dinucleotides (CDNs) are a significant and expanding class of secondary messengers that influence several vital bacterial physiological functions. Therefore, an understanding of the process by which CDNs are degraded by their cognate PDEs is crucial for comprehending a variety of cellular processes, such as the formation and dissemination of biofilms. As an alternative, it might be beneficial to create and/or identify non-hydrolyzable CDN derivatives to employ them as chemical probes of cyclic-di-GMP (c-di-GMP) signaling. Cyclic-di-inosine monophosphate, or c-di-IMP, is not a naturally occurring signaling molecule in biological systems, but it has strong adjuvant effects on metazoans and functions as an immunological modulator and stimulant. Here we report the first structural details of c-di-IMP and EAL interaction through high-resolution (2.2 Å) crystal structure of VcEAL in complex with c-di-IMP + Ca2+. Comparison of the VcEAL structures bound with cyclic-di-GMP (c-di-GMP), 3\',5\'-cyclic-AMP-GMP (cGAMP) and c-di-IMP and the structural variations at the chemical level between these CDNs provides their structural basis of recognition and rate of hydrolysis.

Yersinia pseudotuberculosis (Yptb) is a pathogenic gram-negative bacterium that can colonize the intestines of different animals. Its infection leads to the activation of the host\'s innate immunity. Both host and bacterial-derived cyclic dinucleotides (CDNs) could activate the innate immune response of host cells. In bacteria, CDNs like c-di-AMP, c-di-GMP, or 3\'3\'-cGAMP can be hydrolyzed by different hydrolases. Recent studies showed that the degradation of those second messengers helps the pathogen evade immune detection. In this study, we identified a hydrolase, YPK_3776, namely CpdB in Yptb. CpdB is predicted to bind bacterial-derived c-di-AMP, c-di-GMP, 3\'3\'-cGAMP and host-derived 2\'3\'-cGAMP. Surprisingly, by using high-performance liquid chromatography (HPLC), we found that CpdB could only degrade bacterial-derived CDNs but not host-derived 2\'3\'-cGAMP. In addition, CpdB has 2\'3\'-cNMP activity. Consistently, the Yptb mutant lacking the cpdB gene exhibited a higher level of intracellular c-di-GMP. Furthermore, the ∆cpdB mutant elicited stronger innate immune responses during Yptb infection in macrophages, suggesting CpdB enables Yptb to evade host immune surveillance. Furthermore, CpdB inhibited the Yptb-induced innate immune response in a STING-dependent manner. Finally, we showed the ∆cpdB infection in mice model exhibited in lower bacterial burden, as compared to wild-type strain infection, indicating CpdB is important for bacterial survival in the host. Together, we identified a cyclic dinucleotide hydrolase CpdB in Yptb that could degrade bacterial-derived CDNs which help the pathogen to evade immune detection via the STING pathway.

Long-term synaptic plasticity at glutamatergic synapses on striatal spiny projection neurons (SPNs) is central to learning goal-directed behaviors and habits. Our studies reveal that SPNs manifest a heterosynaptic, nitric oxide (NO)-dependent form of long-term postsynaptic depression of glutamatergic SPN synapses (NO-LTD) that is preferentially engaged at quiescent synapses. Plasticity is gated by Ca2+ entry through CaV1.3 Ca2+ channels and phosphodiesterase 1 (PDE1) activation, which blunts intracellular cyclic guanosine monophosphate (cGMP) and NO signaling. Both experimental and simulation studies suggest that this Ca2+-dependent regulation of PDE1 activity allows for local regulation of dendritic cGMP signaling. In a mouse model of Parkinson disease (PD), NO-LTD is absent because of impaired interneuronal NO release; re-balancing intrastriatal neuromodulatory signaling restores NO release and NO-LTD. Taken together, these studies provide important insights into the mechanisms governing NO-LTD in SPNs and its role in psychomotor disorders such as PD.

Microsporidia are obligate, intracellular, spore-forming eukaryotic fungi that infect humans and animals. In the treatment of disseminated microsporidiosis albendazole is the choice of drug. In recent years, antiparasitic activity of phosphodiesterase (PDE) enzyme inhibitors has been demonstrated against parasites and fungi, however, there is no information on microsporidia. Vinpocetine is currently used as a cerebral vasodilator drug and also as a dietary supplement to improve cognitive functions. Vinpocetine inhibits PDE1, so we aimed to investigate whether vinpocetine alone or in combination with albendazole has any effect on the spore load of Encephalitozoon intestinalis (E. intestinalis)-infected HEK293 cells. After determining the noncytotoxic concentrations of vinpocetine and albendazole on the host cell by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, HEK293 cells were infected with E. intestinalis spores. Then, two different concentrations of vinpocetine, albendazole, and a combination of both drugs were applied to the cells with an interval of 72 h for 15 days. Spore load of the cells was analyzed by real-time PCR. After the last treatment, spore Deoxyribonucleic Acid (DNA) load was significantly reduced only in the group treated with 14 ng/ml albendazole. It was not different from control in groups treated with 7 ng/ml albendazole and 4-20 µM vinpocetine. However, the combination of vinpocetine significantly increased the effect of albendazole at both concentrations. To our knowledge, this is the first study to investigate the microsporicidal activity of vinpocetine as well as its combinations with albendazole. However, further studies are needed to investigate the mechanism of action and also confirm in vivo conditions.
Encephalitozoon intestinalis, a common cause of microsporidia-associated diseases in humans, albendazole is used in the treatment of E. intestinalis infection, vinpocetine inhibits PDE1 and voltage-gated Ca2+ channels, vinpocetine significantly enhances the effect of albendazole on E. intestinalis spore DNA load.

OBJECTIVE: To identify the physiological role of the acid-base sensing enzyme, soluble adenylyl cyclase (sAC), in red blood cells (RBC) of the model teleost fish, rainbow trout.
METHODS: We used: (i) super-resolution microscopy to determine the subcellular location of sAC protein; (ii) live-cell imaging of RBC intracellular pH (pHi) with specific sAC inhibition (KH7 or LRE1) to determine its role in cellular acid-base regulation; (iii) spectrophotometric measurements of haemoglobin-oxygen (Hb-O2) binding in steady-state conditions; and (iv) during simulated arterial-venous transit, to determine the role of sAC in systemic O2 transport.
RESULTS: Distinct pools of sAC protein were detected in the RBC cytoplasm, at the plasma membrane and within the nucleus. Inhibition of sAC decreased the setpoint for RBC pHi regulation by ~0.25 pH units compared to controls, and slowed the rates of RBC pHi recovery after an acid-base disturbance. RBC pHi recovery was entirely through the anion exchanger (AE) that was in part regulated by HCO3 --dependent sAC signaling. Inhibition of sAC decreased Hb-O2 affinity during a respiratory acidosis compared to controls and reduced the cooperativity of O2 binding. During in vitro simulations of arterial-venous transit, sAC inhibition decreased the amount of O2 that is unloaded by ~11%.
CONCLUSIONS: sAC represents a novel acid-base sensor in the RBCs of rainbow trout, where it participates in the modulation of RBC pHi and blood O2 transport though the regulation of AE activity. If substantiated in other species, these findings may have broad implications for our understanding of cardiovascular physiology in vertebrates.

Tadalafil, a potent phosphodiesterase inhibitor 5 (PDE-5), is commonly used for the management of erectile dysfunction. However, its therapeutic potential extends beyond this indication. This study aimed to investigate the impact of tadalafil on the recovery of testicular parenchyma in male Wistar rats exposed to testicular thermal stress. Fifty-four Wistar rats were subjected to testicular thermal stress and randomly assigned to receive either tadalafil treatment (TAD) or no treatment (control). TAD was administered intraperitoneally at a dose of either 0.9 mg/kg or 1.8 mg/kg. Biometric parameters, histopathological assessment of the testis, serum testosterone levels, oxidative stress, and interleukin levels were evaluated on days 7, 15, and 30 after thermal shock. The animals were euthanized at the end of each experimental period, and samples were collected. TAD treatment maintained testicular weight and reduced the testicular degenerative process up to day 7 post-injury. However, despite TAD therapy, serum testosterone levels were decreased in the treated groups at days 7 and 15 post-thermal stress. TAD also decreased TNF-α and NO levels at different doses but had no effect on IL-6. The treatment with TAD after heat shock demonstrated anti-inflammatory and antioxidant properties but did not prevent the aggravation of testicular lesions in subsequent periods, even with the systematic reduction in TNF-α and NO levels. Therefore, this selective PDE-5 inhibitor, at the dosages used, did not have a positive impact on testosterone levels during the post-thermal stress period, which could compromise the resumption of the spermatogenic process.

We developed a method that utilizes fluorescent labeling of nuclear envelopes alongside cytometry sorting for the selective isolation of Purkinje cell (PC) nuclei. Beginning with SUN1 reporter mice, we GFP-tagged envelopes to confirm that PC nuclei could be accurately separated from other cell types. We then developed an antibody-based protocol to make PC nuclear isolation more robust and adaptable to cerebellar tissues of any genotypic background. Immunofluorescent labeling of the nuclear membrane protein RanBP2 enabled the isolation of PC nuclei from C57BL/6 cerebellum. By analyzing the expression of PC markers, nuclear size, and nucleoli number, we confirmed that our method delivers a pure fraction of PC nuclei. To demonstrate its applicability, we isolated PC nuclei from spinocerebellar ataxia type 7 (SCA7) mice and identified transcriptional changes in known and new disease-associated genes. Access to pure PC nuclei offers insights into PC biology and pathology, including the nature of selective neuronal vulnerability.

UNASSIGNED: Phosphodiesterase 9 (PDE9) has been demonstrated as a potential target for neurological disorders and cardiovascular diseases, such as Alzheimer\'s disease and heart failure. For the last few years, a series of PDE9 inhibitors with structural diversities have been developed and patented by researchers and pharmaceutical companies, providing insights into first-in-class therapies of PDE9 drug candidates.
UNASSIGNED: This review provides an overview of PDE9 inhibitors in patents from 2018 to the present.
UNASSIGNED: Only a few of the current PDE9 inhibitors are highly selective over other PDEs, which limits their application in pharmacological and clinical research. The design and development of highly selective PDE9 inhibitors remain the top priority in future research. The advantages of targeting PDE9 rather than other PDEs in treating neurodegenerative diseases need to be explained thoroughly. Besides, application of PDE9 inhibitor-based combination therapies sheds light on treating diabetes and refractory heart diseases. Finally, PDE9 inhibitors should be further explored in clinical indications beyond neurological disorders and cardiovascular diseases.

Mycobacterium tuberculosis (M. tb) is a significant intracellular pathogen responsible for numerous infectious disease-related deaths worldwide. It uses ESX-1 T7SS to damage phagosomes and to enter the cytosol of host cells after phagocytosis. During infection, M. tb and host mitochondria release dsDNA, which activates the CGAS-STING1 pathway. This pathway leads to the production of type I interferons and proinflammatory cytokines and activates autophagy, which targets and degrades bacteria within autophagosomes. However, the role of type I IFNs in immunity against M. tb is controversial. While previous research has suggested a protective role, recent findings from cgas-sting1 knockout mouse studies have contradicted this. Additionally, a study using knockout mice and non-human primate models uncovered a new mechanism by which neutrophils recruited to lung infections form neutrophil extracellular traps. Activating plasmacytoid dendritic cells causes them to produce type I IFNs, which interfere with the function of interstitial macrophages and increase the likelihood of tuberculosis. Notably, M. tb uses its virulence proteins to disrupt the CGAS-STING1 signaling pathway leading to enhanced pathogenesis. Investigating the CGAS-STING1 pathway can help develop new ways to fight tuberculosis.

Biochemical assays are described to analyze signal transduction by the second messenger cGMP in Dictyostelium. The methods include enzyme assays to measure the activity and regulation of cGMP synthesizing guanylyl cyclases and cGMP-degrading phosphodiesterases. In addition, several methods are described to quantify cGMP levels. The target of cGMP in Dictyostelium is the large protein GbpC that has multiple domains including a Roc domain, a kinase domain, and a cGMP-stimulated Ras-GEF domain. A cGMP-binding assay is described to detect and quantify GbpC.