Target Of Rapamycin Signaling Pathway Research Articles

Effects of dietary tryptophan levels on antioxidant status and immunity for juvenile blunt snout bream (Megalobrama amblycephala) involved in Nrf2 and TOR signaling pathway

Dietary administration of tryptophan has been proved improving growth performance of fish. An 8-week feeding trial was conducted to investigate the effects of dietary tryptophan level on antioxidant capacity and immune response through Nrf2 and TOR signaling pathway. The results showed that, 0.08% tryptophan level significantly increased plasma aspartate aminotransferase (AST), while immunoglobulin M (IgM) and alkaline phosphatase (ALP) were strikingly increased by 0.40% level. The level of plasma complement component 3 (C3), alanine aminotransferase (ALT) and albumin (ALB) were independent of tryptophan supplementation. Total superoxide dismutase (T-SOD), catalase (CAT), total antioxidant capacity (T-AOC) and glutathione (GSH) activity were increased with increasing dietary tryptophan level until 0.40% and then decreased, while the level of malondialdehyde (MDA) showed a reverse trend. 0.19% and 0.28% tryptophan level significantly improved the glutathione peroxidase 1 (GPx-1) activity. Compared with 0.08% dietary tryptophan level, 0.40% level significantly improved nuclear factor erythroid 2-related factor 2 (Nrf2), GPx, manganese superoxide dismutase (Mn-SOD), CAT and transforming growth factor-β (TGF-β) mRNA level, while Kelch-like ECH-associated protein 1 (Keap1) and interleukin 1β (IL-1β) mRNA level were significantly decreased. The relative expression of copper zinc superoxide dismutase (Cu/Zn-SOD), heme oxygenase-1 (HO-1), target of rapamycin (TOR), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), protein kinase B (Akt) and interleukin 10 (IL-10) were significantly improved by 0.28% diet, while the mRNA level of tumor necrosis factor-α (TNF-α) and nuclear factor-kappa B (NF-κB) were increased by 0.08% diet. Interleukin 8 (IL-8) mRNA level was not significantly affected by dietary tryptophan. Based on MDA and T-SOD value, the optimal dietary tryptophan level of juvenile blunt snout bream was determined to be 0.33% (1.03% of dietary protein) and 0.36% (1.13% of dietary protein), respectively, using quadratic regression analysis.

TOR as a Regulatory Target in Rhipicephalus microplus Embryogenesis.

Embryogenesis is a metabolically intensive process carried out under tightly controlled conditions. The insulin signaling pathway regulates glucose homeostasis and is essential for reproduction in metazoan model species. Three key targets are part of this signaling pathway: protein kinase B (PKB, or AKT), glycogen synthase kinase 3 (GSK-3), and target of rapamycin (TOR). While the role of AKT and GSK-3 has been investigated during tick embryonic development, the role of TOR remains unknown. In this study, TOR and two other downstream effectors, namely S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), were investigated in in vitro studies using the tick embryonic cell line BME26. First, we show that exogenous insulin can stimulate TOR transcription. Second, TOR chemical inhibition led to a decrease in BME26 cell viability, loss of membrane integrity, and downregulation of S6K and 4E-BP1 transcription. Conversely, treating BME26 cells with chemical inhibitors of AKT or GSK-3 did not affect S6K and 4E-BP1 transcription, showing that TOR is specifically required to activate its downstream targets. To address the role of TOR in tick reproduction, in vivo studies were performed. Analysis of relative transcription during different stages of tick embryonic development showed different levels of transcription for TOR, and a maternal deposition of S6K and 4E-BP1 transcripts. Injection of TOR double-stranded RNA (dsRNA) into partially fed females led to a slight delay in oviposition, an atypical egg external morphology, decreased vitellin content in eggs, and decreased larval hatching. Taken together, our data show that the TOR signaling pathway is important for tick reproduction, that TOR acts as a regulatory target in Rhipicephalus microplus embryogenesis and represents a promising target for the development of compounds for tick control.

Long non-coding RNA OECC promotes cell proliferation and metastasis through the PI3K/Akt/mTOR signaling pathway in human lung cancer.

Lung cancer is one of the most common malignancies worldwide; however, its detailed molecular mechanism remains largely unknown. Long non-coding RNAs (lncRNAs) have been identified to serve critical roles in tumorigenesis. The aim of the present study was to investigate the role of a newly identified lncRNA, overexpressed in colorectal cancer (OECC), in human lung cancer. It was initially revealed that the relative transcript level of OECC was highly upregulated in clinical human lung cancer tissues as well as in cultured lung cancer cells. Knockdown of OECC with specific short hairpin RNAs in lung cancer cell lines A549 and 95D inhibited colony formation and cell viability, as evidenced using colony formation assays and cell proliferation assays. Furthermore, depletion of OECC in A549 and 95D cells suppressed migration and invasion, which was verified using Transwell assays. RNA-sequence analysis suggested that the phosphoinositide 3-kinase/protein kinase B (Akt)/mammalian target of rapamycin signaling pathway was positively regulated by OECC in lung cancer cells A549. In addition, overexpression of Akt in OECC-depleted A549 and 95D cells reversed the suppression of proliferation and migration caused by OECC depletion. The results of the present study identified lncRNA OECC as a novel regulator of lung cancer progression and provided new clues for the clinical treatment of lung cancer.

LncRNA SNHG6 inhibits cell proliferation and metastasis by targeting ETS1 via the PI3K/AKT/mTOR pathway in colorectal cancer.

The term ‘long non-coding RNAs’ (lncRNAs) refers to all non-protein coding transcripts >200 nucleotides in length. Dysregulation of lncRNAs has been identified in colorectal cancer, which is one of the more serious types of cancer worldwide, third place in the mortality rates, and is associated with poor prognoses. Novel evidence suggests that lncRNAs serve an important role in regulating the development and progression of colorectal cancer. In the present study, it was demonstrated that SNHG6 expression was downregulated in colorectal cancer tissues by reverse transcription quantitative polymerase chain reaction assays; however, ETS1 expression levels were upregulated. Overexpression of SNHG6 not only inhibited the proliferation of colon cancer cells in vitro by inducing apoptosis, but also inhibited cell proliferation, invasion and migration. The overexpression of SNHG6 inhibited colon cell viability and proliferation by targeting ETS1 through the phosphoinositide 3-kinase/protein kinase B/mechanistic target of rapamycin signaling pathway. These results suggested that SNHG6 may directly suppress ETS1, which may be one of potential mechanisms through which it inhibits the viability and proliferation of colorectal cancer cells, and it provides novel insight into the carcinogenesis of colorectal cancer. In addition, it may assist in the development of a treatment approach for ETS1-activated colorectal cancer.

Short communication: Relationship between lysine/methionine ratios and glucose levels and their effects on casein synthesis via activation of the mechanistic target of rapamycin signaling pathway in bovine mammary epithelial cells

The synthesis of protein requires the availability of specific AA and a large supply of energy in bovine mammary epithelial cells (BMEC). Whether an interaction exists between Lys/Met ratio and glucose level on milk protein synthesis and its potential regulatory mechanism is unclear. We investigated the effects of different Lys/Met ratios and glucose levels on casein synthesis-related gene expression in BMEC to elucidate the underlying molecular mechanisms. Primary BMEC were subjected to 4 treatments for 36 h, arranged in a 2 × 2 factorial design with Lys/Met ratios of 3:1 (1.2:0.4 mM, LM3.0; total AA = 8.24 mM) and 2.3:1 (1.4:0.6 mM, LM2.3; total AA = 8.64 mM) and glucose levels of 17.5 mM (high glucose level) and 2.5 mM (low glucose level). No interactions between Lys/Met ratio and glucose level on cell viability, cell cycle progression, mRNA, or protein expression levels were found. High glucose level increased cell proliferation and promoted cell cycle transition from intermediate phase (G1 phase) to synthesis (S phase) by approximately 50%, whereas Lys/Met ratio had no effect. Both mRNA and protein abundance of αS1-casein and β-casein were positively affected by LM3.0, whereas a high glucose level increased protein abundance of αS1-casein and β-casein and increased gene expression of CSN1S1 but not of CSN2. Furthermore, high glucose increased the mRNA abundance of ELF5 and decreased that of GLUT8, enhanced protein expression of total and phosphorylated mechanistic target of rapamycin (mTOR), and decreased phosphorylated AMP-activated protein kinase (AMPK) levels. Treatment LM3.0 had a stimulatory effect on total and phosphorylated mTOR but did not affect AMPK phosphorylation. The mRNA levels of JAK2, ELF5, and RPS6KB1 were upregulated and mRNA levels of EIF4EBP1 were downregulated with LM3.0 compared with LM2.3. Our results indicate that casein synthesis was regulated by Lys/Met ratio via JAK2/ELF5, mTOR, and its downstream RPS6KB1 and EIF4EBP1 signaling. In contrast, glucose regulated casein synthesis through promoting cell proliferation, accelerating cell cycle progression, and activating the ELF5 and AMPK/mTOR signaling pathways. Within the range of substrate levels in the present study, a change in Lys/Met ratio had a stronger effect on abundance of αS1-casein and β-casein than a change in glucose level.

Branched-Chain Amino Acids and Seizures: A Systematic Review of the Literature.

Up to 40% of patients with epilepsy experience seizures despite treatment with antiepileptic drugs; however, branched-chain amino acid (BCAA) supplementation has shown promise in treating refractory epilepsy. The purpose of this systematic review was to evaluate all published studies that investigated the effects of BCAAs on seizures, emphasizing therapeutic efficacy and possible underlying mechanisms. On 31 January, 2017, the following databases were searched for relevant studies: MEDLINE (OvidSP), EMBASE (OvidSP), Scopus (Elsevier), the Cochrane Library, and the unindexed material in PubMed (National Library of Medicine/National Institutes of Health). The searches were repeated in all databases on 18 February, 2019. We only included full-length preclinical and clinical studies that were published in the English language that examined the effects of BCAA administration on seizures. Eleven of 2045 studies met our inclusion criteria: ten studies were conducted in animal models and one study in human subjects. Seven seizure models were investigated: the strychnine (one study), pentylenetetrazole (two studies), flurothyl (one study), picrotoxin (two studies), genetic absence epilepsy in rats (one study), kainic acid (two studies), and methionine sulfoximine (one study) paradigms. Three studies investigated the effect of a BCAA mixture whereas the other studies explored the effects of individual BCAAs on seizures. In most animal models and in humans, BCAAs had potent anti-seizure effects. However, in the methionine sulfoximine model, long-term BCAA supplementation worsened seizure propagation and caused neuron loss, and in the genetic absence epilepsy in rats model, BCAAs exhibited pro-seizure effects. The contradictory effects of BCAAs on seizure activity likely reflect differences in the complex mechanisms that underlie seizure disorders. Some of these mechanisms are likely mediated by BCAA's effects on glucose, glutamate, glutamine, and ammonia metabolism, activation of the mechanistic target of rapamycin signaling pathway, and their effects on aromatic amino acid transport and neurotransmitter synthesis. We propose that a better understanding of mechanisms by which BCAAs affect seizures and neuronal viability is needed to advance the field of BCAA supplementation in epilepsy.

Dysregulated expression of the key effectors of the mammalian target of rapamycin signalling pathway in cutaneous papillomas of dogs.

Cutaneous papillomas (CP) are one of the most common skin neoplasms in dogs. Different murine models have shown that persistent activation of the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway has a central role in the development and progression of CP. The purpose of this study were to evaluate the immunohistochemical expression pattern of two key molecules involved in the PI3K/Akt/mTOR signalling pathway, pAktSer473 , and pS6Ser235/236 , on 36 canine specimens of CP using a tissue microarray. The results show that the PI3K/Akt/mTOR signalling pathway is persistently activated in CP of dogs, pointing to this pathway as a potential therapeutic target.

Effects of dietary protein level on growth performance, digestive enzyme activity, and gene expressions of the TOR signaling pathway in fingerling Pelteobagrus fulvidraco.

An 8-week feeding trial was conducted to investigate effects of dietary protein levels (37, 40, and 43%) on the growth performance, feed utilization, digestive enzyme activity, and gene expressions of target of rapamycin (TOR) signaling pathway in fingerling yellow catfish. One hundred and eighty fingerlings (average weight 0.77 ± 0.03g) were equally distributed across four replicate tanks for each of the three treatments, with 15 fish per tank. No difference (P > 0.05) was observed in initial body weight, survival rate (SR), hepatosomatic index (HSI), viscera index (VSI), dressing percentage (DP), and condition factor (CF) among all the treatments. The diet containing 40% protein increased significantly (P < 0.05) final body weight, weight gain rate (WGR), specific growth rate (SGR), protein efficiency ratio (PER), nitrogen retention (NRE), and energy retention (ERE) in fish. The highest protease activity in the stomach and intestine was observed in the P40 group (P < 0.05), while amylase and lipase were not significantly different (P > 0.05). The transcriptional levels of IGF-1, IGF-1R, and Akt were significantly (P < 0.05) higher in fish fed P40 or P43 than those of fish fed P37. TOR and S6K1 mRNA expressions were significantly (P < 0.05) increased in the P40 groups. Hence, the diet containing 40% protein would be suitable for the optimum growth and effective protein utilization of fingerling Pelteobagrus fulvidraco. In vitro, the transcriptional levels of IGF-1, IGF-1R, Akt, TOR, and S6K1 in hepatocyte supplemented with a 40-μM mixed amino acids were significantly (P < 0.05) higher compared to other treatments. No difference (P > 0.05) was observed in eukaryotic translation initiation factor 4E-binding protein 1 in vivo and in vitro among all the treatments. Effects of dietary protein level on growth performance likely are involved in the activation of TOR signaling pathway in fingerling Pelteobagrus fulvidraco.

Hypoxia-induced long non-coding RNA DARS-AS1 regulates RBM39 stability to promote myeloma malignancy.

Multiple myeloma is a malignant plasma-cell disease, which is highly dependent on the hypoxic bone marrow microenvironment. However, the underlying mechanisms of hypoxia contributing to myeloma genesis are not fully understood. Here, we show that long non-coding RNA DARS-AS1 in myeloma is directly upregulated by hypoxia inducible factor (HIF)-1. Importantly, DARS-AS1 is required for the survival and tumorigenesis of myeloma cells both in vitro and in vivo. DARS-AS1 exerts its function by binding RNA-binding motif protein 39 (RBM39), which impedes the interaction between RBM39 and its E3 ubiquitin ligase RNF147, and prevents RBM39 from degradation. The overexpression of RBM39 observed in myeloma cells is associated with poor prognosis. Furthermore, knockdown of DARS-AS1 inhibits the mammalian target of rapamycin signaling pathway, an effect that is reversed by RBM39 overexpression. We reveal that a novel HIF-1/DARS-AS1/RBM39 pathway is implicated in the pathogenesis of myeloma. Targeting DARS-AS1/RBM39 may, therefore, represent a novel strategy to combat myeloma.

Isoproterenol induces an increase in muscle fiber size by the proliferation of Pax7-positive cells and in a mTOR-independent mechanism.

β‐Adrenergic signaling regulates many physiological processes in skeletal muscles. A wealth of evidence has shown that β‐agonists can increase skeletal muscle mass in vertebrates. Nevertheless, to date, the specific role of β‐adrenergic receptors in different cell phenotypes (myoblasts, fibroblasts, and myotubes) and during the different steps of embryonic skeletal muscle differentiation has not been studied. Therefore, here we address this question through the analysis of embryonic chick primary cultures of skeletal muscle cells during the formation of multinucleated myotubes. We used isoproterenol (ISO), a β‐adrenergic receptor agonist, to activate the β‐adrenergic signaling and quantified several aspects of muscle differentiation. ISO induced an increase in myoblast proliferation, in the percentage of Pax7‐positive myoblasts and in the size of skeletal muscle fibers, suggesting that ISO activates a hyperplasic and hypertrophic muscle response. Interestingly, treatment with ISO did not alter the number of fibroblast cells, suggesting that ISO effects are specific to muscle cells in the case of chick myogenic cell culture. We also show that rapamycin, an inhibitor of the mammalian target of rapamycin signaling pathway, did not prevent the effects of ISO on chick muscle fiber size. The collection of these results provides new insights into the role of β‐adrenergic signaling during skeletal muscle proliferation and differentiation and specifically in the regulation of skeletal muscle hyperplasia and hypertrophy.

Isoleucine Regulates the Synthesis of Pancreatic Enzymes via the Activation of mRNA Expression and Phosphorylation in the Mammalian Target of Rapamycin Signalling Pathways in Pancreatic Tissues.

This study aimed to investigate the effects of isoleucine (Ile) on the synthesis and secretion of digestive enzymes and cellular signalling in the pancreatic tissue of dairy goats. The pancreatic tissues were incubated in buffer containing 0, 0.40, 0.80, and 1.60 mM Ile. High levels of Ile significantly increased the buffer release and total concentration of ɑ-amylase in the tissues (P < 0.001). The total trypsin and chymotrypsin concentrations in each of the Ile groups were significantly higher than those in the control group (P < 0.05); however, lipase was not affected. High levels of Ile significantly increased ɑ-amylase mRNA expression (P < 0.001) but had no effect on the mRNA expression of trypsin, chymotrypsin, or lipase. Ile did not affect S6K1 phosphorylation levels. High levels of Ile significantly increased the expression of the γ isoform of 4EBP1 (P < 0.001), which indicated that the phosphorylation of 4EBP1 was significantly increased. The phosphorylation level of eEF2 gradually decreased with the addition of Ile (P < 0.001). These results suggested that high doses of Ile can regulate the excretion of enzymes, especially ɑ-amylase, in the pancreatic tissues of dairy goats by modulating mTOR signalling, and this regulation is independent of the mTOR-S6K1 pathway.

The Expression of Yes-Associated Protein (YAP) Maintains Putative Cancer Stemness and Is Associated with Poor Prognosis in Intrahepatic Cholangiocarcinoma

Intrahepatic cholangiocarcinoma (ICC) is resistant to most chemotherapeutic agents. Yes-associated protein (YAP) is related to tumor progression; however, its role in ICC remains unknown. We investigated the mechanism underlying YAP-mediated cancer progression by focusing on the property of cancer stem cells (CSCs) in ICC. Immunohistochemistry results revealed the positive YAP expression in 37 of 52 resected ICC cases. Those with positive YAP expression showed poor prognosis in Kaplan-Meier analysis (P=0.023). YAP expression was associated with vimentin and the putative CSC marker, hepatic oval cell marker 6 (OV-6). The knockdown of YAP expression using specific siRNAs in ICC cells decreased octamer-binding transcription factor 4 (OCT4) expression in Western blot analyses and OV-6 and CD133 expression in flow cytometry analysis. Verteporfin, a YAP inhibitor, decreased N-cadherin and OCT4 expression in Western blot analyses. Invitro sphere formation and anoikis resistance assays revealed the impairment in CSC property and anoikis resistance in response to the decrease in YAP expression. Verteporfin treatment activated the protein kinase B/mechanistic target of rapamycin signaling pathway and dramatically impaired IL-6-stimulated STAT3 phosphorylation in ICC cells. The combination of verteporfin and rapamycin, an inhibitor of mechanistic target of rapamycin phosphorylation, inhibited cell proliferation and tumor growth. In conclusion, verteporfin regulates multiple signaling pathways and, in combination with rapamycin, might be a promising therapeutic strategy for ICC treatment.

Inhibitory effect of irisin on vasular smooth muscle cell inflammation and proliferation by regulating autophagy through adenosine monophosphate-actived protein kinase/mammalian target of rapamycin pathway

Objective To explore the underlining mechanisms of irisin inhibiting inflammation and proliferation of vascular smooth muscle cells (VSMCs). Methods The cultured primary rat aortic VSMCs were pre-incubated with irisin and then treated with platelet derived growth factor-BB (PDGF-BB). Enzyme linked immunosorbent assay (ELISA) and methyl thiazol tetrazolium (MTT) assay were used to measure the VSMCs inflammation and proliferation, respectively. The protein expression and phosphorylation of adenosine monophosphate-actived protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway related molecule AMPK/p-AMPK (Thr172), acetyl-CoA carboxylase (ACC)/phosphorylated (p)-ACC (Thr308), mTOR/p-mTOR (Ser2448), ribosomal protein S6 kinase (p70SK6)/p-70SK6 (Thr389), and autophay related molecule microtubule-associated protein 1 light chain 3 (LC3) Ⅰ, LC3Ⅱ and nucleoporin p62 (P62) were detected by Western blotting. Results After treatment with PDGF-BB, VSMCs proliferation dramatically increased, and the inflammatory cytokines IL-1β (37.9±8.5 vs. 10.8±2.9, t=18.620, P<0.01), IL-6 (57.4±14.1 vs. 32.4±8.7, t=6.250, P<0.01), MCP-1 (158.3±39.1 vs. 57.2±12.5, t=14.280, P<0.01), ICAM-1 (371.8±77.3 vs. 160.4±39.8, t=10.850, P<0.01), tumor necrosis factor-α (TNF-α) (16.3±3.8 vs. 7.8±2.4, t=7.390, P<0.01) in the cell supernatant were significantly up-regulated. The protein expression levels of AMPK (0.39±0.11 vs. 0.83±0.18, t=14.250, P<0.01), p-AMPK (0.25±0.03 vs. 0.49±0.12, t=12.370, P<0.01), ACC (0.54±0.16 vs. 1.07±0.24, t=10.670, P<0.01), p-ACC (0.38±0.07 vs. 0.97±0.30, t=18.130, P<0.01), LC3Ⅰ (0.67±0.21 vs. 0.67±0.21, t=5.780, P<0.01) and LC3Ⅱ (0.42±0.13 vs. 0.93±0.38, t=6.820, P<0.01) were down-regulated, mTOR (2.57±0.53 vs. 1.23±0.25, t=21.350, P<0.01), and p-mTOR (1.35±0.0.37 vs. 0.57±0.12, t=8.870, P<0.01), p70SK6 (1.85±0.39 vs. 0.81±0.19, t=7.170, P<0.01), p-70SK6 (0.69±0.22 vs. 0.38±0.11, t=9.260, P<0.01) and P62 (1.24±0.38 vs. 0.58±0.14, t=5.270, P<0.01) were up-regulated. However, pre-incubation with irisin and then stimulation with PDGF-BB could significantly decrease VSMCs proliferation [(122.14±9.42)%, t=4.970, P<0.01], and significantly down-regulate the inflammatory cytokines IL-1β (17.38±4.10, t=6.740, P<0.01), IL-6 (34.9±8.9, t=2.760, P<0.05), MCP-1 (47.28±9.20, t=16.270, P<0.01), ICAM-1 (169.2±55.8, t=7.780, P<0.01) and TNF-α (6.9±2.0, t=8.180, P<0.01) in the cell supernatant. The protein expression levels of AMPK (0.72±0.26, t=2.670, P<0.05), p-AMPK (0.46±0.13, t=3.280, P<0.05), ACC (0.95±0.28, t=5.870, P<0.01), p-ACC (0.78±0.25, t=3.140, P<0.01), LC3Ⅰ (1.34±0.35, t=1.960, P<0.05) and LC3Ⅱ (1.03±0.19, t=7.260, P<0.01) were up-regulated, and mTOR (1.16±0.33, t=6.910, P<0.01), p-mTOR (0.66±0.18, t=6.830, P<0.01), p70SK6 (1.12±0.34, t=2.360, P<0.05), p-70SK6 (0.37±0.12, t=3.120, P<0.01) and P62 (0.68±0.18, t=3.360, P<0.05) were down-regulated. Conclusion Irisin could inhibit the inflammation and proliferation of VSMCs possibly by up-regulating autophagy through activation of the AMPK/mTOR signaling pathway. Key words: Irisin; Adenosine monophosphate-actived protein kinase/mammalian target of rapamycin signaling pathway; Autophagy; Vascualar smooth muscle cell proliferation; Vein graft stenosis

Mutations of the AtYAK1 Kinase Suppress TOR Deficiency in Arabidopsis

The target of rapamycin (TOR) kinase is a conserved energy sensor that regulates growth in response to environmental cues. However, little is known about the TOR signaling pathway in plants. We used Arabidopsis lines affected in the lethal with SEC13 protein 8 (LST8-1) gene, a core element of the TOR complex, to search for suppressor mutations. Two suppressor lines with improved growth were isolated that carried mutations in the Yet Another Kinase 1 (AtYAK1) gene encoding a member of the dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) family. Atyak1 mutations partly rescued the developmental defects of lst8-1-1 mutants and conferred resistance to the TOR inhibitor AZD-8055. Moreover, atyak1 mutations suppressed the transcriptomic and metabolic perturbations as well as the abscisic acid (ABA)hypersensitivity of the lst8-1-1 mutants. AtYAK1 interacted with the regulatory-associated protein of TOR (RAPTOR), a component of the TOR complex, and was phosphorylated by TOR. Thus, our findings reveal that AtYAK1 is a TOR effector that probably needs to be switched off to activate plant growth.

Ferumoxytol and CpG oligodeoxynucleotide 2395 synergistically enhance antitumor activity of macrophages against NSCLC with EGFRL858R/T790M mutation.

Purpose: Drug resistance is a major challenge for epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) treatment of lung cancer. Ferumoxytol (FMT) drives macrophage (MΦ) transformation towards a M1-like phenotype and thereby inhibits tumor growth. CpG oligodeoxynucleotide 2395 (CpG), a toll-like receptor 9 (TLR9) agonist, is an effective therapeutic agent to induce anticancer immune responses. Herein, the effect of co-administered FMT and CpG on MΦ activation for treating non-small cell lung cancer (NSCLC) was explored.Methods: The mRNA expression levels of M1-like genes in RAW 264.7 MΦ cells stimulated by FMT, CpG and FMT and CpG (FMT/CpG) were evaluated by quantitative reverse transcription PCR (qRT-PCR). Then, the effects of FMT/CpG-pretreated MΦ supernatant on apoptosis and proliferation of H1975 cells were detected by flow cytometry, and the expression of EGFR and its downstream signaling pathway in H1975 cells were explored by western blotting. Finally, a H1975 cell xenograft mouse model was used to study the anti-tumor effect of the combination of FMT and CpG in vivo.Results: FMT and CpG synergistically enhanced M1-like gene expression in MΦ, including tumor necrosis factor-α, interleukin (IL)-12, IL-1α, IL-1β, IL-6 and inducible nitric oxide synthase (iNOS). FMT/CpG-pretreated MΦ supernatant inhibited proliferation and induced apoptosis of H1975 cells, accompanied by down-regulation of cell cycle-associated proteins and up-regulation of apoptosis-related proteins. Further studies indicated that the FMT/CpG-pretreated MΦ supernatant suppressed p-EGFR and its downstream AKT/mammalian target of rapamycin signaling pathway in H1975 cells. Furthermore, FMT/CpG suppressed tumor growth in mice accompanied by a decline in the EGFR-positive tumor cell fraction and increased M1 phenotype macrophage infiltration.Conclusion: FMT acted synergistically with CpG to activate MΦ for suppressed proliferation and promoted apoptosis of NSCLC cells via EGFR signaling. Thus, combining FMT and CpG is an effective strategy for the treatment of NSCLC with EGFRL858R/T790M mutation.

Replacing fish meal with cottonseed meal protein hydrolysate affects amino acid metabolism via AMPK/SIRT1 and TOR signaling pathway of Megalobrama amblycephala

This study was conducted to evaluate effects of replacing fish meal with cottonseed meal protein hydrolysate (CPH) on amino acid metabolism of Megalobrama amblycephala. Fish (38.66 ± 0.08 g) were randomly divided into five groups and fed five isonitrogenous (320 g kg−1 crude protein) and isocaloric (17.8 MJ kg−1 gross energy) diets replacing fish meal by CPH 0% (CPH 0), 1% (CPH 1), 3% (CPH 3), 5% (CPH 5) and 7% (CPH 7). Dietary 3% CPH had no effect on plasma aspartate aminotransferase (AST), alanine aminotransferase (ALT), succinate dehydrogenase (SDH) and xanthine oxidase (XOD) contents and liver SDH and XOD (P > 0.05), but significantly increased hepatic AST and ALT contents (P < 0.05). The plasma threonine (Thr), valine (Val), lysine (Lys), histidine (His), total essential amino acids and total amino acids contents of fish fed CPH5 and CPH7 diets significantly decreased (P < 0.05) compared with the control diet. Meanwhile, threonine (Thr), valine (Val), isoleucine (Ile) and leucine (Leu) contents in muscle significantly decreased (P < 0.05) when fish fed CPH7 diet. Diets with 5% and 7% CPH significantly decreased target of rapamycin (TOR) and S6 kinase-polypeptide 1 (S6K1) mRNA expressions levels in liver and gut, but increased eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2) mRNA expression levels (P < 0.05), as well as increased hepatic AMP-activated protein kinase α1 (AMPKa-1), AMP-activated protein kinase α2 (AMPKa-2) and sirtuin-1 (SIRT-1) mRNA expressions levels. Furthermore, the AMP-activated protein kinase α (t-AMPKα) and phospho-AMPKα (p-AMPKα) protein contents were increased significantly in fish fed with 5% and 7% CPH over the control group (P < 0.05). Overall, replacing fish meal with CPH at high level (5 and 7%) decreased the amino acid metabolism and growth performance, as well as inhibited ATP consumption via inhibiting TOR signaling pathway and activating AMPK/SIRT1 pathway.

Modelling of Neuronal Ceroid Lipofuscinosis Type 2 in Dictyostelium discoideum Suggests That Cytopathological Outcomes Result from Altered TOR Signalling.

The neuronal ceroid lipofuscinoses comprise a group of neurodegenerative disorders with similar clinical manifestations whose precise mechanisms of disease are presently unknown. We created multiple cell lines each with different levels of reduction of expression of the gene coding for the type 2 variant of the disease, Tripeptidyl peptidase (Tpp1), in the cellular slime mould Dictyostelium discoideum. Knocking down Tpp1 in Dictyostelium resulted in the accumulation of autofluorescent material, a characteristic trait of Batten disease. Phenotypic characterisation of the mutants revealed phenotypic deficiencies in growth and development, whilst endocytic uptake of nutrients was enhanced. Furthermore, the severity of the phenotypes correlated with the expression levels of Tpp1. We propose that the phenotypic defects are due to altered Target of Rapamycin (TOR) signalling. We show that treatment of wild type Dictyostelium cells with rapamycin (a specific TOR complex inhibitor) or antisense inhibition of expression of Rheb (Ras homologue enriched in the brain) (an upstream TOR complex activator) phenocopied the Tpp1 mutants. We also show that overexpression of Rheb rescued the defects caused by antisense inhibition of Tpp1. These results suggest that the TOR signalling pathway is responsible for the cytopathological outcomes in the Dictyostelium Tpp1 model of Batten disease.

The IRE1 endoplasmic reticulum stress sensor activates natural killer cell immunity in part by regulating c-Myc.

Natural killer (NK) cells are critical mediators of host immunity to pathogens. Here, we demonstrate that the ER stress sensor inositol-requiring enzyme 1 (IRE1α) and its substrate transcription factor X-box-binding protein 1 (XBP1) drive NK cell responses against viral infection and tumors in vivo. IRE1α-XBP1 were essential for expansion of activated mouse and human NK cells and are situated downstream of the mTOR signaling pathway. Transcriptome and chromatin immunoprecipitation analysis revealed c-Myc as a novel and direct downstream target of XBP1 for regulation of NK cell proliferation. Genetic ablation or pharmaceutical blockade of IRE1α downregulated c-Myc, and NK cells with c-Myc haploinsufficency phenocopied IRE1α-XBP1 deficiency. c-Myc overexpression largely rescued the proliferation defect in IRE1α−/− NK cells. Like c-Myc, IRE1α-XBP1 also promotes oxidative phosphorylation in NK cells. Overall, our study identifies a novel IRE1α-XBP1-cMyc axis in NK cell immunity, providing new insight into host protection against infection and cancer.

Cottonseed meal protein hydrolysate stimulates feed intake and appetite in Chinese mitten crab, Eriocheir sinensis

One hundred and twenty crabs (initial weight: 51.32 ± 0.06 g) were fed three experimental diets with cottonseed meal protein hydrolysate (CPH) at 0, 6 and 12 g/kg for 60 days. The results showed that CPH0.6 diet significantly improved the 1-hr feed intake, protease activity and weight gain of crabs. In thoracic ganglia, 4E-binding protein (4E-BP) expression level of crab fed the diets containing CPH was significantly higher than CPH0 group. In cerebral ganglia, protein kinase B and 4E-BP expression levels of crab fed CPH0.6 diets were significantly lower than CPH0 group. In mid-intestine, target of rapamycin (TOR) relative expression level of crabs fed the diets containing CPH was significantly lower than CPH0 group. 4E-BP expression level showed an opposite pattern. In thoracic ganglia, cerebral ganglia and mid-intestine, leptin receptor expression level of crabs fed CPH0.6 diet was significantly lower than CPH0 group. Neuropeptide Y and neuropeptide Y receptor expression level of crab fed CPH0.6 diet was significantly higher than CPH0 group. In conclusion, our results indicated that dietary supplementation of 6 g/kg CPH could stimulate appetite and quicken feeding rate via the TOR signalling pathway.

Activation of mammalian target of rapamycin signaling pathway in placental tissues of gravidas with gestational diabetes mellitus

Objective To investigate the activation of mammalian target of rapamycin (mTOR) signaling pathway in placental tissues of gestational diabetes mellitus (GDM) women and in JEG3 cells after high glucose treatment. Methods Placental tissues were collected from 60 singleton pregnant women at term, who underwent elective cesarean section in Peking University First Hospital from December 2016 to June 2017, including 30 GDM women (GDM group) and 30 normal glucose tolerance (NGT) women (NGT group). Expressions of mTOR, Rictor, Raptor, S6 kinase (S6K) and phosphorylated-S6K (p-S6K), which were mTOR signaling pathway-related molecules in those placental tissues were detected by real-time fluorescence quantitative polymerase chain reaction, Western blotting and immunohistochemistry. At the same time, JEG3 cells were treated with glucose of different concentrations (5.5, 10.0, 15.0, 25.0 and 50.0 mmol/L) for 24 h, and the expressions of mTOR, S6K and p-S6K were detected by Western blotting. Two independent samples t-test, one-way analysis of variance test and Spearman correlation analysis were used for statistical analysis. Results Compared with the NGT group, the pre-pregnancy body mass index(BMI), neonatal birth weight, fasting blood glucose(FBG), and 1 h and 2 h post-load blood glucose in oral glucose tolerance test (OGTT) of the GDM group were significantly increased [pre-pregnant BMI: (21.6±2.7) vs (23.4±3.5) kg/m2, t=-2.192; neonatal birth weight: (3 337.5±347.7) vs (3 618.3±580.9) g, t=-2.727; FBG: (4.6±0.4) vs (5.2±0.8) mmol/L, t=-3.947; 1 h glucose level: (7.4±1.2) vs (9.6±1.9) mmol/L, t=-5.332; 2 h glucose level: (6.3±1.0) vs (8.1±1.5) mmol/L, t=-5.314; all P<0.05]. The mRNA expressions of mTOR, Rictor and Raptor were significantly higher in the GDM group than in the NGT group (0.051±0.015 vs 0.031±0.011, t=-5.635; 0.038±0.017 vs 0.026±0.010, t=-3.485; 0.036±0.012 vs 0.025±0.011, t=-3.312; all P<0.05). The expression of mTOR, Rictor, Raptor and p-S6K protein in the GDM group were enhanced as compared with those in the NGT group (0.834±0.432 vs 0.386±0.361, t=-2.249; 0.589±0.236 vs 0.262±0.075, t=-3.726; 0.767±0.345 vs 0.323±0.109, t=-3.472; 1.847±1.025 vs 0.834±0.432, t=-2.575; all P<0.05). The results of immunohistochemistry also showed that the p-S6K in the placental tissues of the GDM group was higher than that of the NGT group (0.29±0.09 vs 0.18±0.08, t=-3.122, P=0.004). The expressions of mTOR protein (0.190±0.085, 0.301±0.089, 0.419±0.065, 0.562±0.108, 0.412±0.058, F=18.351, P<0.05) and p-S6K protein (0.753±0.150, 1.146±0.289, 2.148±0.188, 2.248±0.115, 2.134±0.214, F=66.242, P<0.05) in JEG3 cells treated with different concentrations of glucose (5.5, 10.0, 15.0, 25.0 and 50.0 mmol/L) were significantly different and increased with increasing concentrations of glucose (r=0.314, P=0.035; r=0.457, P=0.002). Conclusions The up-regulated expressions of mTOR signaling pathway-related molecules in GDM placenta and high glucose-treated trophoblast cells (JEG3 cells) indicate a possible correlation between mTOR signaling pathway and GDM. However, the specific mechanisms need further study. Key words: Diabetes, gestational; Placenta; Mammalian target of rapamycin, serine/threonine kinases; Signal transduction