Discovery of 4-amino-1,6-dihydro-7H-pyrrolo[2,3-d]pyridazin-7-one derivatives as potential receptor-interacting serine/threonine-protein kinase 1 (RIPK1) inhibitors.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is an important regulatory factor in the necroptosis signaling pathway, and is considered an attractive therapeutic target for treating multiple inflammatory diseases. Herein, we describe the design, synthesis, and structure-activity relationships of 4-amino-1,6-dihydro-7H-pyrrolo [2,3-d]pyridazin-7-one derivatives as RIPK1 inhibitors. Among them, 13c showed favorable RIPK1 kinase inhibition activity with an IC50 value of 59.8 nM, and high RIPK1 binding affinity compared with other regulatory kinases of necroptosis (RIPK1 Kd = 3.5 nM, RIPK3 Kd = 1700 nM, and MLKL Kd > 30,000 nM). 13c efficiently blocked TNFα-induced necroptosis in both human and murine cells (EC50 = 1.06-4.58 nM), and inhibited TSZ-induced phosphorylation of the RIPK1/RIPK3/MLKL pathway. In liver microsomal assay studies, the clearance rate and half-life of 13c were 18.40 mL/min/g and 75.33 min, respectively. 13c displayed acceptable pharmacokinetic characteristics, with oral bioavailability of 59.55%. In TNFα-induced systemic inflammatory response syndrome, pretreatment with 13c could effectively protect mice from loss of body temperature and death. Overall, these compounds are promising candidates for future optimization studies.

Activation of RMTg projections to the VTA reverse cocaine-induced molecular adaptation in the reward system.

The rostromedial tegmental nucleus (RMTg) plays a crucial role in regulating reward-related behavior by exerting inhibitory control over the ventral tegmental area (VTA). This modulation of dopamine neuron activity within the VTA is essential for maintaining homeostasis in the reward system. Recently we have shown that activation of RMTg projections to the VTA during the acquisition of cocaine-conditioned place preference (CPP) reduces the rewarding properties of cocaine and decreases VTA dopamine neuron activity. By inhibiting dopamine neurons in the VTA, we hypothesized that RMTg projections hold the potential to restore reward system homeostasis disrupted by repeated cocaine use, and attenuate molecular adaptations in the reward system, including alterations in signaling pathways. Our study demonstrates that enhancing the GABAergic inputs from the RMTg to the VTA can mitigate cocaine-induced molecular changes in key regions, namely the VTA, nucleus accumbens (NAc), and prefrontal cortex (PFC). Specifically, we found that cocaine-induced alteration in the phosphorylation state of ERK (pERK) and GluA1 on serine 845 (S845) and serine 831 (S831), that play a major role in plasticity by controlling the activity and trafficking of AMPA receptors, were significantly reversed following optic stimulation of RMTg afferents to the VTA. These findings highlight the therapeutic potential of targeting the RMTg-VTA circuitry for mitigating cocaine reward. Ultimately, this research may pave the way for novel therapeutic interventions that restore balance in the reward system and alleviate the detrimental effects of cocaine.

Inhibitory actions of oxyresveratrol on the PI3K/AKT signaling cascade in cervical cancer cells.

The phosphatidyl inositol 3-kinase (PI3K)/AKT signaling plays a critical role in cancer cell proliferation, migration, and invasion. This signal transduction axis in HPV-positive cervical cancer has been proved to be directly activated by E6/E7 proteins of the virus enhancing cervical cancer progression. Hence, the PI3K/AKT pathway is one of the key therapeutic targets for HPV-positive cervical cancer. Here we discovered that oxyresveratrol (Oxy) at noncytotoxic concentration specifically suppressed the phosphorylation of AKT but not ERK1/2. This potent inhibitory effect of Oxy was still observed even when cells were stimulated with fetal bovine serum. Inhibition of AKT phosphorylation at serine 473 by Oxy resulted in a significant decrease in serine 9 phosphorylation of GSK-3ß, a downstream target of AKT. Dephosphorylation of GSK-3ß at this serine residue activates its function in promoting the degradation of MCL-1, an anti-apoptotic protein. Results clearly demonstrated that in association with GSK-3ß activation, Oxy preferentially downregulated the expression of anti-apoptotic protein MCL-1. Furthermore, results from the functional analyses revealed that Oxy inhibited cervical cancer cell proliferation, at least in part through suppressing nuclear expression of Ki-67. Besides, the compound retarded cervical cancer cell migration even the cells were exposed to a potent enhancer of epithelial-mesenchymal transition, TGF-ß1. In consistent with these data, Oxy reduced the expression of ß-catenin, N-cadherin, and vimentin. In conclusion, the study disclosed that Oxy specifically inhibits the AKT/GSK-3ß/MCL-1 axis resulting in reduction in cervical cancer cell viability, proliferation, and migration.

P2X receptor- and postsynaptic NMDA receptor-mediated long-lasting facilitation of inhibitory synapses in the rat insular cortex.

Adenosine triphosphate (ATP) changes the efficacy of synaptic transmission. Despite recent progress in terms of the roles of purinergic receptors in cerebrocortical excitatory synaptic transmission, their contribution to inhibitory synaptic transmission is unknown. To elucidate the effects of α,ß-methylene ATP (αß-mATP), a selective agonist of P2X receptors (P2XRs), on inhibitory synaptic transmission in the insular cortex (IC), we performed whole-cell patch-clamp recording from IC pyramidal neurons (PNs) and fast-spiking neurons (FSNs) in either sex of VGAT-Venus transgenic rats. αß-mATP increased the amplitude of miniature IPSCs (mIPSCs) under conditions in which NMDA receptors (NMDARs) are recruitable. αß-mATP-induced facilitation of mIPSCs was sustained even after the washout of αß-mATP, which was blocked by preincubation with fluorocitrate. The preapplication of NF023 (a P2X1 receptor antagonist) or AF-353 (a P2X3 receptor antagonist) blocked αß-mATP-induced mIPSC facilitation. Intracellular application of the NMDAR antagonist MK801 blocked the facilitation. d-serine, which is an intrinsic agonist of NMDARs, mimicked αß-mATP-induced mIPSC facilitation. The intracellular application of BAPTA a Ca2+ chelator, or the bath application of KN-62, a CaMKII inhibitor, blocked αß-mATP-induced mIPSC facilitation, thus indicating that mIPSC facilitation by αß-mATP required postsynaptic [Ca2+]i elevation through NMDAR activation. Paired whole-cell patch-clamp recordings from FSNs and PNs demonstrated that αß-mATP increased the amplitude of unitary IPSCs without changing the paired-pulse ratio. These results suggest that αß-mATP-induced IPSC facilitation is mediated by postsynaptic NMDAR activations through d-serine released from astrocytes. Subsequent [Ca2+]i increase and postsynaptic CaMKII activation may release retrograde messengers that upregulate GABA release from presynaptic inhibitory neurons, including FSNs. (250/250 words).

Discovery of hydroxamate as a promising scaffold dually inhibiting metallo- and serine-ß-lactamases.

The bacterial infection mediated by ß-lactamases MßLs and SßLs has grown into an emergent health threat, however, development of a molecule that dual inhibits both MßLs and SßLs is challenging. In this work, a series of hydroxamates 1a-g, 2a-e, 3a-c, 4a-c were synthesized, characterized by 1H and 13C NMR and confirmed by HRMS. Biochemical assays revealed that these molecules dually inhibited MßLs (NDM-1, IMP-1) and SßLs (KPC-2, OXA-48), with an IC50 value in the range of 0.64-41.08 and 1.01-41.91 µM (except 1a and 1d on SßLs, IC50 > 50 µM), and 1f was found to be the best inhibitor with an IC50 value in the range of 0.64-1.32 and 0.57-1.01 µM, respectively. Mechanism evaluation indicated that 1f noncompetitively and irreversibly inhibited NDM-1 and KPC-2, with Ki value of 2.5 and 0.55 µM, is a time- and dose-dependent inhibitor of both MßLs and SßLs. MIC tests shown that all hydroxamates increased the antimicrobial effect of MER on E. coli-NDM-1 and E. coli-IMP-1 (expect 1b, 1d, 1g and 2d), resulting in a 2-8-fold reduction in MICs of MER, 1e-g, 2b-d, 3a-c and 4b-c decreased 2-4-fold MICs of MER on E. coli-KPC-2, and 1c, 1f-g, 2a-c, 3b, 4a and 4c decreased 2-16-fold MICs of MER on E. coli-OXA-48. Most importantly, 1f-g, 2b-c, 3b and 4c exhibited the dual synergizing inhibition against both E. coli-MßLs and E. coli-SßLs tested, resulting in a 2-8-fold reduction in MICs of MER, and 1f was found to have the best effect on the drug-resistant bacteria tested. Also, 1f shown synergizing antimicrobial effect on five clinical isolates EC04, EC06, EC08, EC10 and EC24 that produce NDM-1, resulting in a 2-8-fold reduction in MIC of MER, but its effect on E. coli and K. pneumonia-KPC-NDM was not to be observed using the same dose of inhibitor. Mice tests shown that the monotherapy of 1f or 4a in combination with MER significantly reduced the bacterial load of E. coli-NDM-1 and E. coli-OXA-48 cells in liver and spleen, respectively. The discovery in this work offered a promising bifunctional scaffold for creating the specific molecules that dually inhibit MßLs and MßLs, in combating antibiotic-resistant bacteria.

D-serine reconstitutes synaptic and intrinsic inhibitory control of pyramidal neurons in a neurodevelopmental mouse model for schizophrenia.

The hypothesis of N-methyl-D-aspartate receptor (NMDAR) dysfunction for cognitive impairment in schizophrenia constitutes the theoretical basis for the translational application of NMDAR co-agonist D-serine or its analogs. However, the cellular mechanism underlying the therapeutic effect of D-serine remains unclear. In this study, we utilize a mouse neurodevelopmental model for schizophrenia that mimics prenatal pathogenesis and exhibits hypoexcitability of parvalbumin-positive (PV) neurons, as well as PV-preferential NMDAR dysfunction. We find that D-serine restores excitation/inhibition balance by reconstituting both synaptic and intrinsic inhibitory control of cingulate pyramidal neurons through facilitating PV excitability and activating small-conductance Ca2+-activated K+ (SK) channels in pyramidal neurons, respectively. Either amplifying inhibitory drive via directly strengthening PV neuron activity or inhibiting pyramidal excitability via activating SK channels is sufficient to improve cognitive function in this model. These findings unveil a dual mechanism for how D-serine improves cognitive function in this model.

Effects of the Toxic Non-Protein Amino Acid ß-Methylamino-L-Alanine (BMAA) on Intracellular Amino Acid Levels in Neuroblastoma Cells.

The cyanobacterial non-protein amino acid (AA) ß-Methylamino-L-alanine (BMAA) is considered to be a neurotoxin. BMAA caused histopathological changes in brains and spinal cords of primates consistent with some of those seen in early motor neuron disease; however, supplementation with L-serine protected against some of those changes. We examined the impact of BMAA on AA concentrations in human neuroblastoma cells in vitro. Cells were treated with 1000 µM BMAA and intracellular free AA concentrations in treated and control cells were compared at six time-points over a 48 h culture period. BMAA had a profound effect on intracellular AA levels at specific time points but in most cases, AA homeostasis was re-established in the cell. The most heavily impacted amino acid was serine which was depleted in BMAA-treated cells from 9 h onwards. Correction of serine depletion could be a factor in the observation that supplementation with L-serine protects against BMAA toxicity in vitro and in vivo. AAs that could potentially be involved in protection against BMAA-induced oxidation such as histidine, tyrosine, and phenylalanine were depleted in cells at later time points.

[Effects and mechanisms of total flavones of Abelmoschus manihot in inhibiting podocyte necroptosis and renal fibrosis in diabetic kidney disease].

Previous studies have shown that high blood glucose-induced chronic microinflammation can cause inflammatory podocyte injury in patients with diabetic kidney disease(DKD). Therein, necroptosis is a new form of podocyte death that is closely associated with renal fibrosis(RF). To explore the effects and mechanisms in vivo of total flavones of Abelmoschus manihot(TFA), an extract from traditional Chinese herbal medicine Abelmoschus manihot for treating kidney diseases, on podocyte necroptosis and RF in DKD, and to further reveal its scientific connotation with multi-pathway and multi-target, the authors randomly divided all rats into four groups: a namely normal group, a model group, a TFA group and a rapamycin(RAP) group. After the modified DKD rat models were successfully established, four group rats were given double-distilled water, TFA suspension and RAP suspension, respectively by gavage every day. At the end of the 4th week of drug treatment, all rats were sacrificed, and the samples of their urine, blood and kidneys were collected. And then, the various indicators related to podocyte necroptosis and RF in the DKD model rats were observed, detected and analyzed, respectively. The results indicated that, general condition, body weight(BW), serum creatinine(Scr), urinary albumin(UAlb), and kidney hypertrophy index(KHI) in these modified DKD model rats were both improved by TFA and RAP. Indicators of RF, including glomerular histomorphological characteristics, fibronectin(FN) and collagen type Ⅰ(collagen Ⅰ) staining extent in glomeruli, as well as the protein expression levels of FN, collagen Ⅰ, transforming growth factor-ß1(TGF-ß1) and Smad2/3 in the kidneys were improved respectively by TFA and RAP. Podocyte damage, including foot process form and the protein expression levels of podocin and CD2AP in the kidneys was improved by TFA and RAP. In addition, tumor necrosis factor-α(TNF-α)-mediated podocyte necroptosis in the kidneys, including the morphological characteristics of podocyte necroptosis, the extent and levels of the protein expression of TNF-α and phosphorylated mixed lineage kinase domain like pseudokinase(p-MLKL) was improved respectively by TFA and RAP. Among them, RAP had the better effect on p-MLKL. More importantly, the activation of the receptor interacting serine/threonine protein kinase 1(RIPK1)/RIPK3/MLKL signaling axis in the kidneys, including the expression levels of its key signaling molecules, such as phosphorylated receptor interacting serine/threonine protein kinase 1(p-RIPK1), p-RIPK3, p-MLKL and cysteinyl aspartate specific proteinase-8(caspase-8) was improved respectively by TFA and RAP. Among them, the effect of TFA on p-RIPK1 was superior. On the whole, in this study, the authors demonstrated that TFA alleviates podocyte necroptosis and RF in DKD through inhibiting the activation of the TNF-α-mediated RIPK1/RIPK3/MLKL signaling axis in diabetic kidneys. The authors' findings provide new pharmacological evidence to reveal the scientific connotation of TFA in treating RF in DKD in more depth.

Knockdown of BUB1 inhibits tumor necrosis factor-α-induced proliferation and migration of rheumatoid arthritis synovial fibroblasts by regulating PI3K/Akt pathway.

BACKGROUND: Rheumatoid arthritis (RA) is a common disease with joint cartilage destruction. BUB1 Mitotic Checkpoint Serine/Threonine Kinase (BUB1) is abnormally expressed in synovial tissues of RA patients, but its effect on RA remains unclear. In this study, we explored the role of BUB1 in RA. METHODS: An RA cell model was constructed by treating MH7A cells with tumor necrosis factor-α (TNF-α). The levels of BUB1, GAPDH, phosphorylated phosphatidylinositol 3 kinase (p-PI3K)/PI3K, and phosphorylated serine/threonine kinase (p-Akt)/Akt in MH7A cells were examined by Western blot. The MH7A cell proliferation was examined by colony formation assay. Wound healing assay and transwell assay were carried out to detect MH7A cell migration and invasion. The mRNA levels of proinflammatory cytokines were assessed by quantitative reverse transcription polymerase chain reaction. RESULTS: The results showed that knockdown BUB1 inhibited TNF-α-induced MH7A cell proliferation, migration, and invasion. Silencing BUB1 repressed the PI3K/Akt pathway in TNF-α-induced MH7A cells. We also found that the TNF-α-induced MH7A cell proliferation, migration, and invasion were repressed by si-BUB1 transfection, whereas these effects were attenuated by 740Y-P (an activator of the PI3K pathway) co-treatment. Knockdown of BUB1 reduced the expression of the proinflammatory cytokines. CONCLUSION: Knockdown BUB1 repressed TNF-α-induced MH7A cell proliferation, migration and invasion through the PI3K/Akt pathway.

Luvadaxistat: A Novel Potent and Selective D-Amino Acid Oxidase Inhibitor Improves Cognitive and Social Deficits in Rodent Models for Schizophrenia.

N-methyl-D-aspartate (NMDA) receptor hypofunctionality is a well-studied hypothesis for schizophrenia pathophysiology, and daily dosing of the NMDA receptor co-agonist, D-serine, in clinical trials has shown positive effects in patients. Therefore, inhibition of D-amino acid oxidase (DAAO) has the potential to be a new therapeutic approach for the treatment of schizophrenia. TAK-831 (luvadaxistat), a novel, highly potent inhibitor of DAAO, significantly increases D-serine levels in the rodent brain, plasma, and cerebrospinal fluid. This study shows luvadaxistat to be efficacious in animal tests of cognition and in a translational animal model for cognitive impairment in schizophrenia. This is demonstrated when luvadaxistat is dosed alone and in conjunction with a typical antipsychotic. When dosed chronically, there is a suggestion of change in synaptic plasticity as seen by a leftward shift in the maximum efficacious dose in several studies. This is suggestive of enhanced activation of NMDA receptors in the brain and confirmed by modulation of long-term potentiation after chronic dosing. DAAO is highly expressed in the cerebellum, an area of increasing interest for schizophrenia, and luvadaxistat was shown to be efficacious in a cerebellar-dependent associative learning task. While luvadaxistat ameliorated the deficit seen in sociability in two different negative symptom tests of social interaction, it failed to show an effect in endpoints of negative symptoms in clinical trials. These results suggest that luvadaxistat potentially could be used to improve cognitive impairment in patients with schizophrenia, which is not well addressed with current antipsychotic medications.

Ergometrine stimulates histamine H2 receptors in the isolated human atrium.

Ergometrine (6aR,9R)-N-((S)-1-hydroxypropan-2-yl)-7-methyl-4,6,6a,7,8,9-hexa-hydro-indolo-[4,3-fg]chinolin-9-carboxamide or lysergide acid ß-ethanolamide or ergonovine) activates several types of serotonin and histamine receptors in the animal heart. We thus examined whether ergometrine can activate human serotonin 5-HT4 receptors (h5-HT4R) and/or human histamine H2 receptors (hH2R) in the heart of transgenic mice and/or in the human isolated atrium. Force of contraction or beating rates were studied in electrically stimulated left atrial or spontaneously beating right atrial preparations or spontaneously beating isolated retrogradely perfused hearts (Langendorff setup) of mice with cardiac specific overexpression of the h5-HT4R (5-HT4-TG) or of mice with cardiac specific overexpression of the hH2R (H2-TG) or in electrically stimulated human right atrial preparations obtained during cardiac surgery. Western blots to assess phospholamban (PLB) phosphorylation on serine 16 were performed. Ergometrine exerted concentration- and time-dependent positive inotropic effects and positive chronotropic effects in atrial preparations starting at 0.3 µM and reaching a plateau at 10 µM in H2-TGs (n = 7). This was accompanied by an increase in PLB phosphorylation at serine 16. Ergometrine up 10 µM failed to increase force of contraction in left atrial preparations from 5-HT4-TGs (n = 5). Ten micrometer ergometrine increased the force of contraction in isolated retrogradely perfused spontaneously beating heart preparations (Langendorff setup) from H2-TG but not 5-HT4-TG. In the presence of the phosphodiesterase inhibitor cilostamide (1 µM), ergometrine at 10 µM exerted positive inotropic effects in isolated electrically stimulated human right atrial preparations, obtained during cardiac surgery, and these effects were eliminated by 10 µM of the H2R antagonist cimetidine but not by 10 µM of the 5-HT4R antagonist tropisetron. Furthermore, ergometrine showed binding to human histamine H2 receptors (at 100 µM and 1 mM) using HEK cells in a recombinant expression system (pKi < 4.5, n = 3). In conclusion, we suggest that ergometrine is an agonist at cardiac human H2Rs.

Serine Threonine-Protein Kinase-Derived IW13 Improves Lipid Metabolism via C/EBP-α/SREBP1/FAS Signaling Pathways in HFD-Induced Zebrafish In Vivo Larval Model.

Obesity is linked to the development of major metabolic disorders such as type 2 diabetes, cardiovascular disease, and cancer. Recent research has focused on the molecular link between obesity and oxidative stress. Obesity impairs antioxidant function, resulting in dramatically increased reactive oxygen levels and apoptosis. In this study, we investigated the effect of IW13 peptide on inhibiting lipid accumulation and regulating the antioxidant mechanism to normalize the lipid metabolism in HFD induced zebrafish larvae. Our results showed that co-treatment with IW13 peptide showed a protective effect in HFD zebra fish larvae by increasing the survival and heart rate. However, IW13 peptide co-treatment reduced triglycerides and cholesterol levels while also restoring the SOD and CAT antioxidant enzymes. In addition, IW13 co-treatment inhibited the formation of lipid peroxidation and superoxide anion by regulating the glutathione level. Also, the results showed that IW13 specifically downregulated the expression of the lipogenic-specific genes (C/EBP-α, SREBP1, and FAS). The findings exhibited that the IW13 peptide with effective antioxidant and anti-obesity activity could act as a futuristic drug to treat obesity and oxidative stress-related diseases.

The Acute Effects and Mechanism of Ketamine on Nicotine-Induced Neurogenic Relaxation of the Corpus Cavernosum in Mice.

The present study aimed to investigate the acute effects and the mechanism of ketamine on nicotine-induced relaxation of the corpus cavernosum (CC) in mice. This study measured the intra-cavernosal pressure (ICP) of male C57BL/6 mice and the CC muscle activities using an organ bath wire myograph. Various drugs were used to investigate the mechanism of ketamine on nicotine-induced relaxation. Direct ketamine injection into the major pelvic ganglion (MPG) inhibited MPG-induced increases in ICP. D-serine/L-glutamate-induced relaxation of the CC was inhibited by MK-801 (N-methyl-D-aspartate (NMDA) receptor inhibitor), and nicotine-induced relaxation was enhanced by D-serine/L-glutamate. NMDA had no effect on CC relaxation. Nicotine-induced relaxation of the CC was suppressed by mecamylamine (a non-selective nicotinic acetylcholine receptor antagonist), lidocaine, guanethidine (an adrenergic neuronal blocker), Nw-nitro-L-arginine (a non-selective nitric oxide synthase inhibitor), MK-801, and ketamine. This relaxation was almost completely inhibited in CC strips pretreated with 6-hydroxydopamine (a neurotoxic synthetic organic compound). Ketamine inhibited cavernosal nerve neurotransmission via direct action on the ganglion and impaired nicotine-induced CC relaxation. The relaxation of the CC was dependent on the interaction of the sympathetic and parasympathetic nerves, which may be mediated by the NMDA receptor.

Serine-/Cysteine-Based sp2-Iminoglycolipids as Novel TLR4 Agonists: Evaluation of Their Adjuvancy and Immunotherapeutic Properties in a Murine Model of Asthma.

Glycolipids with TLR4 agonistic properties can serve either as therapeutic agents or as vaccine adjuvants by stimulating the development of proinflammatory responses. Translating them to the clinical setting is hampered by synthetic difficulties, the lack of stability in biological media, and/or a suboptimal profile of balanced immune mediator secretion. Here, we show that replacement of the sugar fragment by an sp2-iminosugar moiety in a prototypic TLR4 agonist, CCL-34, yields iminoglycolipid analogues that retain or improve their biological activity in vitro and in vivo and can be accessed through scalable protocols with total stereoselectivity. Their adjuvant potential is manifested in their ability to induce the secretion of proinflammatory cytokines, prime the maturation of dendritic cells, and promote the proliferation of CD8+ T cells, pertaining to a Th1-biased profile. Additionally, their therapeutic potential for the treatment of asthma, a Th2-dominated inflammatory pathology, has been confirmed in an ovalbumin-induced airway hyperreactivity mouse model.

Nutritional geometry framework of sucrose taste in Drosophila.

Dietary protein (P) and carbohydrate (C) have a major impact on the sweet taste sensation. However, it remains unclear whether the balance of P and C influences the sweet taste sensitivity. Here, we use the nutritional geometry framework (NGF) to address the interaction of protein and carbohydrates on sweet taste using Drosophila as a model. Our results reveal that high-protein, low-carbohydrate (HPLC) diets sensitize to sweet taste and low-protein, high-carbohydrate (LPHC) diets desensitize sweet taste in both male and female flies. We further investigate the underlying mechanisms of the effects of two diets on sweet taste using RNA sequencing. When compared to the LPHC diet, the mRNA expression of genes involved in the metabolism of glycine, serine, and threonine is significantly upregulated in the HPLC diet group, suggesting these amino acids may mediate sweet taste perception. We further find that sweet sensitization occurs in flies fed with the LPHC diet supplemented with serine and threonine. Our study demonstrates that sucrose taste sensitivity is affected by the balance of dietary protein and carbohydrates possibly through changes in serine and threonine.

Enhanced efficiency of calcium-derived oleoyl serine on osteoporosis via Wnt/ß-catenin pathway.

Osteoporosis is a common disease of the elderly that affects millions of patients worldwide. It is mainly characterized by low bone mineral density and increased risk of fracture due to the deterioration of the bone structure, leading to difficulties in functional recovery, reduced quality of life, increased disability risk and mortality in the population. It has already been a major public health problem. Osteoporosis is a chronic disease that is difficult to treat in the elderly population, so it is crucial to develop new drugs for the treatment of osteoporosis. Oleoyl serine, an endogenous fatty acyl amide found in bone, has been shown to have excellent anti-osteoporosis effects, but it is easily hydrolyzed by amidases in vivo. The aim of this study is to determine the anti-osteoporotic effect of calcium-derived oleoyl serine, a novel oleoyl serine derivative and the molecular mechanism underneath. In vitro experiments demonstrated that calcium-derived oleoyl serine suppressed the expression of Fabp4, and Cebpα while Alp, and Runx2 was significantly upregulated compared with the oleoyl serine group and control. With the activation of ß-catenin, calcium-derived oleoyl serine restored the abnormal osteogenesis and lipogenesis, indicating calcium-derived oleoyl serine compared with oleoyl serine has better effects on promoting osteogenesis and suppressing lipogenesis. In vivo experiment agreed with these findings that calcium-derived oleoyl serine promotes osteogenesis and suppresses its lipogenesis to ameliorate osteoporosis via a ß-catenin dependent method. It is a new candidate for treating osteoporosis.

Honokiol attenuates mitochondrial fission and cell apoptosis by activating Sirt3 in intracerebral hemorrhage.

BACKGROUND: Sirtuin-3 (Sirt3) has been documented to protect against mitochondrial dysfunction and apoptosis. Honokiol (HKL) is a Sirt3 pharmacological activator with reported neuroprotective effects in multiple neurological disorders. The present study aimed to explore the neuroprotective effects of HKL and the role of Sirt3 following intracerebral hemorrhage (ICH). METHODS: An in vivo ICH model in rats was established by injecting autologous blood into the right basal ganglia. PC12 cells were stimulated with hemin. For the in vivo investigation, the modified Neurological Severity Scores and the Morris water maze test were performed to assess neurological deficits. Hematoxylin-Eosin and Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining were employed to evaluate the histopathology and apoptosis. Immunohistochemical staining was used to investigate the expression of Sirt3. Adenosine triphosphate (ATP) levels were quantified to assess mitochondrial dysfunction. Cell counting kit-8, lactate dehydrogenase assay, and flow cytometry were used to analyze cell vitality and apoptosis in vitro. Immunofluorescence staining was performed to observe mitochondrial morphology and dynamin-related protein 1 (Drp1) localization to mitochondria. Western blot was applied to quantify the expression of Sirt3, Bax, Bcl-2, cleaved-caspase-3, Drp1, phosphorylation of Drp1 at serine-616, and phosphorylation of Drp1 at serine-637 in vivo and in vitro. RESULTS: HKL treatment alleviated neurological deficits, attenuated the histopathological damage and cell apoptosis, and restored the decreased ATP levels in ICH rats. HKL improved cell survival rate, reduced cell apoptosis, and inhibited mitochondrial fission in PC12 cells. Moreover, both in vivo and in vitro models showed increased phosphorylation of Drp1 at Ser616, and reduced phosphorylation of Drp1 at Ser637. Meanwhile, immunofluorescence co-localization analysis revealed that hemin increased the overlap of Drp1 and mitochondria in PC12 cells. The phosphorylation and mitochondrial translocation of Drp1 were effectively reversed by HKL treatment. Importantly, the selective Sirt3 inhibitor 3-(1H-1,2,3-triazol-4-yl) pyridine suppressed these effects. CONCLUSION: Our findings demonstrated that HKL ameliorated ICH-induced apoptosis and mitochondrial fission by Sirt3, suggesting that HKL has immense prospects for the treatment of ICH.

Reduced attentional lapses in male rats following a combination treatment of low-dose D-serine and atomoxetine.

BACKGROUND: Goal-directed attention involves the selective processing of behaviorally relevant sensory information. This selective processing is thought to be supported by glutamatergic and noradrenergic systems. Pharmacotherapies that simultaneously target these systems could therefore be effective treatments for impaired attention. AIMS: We first tested an N-methyl-D-aspartate (NMDA) receptor co-agonist (D-serine) for effects on attention (processing speed and attentional lapses). NMDA receptor activation is thought to support noradrenergic effects on sensory processing; therefore, we tested a combination treatment comprising D-serine and a norepinephrine reuptake inhibitor (atomoxetine). METHODS: D-serine was first tested in rats performing a two-choice visuospatial discrimination task. Combination treatments comprising relatively low doses of D-serine and atomoxetine were then tested in a separate group. RESULTS: In experiment 1, D-serine reduced the skew of initiation time (IT) distributions (IT devmode) at the highest dose tested (300 mg/kg). In experiment 2, low-dose D-serine (125 mg/kg) had no effect, while low-dose atomoxetine (0.3 mg/kg) reduced IT devmode and slowed movement speed. Importantly, the combination of these relatively low doses of D-serine and atomoxetine reduced IT devmode more than either drug alone without further slowing movement speed. CONCLUSIONS: IT devmode is thought to reflect attentional lapses; therefore, D-serine's effects on IT devmode suggest that NMDA receptors are involved in the preparatory deployment of attention. Greater effects following a combination of D-serine and atomoxetine suggest that preparatory attention can be facilitated by targeting glutamatergic and noradrenergic systems simultaneously. These results could inform the development of improved treatments for individuals with ADHD who experience abnormally high attentional lapses.

RyR2 Serine-2030 PKA Site Governs Ca2+ Release Termination and Ca2+ Alternans.

BACKGROUND: PKA (protein kinase A)-mediated phosphorylation of cardiac RyR2 (ryanodine receptor 2) has been extensively studied for decades, but the physiological significance of PKA phosphorylation of RyR2 remains poorly understood. Recent determination of high-resolution 3-dimensional structure of RyR2 in complex with CaM (calmodulin) reveals that the major PKA phosphorylation site in RyR2, serine-2030 (S2030), is located within a structural pathway of CaM-dependent inactivation of RyR2. This novel structural insight points to a possible role of PKA phosphorylation of RyR2 in CaM-dependent inactivation of RyR2, which underlies the termination of Ca2+ release and induction of cardiac Ca2+ alternans. METHODS: We performed single-cell endoplasmic reticulum Ca2+ imaging to assess the impact of S2030 mutations on Ca2+ release termination in human embryonic kidney 293 cells. Here we determined the role of the PKA site RyR2-S2030 in a physiological setting, we generated a novel mouse model harboring the S2030L mutation and carried out confocal Ca2+ imaging. RESULTS: We found that mutations, S2030D, S2030G, S2030L, S2030V, and S2030W reduced the endoplasmic reticulum luminal Ca2+ level at which Ca2+ release terminates (the termination threshold), whereas S2030P and S2030R increased the termination threshold. S2030A and S2030T had no significant impact on release termination. Furthermore, CaM-wild-type increased, whereas Ca2+ binding deficient CaM mutant (CaM-M [a loss-of-function CaM mutation with all 4 EF-hand motifs mutated]), PKA, and Ca2+/CaMKII (CaM-dependent protein kinase II) reduced the termination threshold. The S2030L mutation abolished the actions of CaM-wild-type, CaM-M, and PKA, but not CaMKII, in Ca2+ release termination. Moreover, we showed that isoproterenol and CaM-M suppressed pacing-induced Ca2+ alternans and accelerated Ca2+ transient recovery in intact working hearts, whereas CaM-wild-type exerted an opposite effect. The impact of isoproterenol was partially and fully reversed by the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide and the CaMKII inhibitor N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide individually and together, respectively. S2030L abolished the impact of CaM-wild-type, CaM-M, and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide-sensitive component, but not the N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide-sensitive component, of isoproterenol.