mTOR Research Articles

Effects of Target of Rapamycin and Phosphatidylinositol 3-Kinase Inhibitors and Other Autophagy-Related Supplements on Life Span in y w Male Drosophila melanogaster.

Various dietary supplements have been shown to extend the life span of Drosophila melanogaster, including several that promote autophagy, such as rapamycin and spermidine. The goal of the study presented here was to test numerous additional potential anti-aging supplements, primarily inhibitors of the target of rapamycin (TOR) and/or phosphatidylinositol 3-kinase (PI3K). Using a single, comparatively long-lived y w test strain, screening was performed in male flies supplemented either throughout adulthood or, in a few cases, beginning in middle or late adult life, with concentrations spanning 4-6 orders of magnitude in most cases. Supplementation with PP242 and deferiprone, an iron chelator, beginning in late adult life had no positive effect on life span. Lifelong supplementation with Ku-0063794, LY294002, PX-866-17OH, Torin2 and WYE-28 had no effect at any dose. Rapamycin, spermidine and wortmannin all had significant life-shortening effects at the highest doses tested. AZD8055, PI-103 hydrochloride and WYE-132 yielded slight beneficial effects at 1-2 doses, but only 100 nM AZD8055 was confirmed to have a minor (1.3%) effect in a replicate experiment, which was encompassed by other control groups within the same study. These compounds had no effect on fly fecundity (egg laying) or fertility (development of progeny to adulthood), but equivalent high doses of rapamycin abolished fertility. The solvent DMSO had no significant effect on life span at the concentrations used to solubilize most compounds in the fly medium, but it drastically curtailed both survival and fertility at higher concentrations. 2-Hydroxypropyl-β-cyclodextrin also failed to extend the life span when provided throughout adulthood or beginning in mid-adult life. Collectively, the results suggest that inhibition of the TOR/PI3K pathway and autophagy through dietary intervention is not a straightforward anti-aging strategy in Drosophila and that further extension of life is difficult in comparatively long-lived flies.

Far-infrared irradiation inhibits proliferation of human upper airway epithelial cells via protein phosphatase 2A-promoted dephosphorylation of p70 S6 kinase.

Far-infrared (FIR) ray, an invisible electromagnetic radiation with a wavelength of 3‒1000μm, elicits various biological effects. Excessive proliferation of human upper airway epithelial cells (HUAEpCs) contributes to the development and exacerbation of nasal narrowing diseases, including nasal polyposis and chronic rhinosinusitis with nasal polyps (CRSwNP). Here, we investigated the molecular mechanisms through which FIR irradiation inhibits the proliferation of HUAEpCs. FIR irradiation significantly inhibited the proliferation of NCI-H292 cells without alteration in cell viability. The anti-proliferative effect of FIR radiation was accompanied by decreased phosphorylation of p70S6K at Thr389 (p-p70S6K-Thr389), without changes in the phosphorylation of mammalian target of rapamycin and adenosine monophosphate-activated protein kinase (AMPK). Overexpression of p70S6K-T389E mutant gene, not dominant negative-AMPKα1 gene, significantly reversed FIR irradiation-inhibited p-p70S6K-Thr389 and cell proliferation. Cotreatment with okadaic acid or knockdown of protein phosphatase 2A catalytic subunit (PP2Ac) gene expression significantly reversed FIR irradiation-decreased p-p70S6K-Thr389 and cell proliferation. FIR irradiation remarkably promoted the physical association of p70S6K and PP2Ac without change in total PP2Ac expression. Hyperthermal stimulus (39°C) did not alter p-p70S6K-Thr389 and cell proliferation. In line with NCI-H292 cell results, FIR irradiation, not hyperthermal stimulus, significantly decreased p-p70S6K-Thr389 and cell proliferation in primary human nasal turbinate and polyp epithelial cells. These results demonstrated that FIR irradiation decreased the proliferation of HUAEpCs through PP2A-mediated inhibition of p70S6K phosphorylation, independent of its hyperthermal effect. Our data suggest that FIR therapy can be used to treat upper airway narrowing epithelial hyperplastic diseases, including nasal polyposis and CRSwNP.

Somatic variant analysis of resected brain tissue in epilepsy surgery patients.

We studied the distribution of germline and somatic variants in epilepsy surgery patients with (suspected) malformations of cortical development (MCD) who underwent surgery between 2015 and 2020 at University Medical Center Utrecht (the Netherlands) and pooled our data with four previously published cohort studies. Tissue analysis yielded a pathogenic variant in 203 of 663 (31%) combined cases. In 126 of 379 (33%) focal cortical dysplasia (FCD) type II cases and 23 of 37 (62%) hemimegalencephaly cases, a pathogenic variant was identified, mostly involving the mTOR signaling pathway. Pathogenic variants in 10 focal epilepsy genes were found in 48 of 178 (27%) FCDI/mild MCD/mMCD with oligodendroglial hyperplasia and epilepsy cases; 36 of these (75%) were SLC35A2 variants. Six of 69 (9%) patients without a histopathological lesion had a pathogenic variant in SLC35A2 (n = 5) or DEPDC5 (n = 1). A germline variant in blood DNA was confirmed in all cases with a pathogenic variant in tissue, with a variant allele frequency (VAF) of ~50%. In seven of 114 patients (6%) with a somatic variant in tissue, mosaicism in blood was detected. More than half of pathogenic somatic variants had a VAF < 5%. Further analysis of the correlation between genetic variants and surgical outcomes will improve patient counseling and may guide postoperative treatment decisions.

Effect of acupuncture on AMPK/ULK1 signaling pathway of hippocampus in chronic stress-induced depression rats

To observe the effect of manual acupuncture stimulation on changes of behavior and hippocampal signaling pathways of AMP-activated kinase (AMPK)/UNC-51-like kinase 1 (ULK1) in rats with chronic unpredictable mild stress (CUMS)-induced depression, so as to explore its molecular mechanism underlying improvement of depression. Thirty-two male SD rats were randomly divided into normal control, model, acupuncture and medication (fluoxetine) groups, with 8 rats in each group. The depression model was established by using CUMS (fasting, water depredation, restraint, swimming in cold water, crowding, stroboscope stimulation, and tail clipping, each of which was used once a week) for 6 weeks. Manual acupuncture stimulation was applied to "Baihui"(GV20) and "Yintang"(EX-HN3) for 10 min, once daily, 6 times a week for 6 weeks before every-day modeling. Rats of the medication group received gavage of fluoxetine (10 mg/kg) once a day for 6 weeks before every-day modeling. The percentage of sucrose preference, and the percentages of open arm times and open arm time in the elevated plus maze experiments were detected. The expression levels of AMPK, phosphorylated (p)-AMPK, ULK1, p-ULK1, mammalian target of rapamycin (mTOR) and p-mTOR proteins in the hippocampus tissue were detected by Western blot, and the expression of autophagy effecting protein (Beclin-1) and the selective autophagy receptors (p62) mRNA in the hippocampal tissue was detected using real-time fluorescence quantitative PCR, respectively. Compared with the normal group, the percentage of sucrose preference, percentage of times of entering the open arm and percentage of open arm time, the expression levels of p-AMPK /AMPK ratio, p-ULK1 /ULK1 ratio of proteins, and Beclin-1 mRNA were significantly decreased (P<0.01, P<0.05), and the expression levels of p-mTOR /mTOR ratio and p62 mRNA were significantly increased (P<0.01, P<0.05) in the model group. In comparison with the model group, the percentage of sucrose preference, percentage of open arm times and open arm time, the expression levels of p-AMPK /AMPK ratio, p-ULK1/ULK1 ratio of proteins, and Beclin-1 mRNA were significantly increased in both acupuncture and medication groups (P<0.01, P<0.05), while the expression levels of p-mTOR /mTOR ratio of proteins, and p62 mRNA were markedly decreased only in the medication group (P<0.05). Acupuncture treatment can alleviate depression-like behavior in CUMS rats, which may be related to its functions in up-regulating AMPK/ULK1 signaling pathway to induce cellular autophagy in the hippocampus.

Intermittent cold stimulation acclimates broilers to acute cold stress by affecting cardiac lipid metabolism.

This study aimed to investigate whether intermittent cold stimulation can induce adaptation in broilers to acute cold stress (ACS) by regulating the lipid metabolism of hearts. CS0 were kept at normal rearing temperature, while CS3 and CS5 were exposed to 3°C for 3 and 5 hours, respectively, on alternate days lower than CS0 from 15d to 35d. On 50d, broilers in three groups were exposed to ACS at 10°C for 12 hours (Y12). The levels of corticosterone (CORT) and liothyronine (T3), mRNA and protein levels of heart AMP-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) pathway genes were assessed at 36d, 50d and Y12. At 36d, mRNA levels of AMPKα, acyl-CoA oxidase (ACO), mTOR, sterol-regulatory element binding protein (SREBP), stearoyl-coA desaturase (SCD), acetyl-coA carboxylase (ACC), fatty acid synthase (FAS) and protein level of peroxisome proliferators-activated receptor α (PPARα) in CS3 and CS5 were significantly lower than those in CS0 (p<0.05). At 50d, compared to CS0, mRNA levels of PPARα, carnitine palmitoyltransferase1 (CPT1), ACO, tuberous sclerosis complex (TSC), SREBP and SCD, as well as protein levels of p-AMPKα/AMPKα, PPARα and SREBP were significantly increased in CS5 (p<0.05). At Y12, the levels of T3 in CS3 and CS5 were significantly higher than those in CS0 (p<0.05), mRNA levels of CPT1, ACO, SREBP, SCD and protein levels of p-AMPKα/AMPKα, SREBP, and FAS were significantly higher in CS5 than in CS0 and CS3 (p<0.05). However, compared to 50d, at Y12, mRNA levels of AMPKα, CPT1 and ACO in CS3 and CS5 significantly decreased (p<0.05), while protein levels of p-AMPKα/AMPKα significantly increased (p<0.05). This study suggested that intermittent cold stimulation at 3°C lower than normal rearing temperature for 5h could help broilers adapt to the ACS by promoting heart lipid metabolism.

The Research Progress of Metformin Regulation of Metabolic Reprogramming in Malignant Tumors.

Metabolism reprogramming is a crucial hallmark of malignant tumors. Tumor cells demonstrateenhanced metabolic efficiency, converting nutrient inputs into glucose, amino acids, and lipids essential for theirmalignant proliferation and progression. Metformin, a commonly prescribed medication for type 2 diabetes mellitus,has garnered attention for its potential anticancer effects beyond its established hypoglycemic benefits. This review adopts a comprehensive approach to delineate the mechanisms underlying metabolite abnormalities within the primary metabolic processes of malignant tumors. This review examines the abnormal activation of G protein-coupled receptors (GPCRs) in these metabolic pathways, encompassing aerobic glycolysis with increased lactate production in glucose metabolism, heightened lipid synthesis and cholesterol accumulation in lipid metabolism, and glutamine activation alongside abnormal protein post-translational modifications in amino acid and protein metabolism. Furthermore, the intricate metabolic pathways and molecular mechanisms through which metformin exerts its anticancer effects are synthesized and analyzed, particularly its impacts on AMP-activated protein kinase activation and the mTOR pathway. The analysis reveals a multifaceted understanding of how metformin can modulate tumor metabolism, targeting key nodes in metabolic reprogramming essential for tumor growth and progression. The review compiles evidence that supports metformin's potential as an adjuvant therapy for malignant tumors, highlighting its capacity to interfere with critical metabolic pathways. In conclusion, this review offers a comprehensive overview of the plausible mechanisms mediating metformin's influence on tumor metabolism, fostering a deeper comprehension of its anticancer mechanisms. By expanding the clinical horizons of metformin and providing insight into metabolism-targeted tumor therapies, this review lays the groundwork for future research endeavors aimed at refining and advancing metabolic intervention strategies for cancer treatment.

Efficient, High-Quality Engineering of Therapeutic Extracellular Vesicles on an Integrated Nanoplatform.

Engineered extracellular vesicles (EVs) have been recognized as important therapeutics for gene and cell therapy. To achieve clinically desired therapy, technologies for EV engineering have high demands on the efficacy in producing EVs and their qualities, which, however, remain challenging to conventional routes due to their limited control on therapeutic payload delivery, EV secretion, and extracellular microenvironments. Here, we report a nanoplatform (denoted as PURE) that enables efficient electro-transfection while stimulating cells to produce high-quality EVs carrying functional RNAs. PURE further employs an ammonium removal zone to maintain the physiological conditions of the extracellular microenvironment and an EV uptake zone that efficiently (87.1%) captures EVs in situ with porous hydrogels. The platform achieved about a 12-fold higher yield of engineered EVs and a 146-fold abundance of desired therapeutics compared to those naturally secreted from cells. The PURE-engineered miR-130a-EVs were validated for effectively upregulating the mTOR signaling pathway in both in vitro and in vivo. Their therapeutic capability was then verified by enhancing the in vitro activation of primordial follicles. In vivo applications further highlighted the therapeutic effects of miR-130a-EVs in restoring ovary function in aged mice. The PURE platform represents a strategy for the clinical translation of EV-mediated therapy.

Oncogene Downregulation by Mahanine Suppresses Drug-Sensitive and Drug-Resistant Lung Cancer and Inhibits Orthotopic Tumor Progression.

Background/Objectives: Lung cancer is one of the deadliest cancers, and drug resistance complicates its treatment. Mahanine (MH), an alkaloid from Murraya koenigii has been known for its anti-cancer properties. However, its effectiveness and mechanisms in treating non-small cell lung cancer (NSCLC) remain largely unexplored. The present study aimed to investigate MH's effect on drug-sensitive and drug-resistant NSCLC and its potential mechanism of action. Methods: We isolated MH from M. koenigii leaves and the purity (99%) was confirmed by HPLC, LC-MS and NMR. The antiproliferative activity of MH was determined using MTT and colony formation assays against drug-sensitive (A549 and H1299) and Taxol-resistant lung cancer cells (A549-TR). Western blot analysis was performed to determine MH's effects on various molecular targets. Anti-tumor activity of MH was determined against lung tumors developed in female NOD Scid mice injected with A549-Fluc bioluminescent cells (1.5 × 106) intrathoracically. Results: MH dose-dependently reduced the proliferation of all lung cancer cells (A549, H1299 and A549-TR), with IC50 values of 7.5, 5, and 10 µM, respectively. Mechanistically, MH arrested cell growth in the G0/G1 and G2/M phases of the cell cycle by inhibiting cyclin-dependent kinase 4/6 (CDK4/6) and cell division control 2 (CDC2) and induced apoptosis through the downregulation of B-cell leukemia/lymphoma 2 (BCL2) and B-cell lymphoma-extra large (BCL-XL). The apoptotic induction capacity of MH can also be attributed to its ability to inhibit pro-oncogenic markers, including mesenchymal-epithelial transition factor receptor (MET), phosphorylated protein kinase B (p-AKT), phosphorylated mammalian target of rapamycin (p-mTOR), survivin, rat sarcoma viral oncogene (RAS), myelocytomatosis oncogene (cMYC), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) levels. In vivo, MH (25 mg/kg b. wt.) significantly (p < 0.001) inhibited the growth of A549 lung cancer orthotopic xenografts in NOD Scid mice by 70%. Conclusions: Our study provides new mechanistic insights into MH's therapeutic potential against NSCLC.

PI3K/AKT signaling and neuroprotection in ischemic stroke: molecular mechanisms and therapeutic perspectives.

It has been reported that the PI3K/AKT signaling pathway plays a key role In the pathogenesis of ischemic stroke. As a result, the development of drugs targeting the PI3K/AKT signaling pathway has attracted increasing attention from researchers. This article reviews the pathological mechanisms and advancements in research related to the signaling pathways in ischemic stroke, with a focus on the PI3K/AKT signaling pathway.The key findings include the following: (1) The complex pathological mechanisms of ischemic stroke can be categorized into five major types: excitatory amino acid toxicity, Ca2+ overload, inflammatory response, oxidative stress, and apoptosis. (2) The PI3K/AKT-mediated signaling pathway is closely associated with the occurrence and progression of ischemic stroke, which primarily involves the NF-kB, NRF2, BCL-2, mTOR, and endothelial NOS signaling pathways. (3) Natural products, including flavonoids, quinones, alkaloids, phenylpropanoids, phenols, terpenoids, and iridoids, show great potential as candidate substances for the development of innovative anti-stroke medications. (4) Recently, novel therapeutic techniques, such as electroacupuncture and mesenchymal stem cell therapy, have demonstrated the potential to improve stroke outcomes by activating the PI3K/AKT signaling pathway, providing new possibilities for the treatment and rehabilitation of patients with ischemic stroke. Future investigations should focus on the direct regulatory mechanisms of drugs targeting the PI3K/AKT signaling pathway and their clinical translation to develop innovative treatment strategies for ischemic stroke.

METTL3 affects the biological function of lung adenocarcinoma through the FGF2/PI3K/AKT /mTOR pathway.

This study aims to investigate the role of the m6A regulatory factor METTL3 in LUAD. By examining the expression of METTL3 in LUAD and conducting cellular functional experiments, the biological functions of METTL3 were discussed. mRNA-seq and MeRIP-qPCR were used to identify downstream target genes and pathways. The expression level of METTL3 in LUAD is lower than that in the control group. The downregulation of METTL3 promoted the proliferation, migration, and invasion of LUAD cells, while overexpression of METTL3 results in the opposite effects. Furthermore, we found that FGF2 was negatively regulated by METTL3. Inhibiting FGF2 reversed the tumor-promoting effects caused by METTL3 downregulation in LUAD cells. Silencing METTL3 enhanced the stability of FGF2 mRNA. Silencing FGF2 resulted in reduced activity of the PI3K/AKT/mTOR signaling pathway in METTL3 knockdown LUAD cells. In summary, our findings unveil an intricate signaling network involving METTL3/FGF2/PI3K/AKT/mTOR in LUAD and provide valuable insights into the molecular mechanisms underlying tumor progression, thus holding significant implications for targeted therapy and advancing LUAD research.

Somatostatin interneuron fate-mapping and structure in a Pten knockout model of epilepsy.

Disruption of inhibitory interneurons is common in the epileptic brain and is hypothesized to play a pivotal role in epileptogenesis. Abrupt disruption and loss of interneurons is well-characterized in status epilepticus models of epilepsy, however, status epilepticus is a relatively rare cause of epilepsy in humans. How interneuron disruption evolves in other forms of epilepsy is less clear. Here, we explored how somatostatin (SST) interneuron disruption evolves in quadruple transgenic Gli1-CreERT2, Ptenfl/fl, SST-FlpO, and frt-eGFP mice. In these animals, epilepsy develops following deletion of the mammalian target of rapamycin (mTOR) negative regulator phosphatase and tensin homolog (Pten) from a subset of dentate granule cells, while downstream Pten-expressing SST neurons are fate-mapped with green fluorescent protein (GFP). The model captures the genetic complexity of human mTORopathies, in which mutations can be restricted to excitatory neuron lineages, implying that interneuron involvement is later developing and secondary. In dentate granule cell (DGC)-Pten knockouts (KOs), the density of fate-mapped SST neurons was reduced in the hippocampus, but their molecular phenotype was unchanged, with similar percentages of GFP+ cells immunoreactive for SST and parvalbumin (PV). Surviving SST neurons in the dentate gyrus had larger somas, and the density of GFP+ processes in the dentate molecular layer was unchanged despite SST cell loss and expansion of the molecular layer, implying compensatory sprouting of surviving cells. The density of Znt3-immunolabeled puncta, a marker of granule cell presynaptic terminals, apposed to GFP+ processes in the hilus was increased, suggesting enhanced granule cell input to SST neurons. Finally, the percentage of GFP+ cells that were FosB positive was significantly increased, implying that surviving SST neurons are more active. Together, findings suggest that somatostatin-expressing interneurons exhibit a combination of pathological (cell loss) and adaptive (growth) responses to hyperexcitability and seizures driven by upstream Pten KO excitatory granule cells.

Tongsai Granules inhibit autophagy and macrophage-mediated inflammatory response to improve acute exacerbations of chronic obstructive pulmonary disease in rats

To investigate the inhibitory effect of Tongsai Granules (TSG) on macrophage-mediated inflammatory response to alleviate acute exacerbation of chronic obstructive pulmonary disease (AECOPD) in rats and explore the underlying mechanism. Twenty-four rats were divided into control group, AECOPD model group, TSG treatment group, and moxifloxacin+salbutamol (MXF+STL) treatment group. In the rat models of COPD, AECOPD was induced by nasal instillation of Klebsiella pneumoniae on day 3 of week 9 after modeling, and saline, TSG or MXF+STL were administered via gavage on days 1 and 2 and days 4 to 7 of week 9. After the treatments, lung tissues were collected for examining for pathologies and expressions of inflammatory markers, MMP2, and MMP9. In cultured macrophage MH-S cells with LPS stimulation, the effect of TSG-medicated serum on IL-1β, IL-6, TNF-α, COX-2, and iNOS expressions and phosphorylation levels of p38, p-p62, LC3, FoxO3a, and mTOR were evaluated. TSG significantly improved lung pathologies and lung function in AECOPD rats by reducing bronchial wall thickness and mean alveolar linear intercept, increasing alveolar numbers, and reducing pulmonary expression of IL-1β, IL-6, TNF- α, MMP2 and MMP9. In MH-S cells, TSG significantly suppressed LPS-induced expressions of inflammatory cytokines, COX-2 and iNOS. Serum pharmacology coupled with network pharmacology identified 10 chemical components in TSG-medicated serum, and functional analysis of their 466 targets suggested that the therapeutic effect of TSG on AECOPD was mediated primarily by luteolin and quercetin, which regulate the MAPK, mTOR, FoxO, and autophagy pathways. In MH-S cells, luteolin significantly inhibited LPS-induced inflammatory responses and expressions of p-p38, FoxO3a, mTOR, p-p62 and LC3. TSG reduces macrophage-mediated inflammatory responses to alleviate AECOPD in rats possibly by modulating p38, mTOR, and FoxO3a pathways and inhibiting autophagy.

Photosensitizing metal–organic framework nanoparticles combined with tumor-sensitization strategies can enhance the phototherapeutic effect upon medullary thyroid carcinoma

Photodynamic therapy (PDT) utilizing metal-organic frameworks (MOFs) has developed as a new and efficacious treatment for malignant tumors located on the surface of the human body. In order to achieve more effective PDT treatment outcomes, the traditional method has been to increase the intensity of the laser irradiation, but this approach can easily lead to tissue burns. In this study, we developed a new type of nanoparticle, F68-PKI@PCN224, aims to achieve effective PDT upon medullary thyroid carcinoma (MTC) which is an uncommon form of thyroid cancer that originates in the parafollicular cells of the thyroid and the therapeutic outlook for patients with MTC remains unsatisfactory. F68-PKI@PCN224 combines the antitumor features of PDT with mammalian target of rapamycin (mTOR) inhibitor PKI-587 (PKI). The tumor sensitization, slow release, and pH response features of F68-PKI@PCN224 was demonstrated by a series of in vitro and in vivo experiments / assays. F68-PKI@PCN224 achieved the long-term activation and slow releasing of PKI and TCPP in MTC tumor tissues. During the process of generating PDT effects, F68-PKI@PCN224 enhanced the tumor's sensitivity to PDT, direct laser irradiation of MTC cells or subcutaneous tumor tissues. As a result, low-dose phototherapy achieves a higher anti-tumor effect upon F68-PKI@PCN224 compared with TCPP. This study reveals the synergistic effect between tumor sensitization by mTOR inhibitor and PDT and initially unveils the mechanism of action of these nanoparticles.

An emerging role of ferulic acid on sub-adult grass carp (Ctenopharyngodon idellus): Increasing protein digestion and regulating amino acid and oligopeptide transporters expression

This study was conducted to evaluate the effect of supplement ferulic acid on growth performance, digestive ability and transport function of amino acids and oligopeptides in the intestine of sub-adult grass carp (Ctenopharyngodon idellus). The experiment was divided into two parts: a. Digestion experiment: healthy grass carp (678.83±1.00 g) were divided into two groups: control group (0 mg/kg FA) and ferulic acid group (100 mg/kg FA) for 2 weeks; b. Growth experiment: healthy grass carp (678.83±1.00 g) were divided into five treatment groups, the control group (FA0) with no ferulic acid and the experimental group supplemented with ferulic acid 50 (FA50), 100 (FA100), 150 (FA150) and 200 (FA200) mg/kg feed for 9 weeks. The results showed that a. Ferulic acid supplementation increased the apparent digestibility of crude protein and crude fat in grass carp. b. Ferulic acid supplementation improved the growth performance and feed efficiency (FE) of grass carp; increased the activities of trypsin and Na+/K+-ATPase; and up-regulated the levels of mRNAs of some neutral and cationic (non-anionic) amino acid transporter proteins (Solute Carrier Family7member1 (SLC7A7), SLC7A8, etc.) and H+/oligopeptide transporter protein 1 (PepT1) gene and protein levels. In addition, ferulic acid promoted the gene expression levels of protein kinase B (Akt), target of rapamycin (TOR); up-regulated the mRNA levels of tail-side-associated homologous frame transcription factor 2 (Cdx2), cAMP response element-binding protein (CREB), and transcriptional co-activator-specific protein 1 (Sp1), and down-regulated the gene and protein levels of protein kinase CβII (PKCβII). In a word, dietary ferulic acid promoted growth performance, digestion, as well as amino acid and oligopeptide transport capacities. Finally, based on regression analyses of percentage weight gain (PWG), FE, specific growth rate (SGR), intestine fold height, trypsin activity, and Na+/K+-ATPase activity, we recommend optimal supplementation levels of ferulic acid in sub-adult grass carp of 92.18–120.60 mg/kg diet.

Enhancement of Autophagy in Macrophages via the p120-Catenin-Mediated mTOR Signaling Pathway.

Autophagy serves as a critical regulator of immune responses in sepsis. Macrophages are vital constituents of both innate and adaptive immunity. In this study, we delved into the intricate role of p120-catenin (p120) in orchestrating autophagy in macrophages in response to endotoxin stimulation. Depletion of p120 effectively suppressed LPS-induced autophagy in both J774A.1 macrophages and murine bone marrow-derived macrophages. LPS not only elevated the interaction between p120 and L chain 3 (LC3) I/II but also facilitated the association of p120 with mammalian target of rapamycin (mTOR). p120 depletion in macrophages by small interfering RNA reduced LPS-induced dissociation of mTOR and Unc-51-like kinase 1 (ULK1), leading to an increase in the phosphorylation of ULK1. p120 depletion also enhanced LPS-triggered macrophage apoptosis, as evidenced by increased levels of cleaved caspase 3, 7-aminoactinomycin D staining, and TUNEL assay. Notably, inhibiting autophagy reversed the decrease in apoptosis caused by LPS stimulation in macrophages overexpressing p120. Additionally, the ablation of p120 inhibited autophagy and accentuated apoptosis in alveolar macrophages in LPS-challenged mice. Collectively, our findings strongly suggest that p120 plays a pivotal role in fostering autophagy while concurrently hindering apoptosis in macrophages, achieved through modulation of the mTOR/ULK1 signaling pathway in sepsis. This underscores the potential of targeting macrophage p120 as an innovative therapeutic avenue for treating inflammatory disorders.

Combined spatially resolved metabolomics and spatial transcriptomics reveal the mechanism of RACK1-mediated fatty acid synthesis.

Lipid metabolism is altered in rapidly proliferating cancer cells, where fatty acids (FAs) are utilized in the synthesis of sphingolipids and glycerophospholipids to produce cell membranes and signaling molecules. Receptor for activated C-kinase 1 (RACK1; also known as small ribosomal subunit protein) is an intracellular scaffolding protein involved in signaling pathways. Whether such lipid metabolism is regulated by RACK1 is unknown. Here, integrated spatially resolved metabolomics and spatial transcriptomics revealed that accumulation of lipids in cervical cancer (CC) samples correlated with overexpression of RACK1, and RACK1 promoted lipid synthesis in CC cells. Chromatin immunoprecipitation verified binding of sterol regulatory element-binding protein 1 (SREBP1) to acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN) promoters. RACK1 enhanced de novo FA synthesis by upregulating expression of sterol regulatory element binding transcription factor 1 (SREBP1) and lipogenic genes FASN and ACC1. Co-immunoprecipitation and western blotting revealed that RACK1 interacted with protein kinase B (AKT) to activate the AKT/mammalian target of rapamycin (mTOR)/SREBP1 signaling pathway to promote FA synthesis. Cell proliferation and apoptosis experiments suggested that RACK1-regulated FA synthesis is key in the progression of CC. Thus, RACK1 enhanced lipid synthesis through the AKT/mTOR/SREBP1 signaling pathway to promote the growth of CC cells. RACK1 may become a therapeutic target for CC.

Maternal Broccoli Powder Intake Ameliorates Insulin Resistance and Inflammation via AMPK/mTOR Pathway in the Livers of High-Fructose-Fed Male Rat Offspring Exposed to Maternal Protein Restriction.

Sub-optimal prenatal conditions such as maternal undernutrition during pregnancy and lactation posit high risks of adult metabolic diseases. High fructose intake causes insulin resistance and liver inflammation contributing to metabolic diseases. However, food-based preventive measure for these metabolic diseases in the offspring is under-researched. This study aims to investigate the effect of maternal broccoli powder (BP) intake during lactation on insulin resistance and liver inflammation in high-fructose-diet-fed adult male offspring exposed to maternal protein restriction. Pregnant Wistar rats are provided normal protein (NP) or low protein (LP) diets and 0% or 0.74% BP-containing NP diets and 0% or 0.74% BP-containing LP diet during lactation. At weaning, offspring receiving water (W) or 10% fructose solution (Fr) are assigned into six groups: NP/NP/W, NP/NP/Fr, NP/NPBP/Fr, LP/LP/W, LP/LP/Fr, and LP/LPBP/Fr. At week 13, plasma insulin, macrophage infiltration, activated protein kinase (AMPK) and mechanistic target of rapamycin (mTOR) phosphorylation, and autophagy flux markers are examined. LP/LPBP/Fr shows lower insulin levels and Homeostatic model assessment for insulin resistance (HOMA-IR) values than LP/LP/Fr. Liver macrophage infiltration are decreased in LP/LPBP/Fr. LP/LPBP/Fr exhibits upregulated AMPK phosphorylation, downregulated mTOR phosphorylation, and increased Microtubule-associated protein1A/1B-light chain 3B-II (LC3B-II) levels. Maternal BP intake during lactation ameliorates insulin resistance and inflammation in the livers of adult offspring on a high-fructose diet from LP mothers.

The mTOR Pathway: A Common Link Between Alzheimer’s Disease and Down Syndrome

Down syndrome (DS) is a chromosomal condition that causes many systemic dysregulations, leading to several possible age-related diseases including Alzheimer’s disease (AD). This may be due to the triplication of the Amyloid precursor protein (APP) gene or other alterations in mechanistic pathways, such as the mTOR pathway. Impairments to upstream regulators of mTOR, such as insulin, PI3K/AKT, AMPK, and amino acid signaling, have been linked to amyloid beta plaques (Aβ) and neurofibrillary tangles (NFT), the most common AD pathologies. However, the mechanisms involved in the progression of pathology in human DS-related AD (DS-AD) are not fully investigated to date. Recent advancements in omics platforms are uncovering new insights into neurodegeneration. Genomics, spatial transcriptomics, proteomics, and metabolomics are novel methodologies that provide more data in greater detail than ever before; however, these methods have not been used to analyze the mTOR pathways in connection to DS-AD. Using these new techniques can unveil unexpected insights into pathological cellular mechanisms through an unbiased approach.

Potential liquid biopsy markers of exosomal microRNAs in renal interstitial fibrosis blood and urine

ABSTRACT Objective: To explore more and better liquid biopsy markers of exosomal microRNAs (exo-miRNAs) in renal interstitial fibrosis (RIF) and to preliminary investigate the biological functions and signaling pathways involved in these markers. MATERIALS AND METHODS: High-throughput miRNA sequencing was performed on blood and urine exo-miRNAs from three RIF patients and three healthy volunteers, and differential expression analysis and bioinformatic processing were performed. Results: There were 13 differentially expressed exo-miRNA (DEexo-miRNA) between RIF and healthy blood, and 20 DEexo-miRNAs in urine. These were various DEexo-miRNAs in different specimens, and the former included PC-3p-213532_58, hsa-miR-338-5p_R-1, PC-5p-34127_410, pal-miR-9993a-3p_L+2R-1, and hsa-miR-26a-1-3p with intermediate expression levels; the latter involved hsa-miR-126-3p, hsa-miR-217-5p, hsa-miR-199b-3p_R-1, mmu-miR-5106_R-4_1ss1AG, and PC-5p-39041_356, and others, while hsa-miR-378a-3p, hsa-miR-143-3p_R+1, hsa-miR-183-5p, hsa-miR-126-3p, hsa-miR-155-5p_R-1, mmu-miR-5106_R-4_1ss1AG, hsa-miR-126-5p, and hsa-miR-199b-3p_R-1 had high expression levels. Bioinformatics analysis of the up-regulated DEmiRNA with high expression in urine showed that there are 291 target corresponding mRNAs for six of eight DEexo-miRNAs, with mmu-miR-5106_R-4_1ss1AG and hsa-miR-199b-3p_R-1 having no target gene found in TargetScan and miRanda. GO annotation revealed that GO:0005515 (protein binding) had the lowest P value, involving the most genes. KEGG analysis revealed that the signaling included the mTOR signaling pathway, autophagy - animal, etc., and hsa05200 (pathways in cancer) had a lower P value, involving the most genes. Conclusions: Urine is a better sample for RIF exo-miRNA detection than blood. Urinary exosomal hsa-miR-143-3p_R+1, hsa-miR-183-5p, hsa-miR-126-3p, mmu-miR-5106_R-4_1ss1AG, hsa-miR-126-5p, and hsa-miR-199b-3p_R-1 are novel liquid biopsy markers for RIF. These DEexo-miRNA may be associated with the occurrence and development of RIF and may participate in specific biological processes by regulating the expression of their target mRNA. Further research may require exploring the specific functions and mechanisms of these miRNAs in RIF, as well as whether they can serve as diagnostic or therapeutic targets for RIF.

SMAC mimetic BV6 acts in synergy with mTOR inhibitor to increase cisplatin sensitivity in ovarian cancer.

The objective of this study is to observe the antitumor efficacy of the second mitochondria-derived activator of caspases (SMAC) mimetic bivalent smac mimetic (BV6) in combination with target of rapamycin (mTOR) inhibitor on DDP (cisplatin) sensitivity. Ovarian cancer cells were exposed to cisplatin, BV6, DDP + BV6, and DDP + BV6 + mTOR inhibitor Rapamycin. Using proteomics and bioinformatics, protein expression profiles in ovarian cancer were determined. Bagg Albino color nude mice were treated with DDP or BV6 alone or in combination, or BV6 + DDP + Rapamycin. The effects of different treatments on ovarian cancer cells and tumor growth were evaluated in vivo and in vitro. Proteomics and bioinformatics analysis revealed significant changes of protein kinase (AKT)/mTOR pathway. Consistently, western blot data indicated that AKT/mTOR axis was gradually activated in BV6-treated ovarian cancer cells and attenuated the cytotoxic effect of BV6. Functional assays showed that DDP or BV6 treatment alone significantly enhanced the sensitivity and inhibited the migration of ovarian cancer cells, but without any synergistic effects. In addition, combination with BV6 and mTOR inhibitor Rapamycin significantly decreased cell viability and inhibited migration of ovarian cancer cells exposed to DDP. Consistently, the xenograft model showed that co-treatment with Rapamycin with BV6 had significantly suppressed tumor growth and metastasis. Our study demonstrated that SMAC analogue BV6 exhibits a strong anticancer effect on ovarian cancer in vitro and in vivo. Combination with Rapamycin overcomes the activation of mTOR pathway by BV6 and increases the chemosensitivity to DDP. These data suggest a potential application of triple combination with DDP + BV6 + Rapamycin in clinical management of ovarian cancer.