Sufentanil inhibits Pin1 to attenuate renal tubular epithelial cell ischemia-reperfusion injury by activating the PI3K/AKT/FOXO1 pathway.

BACKGROUND: Renal ischemia-reperfusion injury (RIRI) has become a great concern in clinical practice with high morbidity and mortality rates. Sufentanil has protective effects on IRI-induced organ injury. Herein, the effects of sufentanil on RIRI were investigated. METHODS: RIRI cell model was established by hypoxia/reperfusion (H/R) stimulation. The mRNA and protein expressions were assessed using qRT-PCR and western blot. TMCK-1 cell viability and apoptosis were assessed using MTT assay and flow cytometry, respectively. The mitochondrial membrane potential and ROS level were detected by JC-1 mitochondrial membrane potential fluorescent probe and DCFH-DA fluorescent probe, respectively. LDH, SOD, CAT, GSH and MDA levels were determined by the kits. The interaction between FOXO1 and Pin1 promoter was analyzed using dual luciferase reporter gene and ChIP assays. RESULTS: Our results revealed that sufentanil treatment attenuated H/R-induced cell apoptosis, mitochondrial membrane potential (MMP) dysfunction, oxidative stress, inflammation and activated PI3K/AKT/FOXO1 associated proteins, while these effects were reversed by PI3K inhibitor, suggesting that sufentanil attenuated RIRI via activating the PI3K/AKT/FOXO1 signaling pathway. We subsequently found that FOXO1 transcriptionally activated Pin1 in TCMK-1 cells. Pin1 inhibition ameliorated H/R-induced TCMK-1 cell apoptosis, oxidative stress and inflammation. In addition, as expected, the biological effects of sufentanil on H/R-treated TMCK-1 cells were abrogated by Pin1 overexpression. CONCLUSION: Sufentanil reduced Pin1 expression through activation of the PI3K/AKT/FOXO1 signaling to suppress cell apoptosis, oxidative stress and inflammation in renal tubular epithelial cells during RIRI development.

The TOR complex controls ATP levels to regulate actin cytoskeleton dynamics in Arabidopsis.

Energy is essential for all cellular functions in a living organism. How cells coordinate their physiological processes with energy status and availability is thus an important question. The turnover of actin cytoskeleton between its monomeric and filamentous forms is a major energy drain in eukaryotic cells. However, how actin dynamics are regulated by ATP levels remain largely unknown in plant cells. Here, we observed that seedlings with impaired functions of target of rapamycin complex 1 (TORC1), either by mutation of the key component, RAPTOR1B, or inhibition of TOR activity by specific inhibitors, displayed reduced sensitivity to actin cytoskeleton disruptors compared to their controls. Consistently, actin filament dynamics, but not organization, were suppressed in TORC1-impaired cells. Subcellular localization analysis and quantification of ATP concentration demonstrated that RAPTOR1B localized at cytoplasm and mitochondria and that ATP levels were significantly reduced in TORC1-impaired plants. Further pharmacologic experiments showed that the inhibition of mitochondrial functions led to phenotypes mimicking those observed in raptor1b mutants at the level of both plant growth and actin dynamics. Exogenous feeding of adenine could partially restore ATP levels and actin dynamics in TORC1-deficient plants. Thus, these data support an important role for TORC1 in coordinating ATP homeostasis and actin dynamics in plant cells.

Qing Zao Fang (QZF) Alleviates the Inflammatory Microenvironment of the Submandibular Gland in Sjögren's Syndrome Based on the PI3K/Akt/HIF-1α/VEGF Signaling Pathway.

Sjögren's syndrome (SS) which could lead to a disorder of our immune system is a chronic autoimmune disease characterized by invading exocrine glands such as salivary glands and lacrimal glands and other exocrine glands. Its common symptom is dry mouth and dry eyes, often accompanied by a large number of lymphocyte infiltrations and can involve other organs to cause complex clinical manifestations. In this study, we aimed at investigating the effect of QZF in SS, identifying the molecular mechanism in modulating autoimmune response, and determining the important roles of these factors' function as a modulator in the pathogenesis of SS. The NOD mice were utilized to establish the rats' model of Sjögren's syndrome. After 10 weeks' hydroxychloroquine and QZF in different dose interference, submandibular gland tissue was collected. The therapeutic effect of QZF on SS rats was identified, and the results suggest the comparable potential to hydroxychloroquine. In submandibular gland tissue, interleukin- (IL-) 17 was significantly lower in high-dose QZF than that in SS rats and the focal lymphocytes were highly attenuated. Moreover, we found that PI3K/Akt signals were activated and the downstream HIF-1α/VEGF signals were enhanced in SS rats whose protein expression could be inhibited by QZF treatment. In addition, QZF could modulate autophagy in submandibular gland tissue and then inhibit the inflammation response and therefore facilitate the tissue repair.

MiR-493-5p inhibits the malignant development of gliomas via suppressing E2F3-mediated dysfunctions of P53 and PI3K/AKT pathways.

BACKGROUND: Gliomas is a major challenge of current medical system, and thousands of people are struggling in the pain of this disease worldwide. In the last decade, the functions of miRNAs have been revealed by many studies, and the intervention on miRNA dysfunctions has been thought as a promising way to counter cancer. MiR-493-5p has been identified as a tumor inhibitor to suppress the progressions of several tumors while its role in gliomas remains unknown. Hence, the study investigated the expression levels of miR-493-5p in glioma tissues and cell lines. METHODS: CCK-8 assay, transwell assay and flow cytometry assay were used to observe the effects of miR-493-5p on tumor cells. The downstream targets of miR-493-5p were also searched and verified with online databases and dual-luciferase reporter assay. Moreover, the activities of P53 and PI3K/AKT pathways were also explored by western blot to illustrate the regulation mechanism of miR-493-5p on glioma development. RESULTS: The results showed that miR-493-5p was significantly downregulated in pathological tissues and glioma cell lines, and the increased miR-493-5p effectively inhibited the malignant behavior and promoted the apoptosis of glioma cells. CONCLUSIONS: E2F3 was confirmed as a target of miR-493-5p, and the effects of miR-493-5p on the phenotype of glioma cells could be partly reversed by E2F3. Besides, it was also found that miR-493-5p could effectively suppress the expression of E2F3 and then improve the dysfunctions of the P53 and PI3K/AKT pathways.

MicroRNA-802 promotes the progression of osteosarcoma through targeting p27 and activating PI3K/AKT pathway.

PURPOSE: Increasing evidences suggest dysfunctions of microRNAs (miRNAs) are playing important part in tumors. Therefore, the role of miR-802 in osteosarcoma (OS) was exploited. The object was to evaluate the effect of miR-802 and verify its influence on p27 Kip1 (p27) in OS. METHODS: RT-qPCR experiment was used to detect miR-802 and p27 expression in OS tissues and cells. We explored the function of miR-802 through Transwell assays. The phosphoinositide 3-kinase (PI3K)/AKT serine/threonine kinase pathway and epithelial-mesenchymal transition (EMT) was detected by Western blot assays. Luciferase assay was used to testify the target of miR-802. RESULTS: MiR-802 expression was elevated in OS, which was related to poor clinical outcome in OS patients. MiR-802 overexpression promoted OS migration, invasion and EMT. Further, p27 is a direct target of miR-802. P27 elevation counteracted the promotion effect of OS on EMT, migration and invasion induced by miR-802. In addition, miR-802 overexpression inactivated PI3K/AKT pathway via targeting p27 in OS. CONCLUSION: MiR-802 promoted the progress of EMT, migration and invasion in OS via targeting p27. This newly identified miR-802/p27/PI3K/AKT axis may represent potential targets for OS.

Screening for Active Compounds Targeting Human Natural Killer Cell Activation Identifying Daphnetin as an Enhancer for IFN-γ Production and Direct Cytotoxicity.

Natural killer (NK) cells are a potent weapon against tumor and viral infection. Finding active compounds with the capacity of enhancing NK cell effector functions will be effective to develop new anti-cancer drugs. In this study, we initially screened 287 commercially available active compounds by co-culturing with peripheral blood mononuclear cells (PBMCs). We found that five compounds, namely, Daphnetin, MK-8617, LW6, JIB-04, and IOX1, increased the IFN-γ+ NK cell ratio in the presence of IL-12. Further studies using purified human primary NK cells revealed that Daphnetin directly promoted NK cell IFN-γ production in the presence of IL-12 but not IL-15, while the other four compounds acted on NK cells indirectly. Daphnetin also improved the direct cytotoxicity of NK cells against tumor cells in the presence of IL-12. Through RNA-sequencing, we found that PI3K-Akt-mTOR signaling acted as a central pathway in Daphnetin-mediated NK cell activation in the presence of IL-12. This was further confirmed by the finding that both inhibitors of PI3K-Akt and its main downstream signaling mTOR, LY294002, and rapamycin, respectively, can reverse the increase of IFN-γ production and cytotoxicity in NK cells promoted by Daphnetin. Collectively, we identify a natural product, Daphnetin, with the capacity of promoting human NK cell activation via PI3K-Akt-mTOR signaling in the presence of IL-12. Our current study opens up a new potential application for Daphnetin as a complementary immunomodulator for cancer treatments.

The Key Ingredient Acacetin in Weishu Decoction Alleviates Gastrointestinal Motility Disorder Based on Network Pharmacology Analysis.

BACKGROUND: Gastrointestinal motility disorder is a common gastrointestinal disease, which seriously affects life quality. Traditional Chinese medicine (TCM) has been widely used as an alternative therapy for gastrointestinal motility disorder. Acacetin is a natural flavonoid compound that has antioxidant and anti-inflammatory, antidepressant, and anticancer properties. However, the efficacy of Acacetin in the treatment of gastrointestinal motility disorders has not been studied. Our aim was to investigate the mechanism of Acacetin-alleviated gastrointestinal motility disorder and its efficacy based on network pharmacology. METHODS: We performed network pharmacology to predict the active components, match Weishu decoction (WSD) targets in gastrointestinal motility disorders, and investigate its potential pharmacological mechanisms. We performed the GO and KEGG enrichment analysis. In vivo, we investigated the effects of Acacetin in the gastrointestinal motility disorder model. RESULTS: Based on network pharmacological method, the key active ingredient of WSD was identified as Acacetin, and the enrichment signaling pathway was the PI3K-AKT signaling pathway. Acacetin and Mosapride accelerated gastric emptying time, reduced gastric remnant rate, and increased small intestinal propulsion rate. The levels of GAS and MTL were increased after using Acacetin. These results indicated that Acacetin could improve gastrointestinal motility disorders. Among them, high-dose Acacetin showed a better effect. Acacetin could regulate protein and lipid metabolism in mice with gastrointestinal motility disorder. Furthermore, Acacetin could modulate gastrointestinal inflammation and apoptosis. The detection of the PI3K-AKT signaling pathway-related proteins showed that Acacetin improved gastrointestinal motility disorder by inhibiting the activation of the PI3K-AKT signaling pathway. CONCLUSION: The key ingredient Acacetin in WSD could alleviate gastrointestinal motility disorder by inhibiting the activation of the PI3K-AKT signaling pathway based on network pharmacology analysis. The efficacy and safety of Acacetin treatment provide strong experimental support for the clinical treatment of gastrointestinal motility disorder.

MiR-let-7d-3p inhibits granulosa cell proliferation by targeting TLR4 in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a typical reproductive and endocrinological disorder of women at child-bearing age. In this study, we used miRNA sequencing technology and verified miR-let-7d-3p as a vital miRNA in PCOS. RT-qPCR confirmed miR-let-7d-3p was significantly increased in granulosa cells (GCs) of PCOS. Cell counting kit-8 (CCK-8) identified the suppression of miR-let-7d-3p mimic in KGN cell proliferation and PI3K/Akt signaling pathway. Dual luciferase reporter assay proved that Toll-like receptor 4 (TLR4) was a target of miR-let-7d-3p, and TLR4 was significantly down-regulated by miR-let-7d-3p. Furthermore, over-expression of TLR4 promoted KGN cell proliferation and rescued the inhibition of miR-let-7d-3p on KGN cells. In conclusion, miR-let-7d-3p was a crucial miRNA up-regulated in GCs of PCOS, and inhibited cell proliferation by targeting TLR4 gene.

Modeling PTEN overexpression-induced microcephaly in human brain organoids.

The phosphatase and tensin homolog (PTEN) protein, encoded by the PTEN gene on chromosome 10, is a negative regulator of the phosphoinositide 3-kinase (PI3K) signaling pathway. Loss of PTEN has been linked to an array of human diseases, including neurodevelopmental disorders such as macrocephaly and autism. However, it remains unknown whether increased dosage of PTEN can lead to human disease. A recent human genetics study identifies chromosome 10 microduplication encompassing PTEN in patients with microcephaly. Here we generated a human brain organoid model of increased PTEN dosage. We showed that mild PTEN overexpression led to reduced neural precursor proliferation, premature neuronal differentiation, and the formation of significantly smaller brain organoids. PTEN overexpression resulted in decreased AKT activation, and treatment of wild-type organoids with an AKT inhibitor recapitulated the reduced brain organoid growth phenotypes. Together, our findings provide functional evidence that PTEN is a dosage-sensitive gene that regulates human neurodevelopment, and that increased PTEN dosage in brain organoids results in microcephaly-like phenotypes.

Tacrolimus Decreases Cognitive Function by Impairing Hippocampal Synaptic Balance: a Possible Role of Klotho.

The influence of long-term tacrolimus treatment on cognitive function remains to be elucidated. Using a murine model of chronic tacrolimus neurotoxicity, we evaluated the effects of tacrolimus on cognitive function, synaptic balance, its regulating protein (Klotho), and oxidative stress in the hippocampus. Compared to vehicle-treated mice, tacrolimus-treated mice showed significantly decreased hippocampal-dependent spatial learning and memory function. Furthermore, tacrolimus caused synaptic imbalance, as demonstrated by decreased excitatory synapses and increased inhibitory synapses, and downregulated Klotho in a dose-dependent manner; the downregulation of Klotho was localized to excitatory hippocampal synapses. Moreover, tacrolimus increased oxidative stress and was associated with activation of the PI3K/AKT pathway in the hippocampus. These results indicate that tacrolimus impairs cognitive function via synaptic imbalance, and that these processes are associated with Klotho downregulation at synapses through tacrolimus-induced oxidative stress in the hippocampus.

Diagnosis and management of an endometrial cancer patient with Cowden syndrome.

Somatic PTEN alterations are common in endometrial carcinoma (EC), but in rare cases PTEN mutations are associated with inherited syndromes. Here, we present a case of Cowden syndrome-associated EC. We discuss clinical, pathologic and molecular features of her tumor and PTEN-mutated EC, inherited syndromes predisposing to EC and PTEN-targeted therapies.

Network Pharmacology Prediction and Molecular Docking-Based Strategy to Discover the Potential Pharmacological Mechanism of Huai Hua San Against Ulcerative Colitis.

BACKGROUND: Huai Hua San (HHS), a famous Traditional Chinese Medicine (TCM) formula, has been widely applied in treating ulcerative colitis (UC). However, the interaction of bioactives from HHS with the targets involved in UC has not been elucidated yet. AIM: A network pharmacology-based approach combined with molecular docking and in vitro validation was performed to determine the bioactives, key targets, and potential pharmacological mechanism of HHS against UC. MATERIALS AND METHODS: Bioactives and potential targets of HHS, as well as UC-related targets, were retrieved from public databases. Crucial bioactive ingredients, potential targets, and signaling pathways were acquired through bioinformatics analysis, including protein-protein interaction (PPI), as well as the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Subsequently, molecular docking was carried out to predict the combination of active compounds with core targets. Lastly, in vitro experiments were conducted to further verify the findings. RESULTS: A total of 28 bioactive ingredients of HHS and 421 HHS-UC-related targets were screened. Bioinformatics analysis revealed that quercetin, luteolin, and nobiletin may be potential candidate agents. JUN, TP53, and ESR1 could become potential therapeutic targets. PI3K-AKT signaling pathway might play an important role in HHS against UC. Moreover, molecular docking suggested that quercetin, luteolin, and nobiletin combined well with JUN, TP53, and ESR1, respectively. Cell experiments showed that the most important ingredient of HHS, quercetin, could inhibit the levels of inflammatory factors and phosphorylated c-Jun, as well as PI3K-Akt signaling pathway in LPS-induced RAW264.7 cells, which further confirmed the prediction by network pharmacology strategy and molecular docking. CONCLUSION: Our results comprehensively illustrated the bioactives, potential targets, and molecular mechanism of HHS against UC. It also provided a promising strategy to uncover the scientific basis and therapeutic mechanism of TCM formulae in treating diseases.

Key Signaling Pathways Regulate the Development and Survival of Auditory Hair Cells.

The loss of auditory sensory hair cells (HCs) is the most common cause of sensorineural hearing loss (SNHL). As the main sound transmission structure in the cochlea, it is necessary to maintain the normal shape and survival of HCs. In this review, we described and summarized the signaling pathways that regulate the development and survival of auditory HCs in SNHL. The role of the mitogen-activated protein kinase (MAPK), phosphoinositide-3 kinase/protein kinase B (PI3K/Akt), Notch/Wnt/Atoh1, calcium channels, and oxidative stress/reactive oxygen species (ROS) signaling pathways are the most relevant. The molecular interactions of these signaling pathways play an important role in the survival of HCs, which may provide a theoretical basis and possible therapeutic interventions for the treatment of hearing loss.

Oncogenic Mutations in PI3K/AKT/mTOR Pathway Effectors Associate with Worse Prognosis in BRAFV600E -Driven Papillary Thyroid Cancer Patients.

PURPOSE: The extent to which routine genomic sequencing can identify relevant secondary genomic alterations among BRAFV600E -mutant papillary thyroid carcinoma (PTC) is unknown. Such markers would prove highly valuable for prognostic purposes. EXPERIMENTAL DESIGN: We reviewed clinicopathologic data of 225 patients with BRAFV600E -mutant PTC and integrated them with genomic data derived from targeted next-generation sequencing (NGS) on tumor specimens. We defined patient subgroups based on bona fide secondary oncogenic events (separate from BRAFV600E ) and compared their clinical features and outcomes with those without additional oncogenic alterations. RESULTS: Additional oncogenic alterations were identified in 16% of tumors. Patients in the "BRAF+additional mutations" group were more likely to be at high American Thyroid Association (ATA) risk of recurrence (48.6% vs. 17.6%; P = 0.0009), had larger baseline tumor (2.7 vs. 1.9 cm; P = 0.0005) and more advanced stage at presentation (14.3% vs. 1.1% stage 4; P < 0.0001). Importantly, over a 65-month follow-up, disease-specific mortality (DSM) was increased when additional mutations were identified (13.8% vs. 1.4% in the BRAF-only group; P = 0.005). Separately, we identified a subcluster of patients harboring oncogenic mutations in key effectors of the PI3K/AKT/mTOR pathway, which were independently associated with DSM (OR = 47.9; 95% confidence interval, 3.5-1,246.5; P = 0.0043). CONCLUSIONS: Identification of additional PIK3/AKT/mTOR alterations in patients with BRAFV600E -mutant PTC provides important and actionable prognostic risk stratification. These data support genomic profiling of PTC tumors to inform prognosis and clinical strategy.

Apilimod alters TGFß signaling pathway and prevents cardiac fibrotic remodeling.

Rationale: TGFß signaling pathway controls tissue fibrotic remodeling, a hallmark in many diseases leading to organ injury and failure. In this study, we address the role of Apilimod, a pharmacological inhibitor of the lipid kinase PIKfyve, in the regulation of cardiac pathological fibrotic remodeling and TGFß signaling pathway. Methods: The effects of Apilimod treatment on myocardial fibrosis, hypertrophy and cardiac function were assessed in vivo in a mouse model of pressure overload-induced heart failure. Primary cardiac fibroblasts and HeLa cells treated with Apilimod as well as genetic mutation of PIKfyve in mouse embryonic fibroblasts were used as cell models. Results: When administered in vivo, Apilimod reduced myocardial interstitial fibrosis development and prevented left ventricular dysfunction. In vitro, Apilimod controlled TGFß-dependent activation of primary murine cardiac fibroblasts. Mechanistically, both Apilimod and genetic mutation of PIKfyve induced TGFß receptor blockade in intracellular vesicles, negatively modulating its downstream signaling pathway and ultimately dampening TGFß response. Conclusions: Altogether, our findings propose a novel function for PIKfyve in the control of myocardial fibrotic remodeling and the TGFß signaling pathway, therefore opening the way to new therapeutic perspectives to prevent adverse fibrotic remodeling using Apilimod treatment.

Noncanonical Thyroid Hormone Receptor α Action Mediates Arterial Vasodilation.

CONTEXT: Hypothyroidism impairs cardiovascular health and contributes to endothelial dysfunction with reduced vasodilation. How 3,5,3'-triiodothyronine (T3) and its receptors are involved in the regulation of vasomotion is not yet fully understood. In general, thyroid hormone receptors (TRs) either influence gene expression (canonical action) or rapidly activate intracellular signaling pathways (noncanonical action). OBJECTIVE: Here we aimed to characterize the T3 action underlying the mechanism of arterial vasodilation and blood pressure (BP) regulation. METHODS: Mesenteric arteries were isolated from male rats, wild-type (WT) mice, TRα knockout (TRα 0) mice, and from knockin mice with a mutation in the DNA-binding domain (TRα GS). In this mutant, DNA binding and thus canonical action is abrogated while noncanonical signaling is preserved. In a wire myograph system, the isolated vessels were preconstricted with norepinephrine. The response to T3 was measured, and the resulting vasodilation (Δ force [mN]) was normalized to maximum contraction with norepinephrine and expressed as percentage vasodilation after maximal preconstriction with norepinephrine (%NE). Isolated vessels were treated with T3 (1 × 10-15 to 1 × 10-5 mol/L) alone and in combination with the endothelial nitric oxide-synthase (eNOS) inhibitor L-NG-nitroarginine methyl ester (L-NAME) or the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin. The endothelium was removed to determine the contribution of T3 to endothelium-dependent vasodilation. The physiological relevance of T3-induced vasodilation was determined by in vivo arterial BP measurements in male and female mice. RESULTS: T3 treatment induced vasodilation of mesenteric arteries from WT mice within 2 minutes (by 21.5 ±â€…1.7%NE). This effect was absent in arteries from TRα 0 mice (by 5.3 ±â€…0.6%NE, P < .001 vs WT) but preserved in TRα GS arteries (by 17.2 ±â€…1.1%NE, not significant vs WT). Inhibition of either eNOS or PI3K reduced T3-mediated vasodilation from 52.7 ±â€…4.5%NE to 28.5 ±â€…4.1%NE and 22.7 ±â€…2.9%NE, respectively. Removal of the endothelium abolished the T3-mediated vasodilation in rat mesenteric arteries (by 36.7 ±â€…5.4%NE vs 3.5 ±â€…6.2%NE). In vivo, T3 injection led to a rapid decrease of arterial BP in WT (by 13.9 ±â€…1.9 mm Hg) and TRα GS mice (by 12.4 ±â€…1.9 mm Hg), but not in TRα 0 mice (by 4.1 ±â€…1.9 mm Hg). CONCLUSION: These results demonstrate that T3 acting through noncanonical TRα action affects cardiovascular physiology by inducing endothelium-dependent vasodilation within minutes via PI3K and eNOS activation.

A key requirement for synaptic Reelin signaling in ketamine-mediated behavioral and synaptic action.

Ketamine is a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist that produces rapid antidepressant action in some patients with treatment-resistant depression. However, recent data suggest that ∼50% of patients with treatment-resistant depression do not respond to ketamine. The factors that contribute to the nonresponsiveness to ketamine's antidepressant action remain unclear. Recent studies have reported a role for secreted glycoprotein Reelin in regulating pre- and postsynaptic function, which suggests that Reelin may be involved in ketamine's antidepressant action, although the premise has not been tested. Here, we investigated whether the disruption of Reelin-mediated synaptic signaling alters ketamine-triggered synaptic plasticity and behavioral effects. To this end, we used mouse models with genetic deletion of Reelin or apolipoprotein E receptor 2 (Apoer2), as well as pharmacological inhibition of their downstream effectors, Src family kinases (SFKs) or phosphoinositide 3-kinase. We found that disruption of Reelin, Apoer2, or SFKs blocks ketamine-driven behavioral changes and synaptic plasticity in the hippocampal CA1 region. Although ketamine administration did not affect tyrosine phosphorylation of DAB1, an adaptor protein linked to downstream signaling of Reelin, disruption of Apoer2 or SFKs impaired baseline NMDA receptor-mediated neurotransmission. These results suggest that maintenance of baseline NMDA receptor function by Reelin signaling may be a key permissive factor required for ketamine's antidepressant effects. Taken together, our results suggest that impairments in Reelin-Apoer2-SFK pathway components may in part underlie nonresponsiveness to ketamine's antidepressant action.

Effect of Whole Tissue Culture and Basic Fibroblast Growth Factor on Maintenance of Tie2 Molecule Expression in Human Nucleus Pulposus Cells.

Previous work showed a link between Tie2+ nucleus pulposus progenitor cells (NPPC) and disc degeneration. However, NPPC remain difficult to maintain in culture. Here, we report whole tissue culture (WTC) combined with fibroblast growth factor 2 (FGF2) and chimeric FGF (cFGF) supplementation to support and enhance NPPC and Tie2 expression. We also examined the role of PI3K/Akt and MEK/ERK pathways in FGF2 and cFGF-induced Tie2 expression. Young herniating nucleus pulposus tissue was used. We compared WTC and standard primary cell culture, with or without 10 ng/mL FGF2. PI3K/Akt and MEK/ERK signaling pathways were examined through western blotting. Using WTC and primary cell culture, Tie2 positivity rates were 7.0 ± 2.6% and 1.9 ± 0.3% (p = 0.004), respectively. Addition of FGF2 in WTC increased Tie2 positivity rates to 14.2 ± 5.4% (p = 0.01). FGF2-stimulated expression of Tie2 was reduced 3-fold with the addition of the MEK inhibitor PD98059 (p = 0.01). However, the addition of 1 µM Akt inhibitor, 124015-1MGCN, only reduced small Tie2 expression (p = 0.42). cFGF similarly increased the Tie2 expression, but did not result in significant phosphorylation in both the MEK/ERK and PI3K/Akt pathways. WTC with FGF2 addition significantly increased Tie2 maintenance of human NPPC. Moreover, FGF2 supports Tie2 expression via MEK/ERK and PI3K/Akt signals. These findings offer promising tools and insights for the development of NPPC-based therapeutics.

Contribution of Thrombospondin-1 and -2 to Lipopolysaccharide-Induced Acute Respiratory Distress Syndrome.

Thrombospondin (TSP) proteins have been shown to impact T-cell adhesion, migration, differentiation, and apoptosis. Thrombospondin-1 (TSP-1) is specifically upregulated in several inflammatory diseases and can effectively promote lipopolysaccharide- (LPS-) induced inflammation. In contrast, thrombospondin-2 (TSP-2) has been associated with activation of "anti-inflammatory" T-regulatory cells (Tregs). In this study, we investigated the effects of both TSP-1 and TSP-2 overexpression on macrophage polarization and activation in vitro and in vivo. We analyzed the effects of TSP-1 and TSP-2 on inflammation, vascular endothelial permeability, edema, ultrastructural morphology, and apoptosis in lung tissues of an ARDS mouse model and cultured macrophages. Our results demonstrated that TSP-2 overexpression effectively attenuated LPS-induced ARDS in vivo and promoted M2 macrophage phenotype polarization in vitro. Furthermore, TSP-2 played a role in regulating pulmonary vascular barrier leakage by activating the PI3K/Akt pathway. Overall, our findings indicate that TSP-2 can modulate inflammation and could therefore be a potential therapeutic target against LPS-induced ARDS.

Properties of FDA-approved small molecule phosphatidylinositol 3-kinase inhibitors prescribed for the treatment of malignancies.

The discovery of the phosphatidylinositol 3-kinase (PI 3-kinase) pathway was a major advance in understanding eukaryotic signal transduction. The high frequency of PI 3-kinase pathway mutations in many cancers stimulated the development of drugs targeting these oncogenic mutants. The PI 3-kinases are divided into three classes and Class I PI 3-kinases, which catalyze the phosphorylation of phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2) to generate phosphatidylinositol-3,4,5-trisphosphate (PIP3), are the main subject of this review. The class I PI 3-kinases are made up of p110α, p110ß, p110δ, and p110γ catalytic subunits. These catalytic subunits are constitutively bound to regulatory subunits (p85α, p85ß, p55γ, p101, and p87 proteins). The p85/p55 regulatory subunits heterodimerize with p110α or p110δ thereby forming complexes that are regulated chiefly by receptor protein-tyrosine kinases. The p101 and p87 subunits heterodimerize with p110γ to form complexes that are regulated mainly by G protein-coupled receptors (GPCRs). Complexes containing the p110ß subunit are activated by receptor protein-tyrosine kinases as well as GPCRs. Following the generation of PIP3, the AKT and mTOR protein-serine/threonine kinases are activated leading to cell growth, proliferation, and survival. Like protein kinases, the PI 3-kinase domains consist of a bilobed structure connected by a hinge-linker segment. ATP and most PI 3-kinase and protein kinase inhibitors form hydrogen bonds with hinge residues. The small and large lobes of PI 3-kinases and protein kinases have a very similar three-dimensional structure called the protein kinase fold. Both PI 3-kinases and eukaryotic protein kinases possess an activation segment that begins with a DFG triad (Asp-Phe-Gly); the activation segment of protein kinases usually ends with an APE (Ala-Pro-Glu) signature while that of PI 3-kinases ends with a PFxLT (Pro-Phe-Xxx-Leu-Thr) signature. Dormant PI 3-kinases have a collapsed activation loop and active PI 3-kinases have an extended activation loop. The distance between the α-carbon atom of the DFG-D residue at the beginning of the activation loop and that of the PFxLT-F residue at the end of the activation loop in dormant PI 3-kinases is about 13 Å; this distance in active PI 3-kinases is about 18 Å. The protein kinase catalytic loop has an HRD (His-Arg-Asp) signature while that of the PI 3-kinases reverses the order with a DRH triad. Alpelisib is an orally effective FDA-approved PI 3-kinase-α inhibitor used for the treatment of breast cancer. Copanlisib, duvelisib, idelalisib, and umbralisib are PI 3-kinase-δ inhibitors that are approved for the third-line treatment of follicular lymphomas and other hematological disorders. Copanlisib is also a potent inhibitor of PI 3-kinase-α. Of the five approved drugs, all are orally bioavailable except copanlisib. Idelalisib interacts with the active conformation of PI 3-kinase-δ and is classified as a type I inhibitor. Alpelisib and copanlisib interact with inactive PI 3-kinase-α and PI 3-kinase-γ, respectively, and are classified as a type I½ antagonists. Except for umbralisib with a molecular weight of 571.5, all five drugs conform to the Lipinski rule of five for oral effectiveness. Copanlisib, however, must be given intravenously. Alpelisib and copanlisib inhibit PI 3-kinase-α, which is involved in insulin signaling, and both drugs promote insulin-resistance and produce hyperglycemia. The five FDA-approved PI 3-kinase inhibitors produce significant on-target toxicities, more so than many approved protein kinase antagonists. The development of PI 3-kinase inhibitors with fewer toxicities is an important long-term therapeutic goal.