TOR Pathway Research Articles

An mTOR inhibitor discovery system using drug-sensitized yeast.

Inhibition of the target of rapamycin (TOR/mTOR) protein kinase by the drug rapamycin extends lifespan and health span across diverse species. However, rapamycin has potential off-target and side effects that warrant the discovery of additional TOR inhibitors. TOR was initially discovered in Saccharomyces cerevisiae (yeast) which contains two TOR paralogs, TOR1 and TOR2. Yeast lacking functional Tor1 are viable but are hypersensitive to growth inhibition by TORC1 inhibitors, which is a property of yeast that can be exploited to identify TOR inhibitors. Additionally, yeast lacking FK506-sensitive proline rotamase (FPR1) or containing a tor1-1 allele (a mutation in the Fpr1-rapamycin binding domain of Tor1) are robustly and selectively resistant to rapamycin and analogs that allosterically inhibit TOR activity via an FPR1-dependent mechanism. To facilitate the identification of TOR inhibitors, we generated a panel of yeast strains with mutations in TOR pathway genes combined with the removal of 12 additional genes involved in drug efflux. This creates a drug-sensitive strain background that can sensitively and effectively identify TOR inhibitors. In a wild-type yeast strain background, 25µM of Torin1 and 100µM of GSK2126458 (omipalisib) are necessary to observe TOR1-dependent growth inhibition by these known TOR inhibitors. In contrast, 100nM Torin1 and 500nM GSK2126458 (omipalisib) are sufficient to identify TOR1-dependent growth inhibition in the drug-sensitized background. This represents a 200-fold and 250-fold increase in detection sensitivity for Torin1 and GSK2126458, respectively. Additionally, for the TOR inhibitor AZD8055, the drug-sensitive system resolves that the compound results in TOR1-dependent growth sensitivity at 100µM, whereas no growth inhibition is observed in a wild-type yeast strain background. Our platform also identifies the caffeine analog aminophylline as a TOR1-dependent growth inhibitor via selective tor1 growth sensitivity. We also tested nebivolol, isoliquiritigenin, canagliflozin, withaferin A, ganoderic acid A, and taurine and found no evidence for TOR inhibition using our yeast growth-based model. Our results demonstrate that this system is highly effective at identifying compounds that inhibit the TOR pathway. It offers a rapid, cost-efficient, and sensitive tool for drug discovery, with the potential to expedite the identification of new TOR inhibitors that could serve as geroprotective and/or anti-cancer agents.

Deciphering S1P downregulation and sphingolipid homeostasis disruption in fungal keratitis via multi-omics and MALDI-MSI analysis.

The absence of effective treatment strategies in Fungal Keratitis (FK) emphasizes the critical need to understand the pathogenic mechanisms to enhance therapeutic outcomes. Sphingolipids have been proved to play a pivotal role in the pathogenesis of fungal infections, but the specific alteration in sphingolipids and regulatory pathways remain elusive. Our aim is to gain insight into the pathophysiological mechanisms of sphingolipid homeostasis in FK through multi-omics analysis. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) was performed in FK patients and mouse model. Furthermore, time-course RNA-seq was performed and Weighted gene co-expression network analysis (WGCNA) was used to reveal the driver genes in FK. We further investigated the effect of FTY-720, a mimetic of sphingosine 1-phosphate (S1P), on the progression of FK. MALDI-MSI analysis of FK patients revealed a downregulation of sphingolipids, with sphingolipid metabolism identified as the most prominently enriched pathway. These alterations were validated in mouse model, in which S1P, ceramide, ceramide 1-phosphate and sphingomyelin were found to be downregulated. Time-course transcriptomic analysis suggests that degradation of sphingolipids by specific enzymes drives the progression of FK, involving phospholipid degradation, downregulation of TOR pathway, and activation of innate immune response. Consequently, epithelial cell function was inhibited and cell death increased. Importantly, restoring sphingolipid homeostasis by FTY-720 reversed the level of S1P and relieved the progression of FK. In summary, this study reveals that disruption of sphingolipid homeostasis promotes disease progression in FK. Furthermore, restoring sphingolipid homeostasis emerges as a promising strategy to mitigate the progression of FK.

OsFKBP12 transduces the sucrose signal from OsNIN8 to the OsTOR pathway in a loosely binding manner for cell division.

Previously, OsNIN8 initiated a sucrose signal for cell division in radicle and seed development in rice. Here, a set of genes was induced in starved sprouts after sucrose treatment, and 14 genes were screened between ZH11 and nin8 as reporters of sucrose signal. Expressions of reporter depended on levels of OsNIN8 in overexpression and RNAi lines. Further, OsNIN8 interacted with OsFKBP12, a regulator of TOR signal for cell division, and OsFKBP12 interacted with OsTOR (OsTORKD). However, interactions of OsFKBP12 with OsNIN8 or OsTORKD were a loose binding depending on the hydrophobicity of OsFKBP12 C-terminus in Y2H. In addition, OsFKBP12 associating with OsNIN8 was endothermic but with OsNIN8m was exothermic. Knockout OsFKBP12 reappeared nin8 phenotypes and the complementation of the knockout with C-termini of OsFKBP12 worsened the phenotypes. Treatment with TOR inhibitors caused short radicle and OsTOR RNAi repeated low seed-setting of the phenotypes. So, OsFKBP12 transduced sucrose signal from OsNIN8 to the TOR pathway.

Cytochrome c levels link mitochondrial function to plant growth and stress responses through changes in SnRK1 pathway activity.

Energy is required for growth as well as for multiple cellular processes. During evolution, plants developed regulatory mechanisms to adapt energy consumption to metabolic reserves and cellular needs. Reduced growth is often observed under stress, leading to a growth-stress trade-off that governs plant performance under different conditions. In this work, we report that plants with reduced levels of the mitochondrial respiratory chain component cytochrome c (CYTc), required for electron transport coupled to oxidative phosphorylation and ATP production, show impaired growth and increased global expression of stress-responsive genes, similar to those observed after inhibiting the respiratory chain or the mitochondrial ATP synthase. CYTc-deficient plants also show activation of the SnRK1 pathway, which regulates growth, metabolism, and stress responses under carbon starvation conditions, even though their carbohydrate levels are not significantly different from wild-type. Notably, loss-of-function of the gene encoding the SnRK1α1 subunit restores the growth of CYTc-deficient plants, as well as autophagy, free amino acid and TOR pathway activity levels, which are affected in these plants. Moreover, increasing CYTc levels decreases SnRK1 pathway activation, reflected in reduced SnRK1α1 phosphorylation, with no changes in total SnRK1α1 protein levels. Under stress imposed by mannitol, the growth of CYTc-deficient plants is relatively less affected than that of wild-type plants, which is also related to the activation of the SnRK1 pathway. Our results indicate that SnRK1 function is affected by CYTc levels, thus providing a molecular link between mitochondrial function and plant growth under normal and stress conditions.

F56F11.4 Extends Healthspan by Regulation of TOR-Related Pathway in C. elegans

Aging has emerged as a significant issue in human society, warranting intensified research efforts. Despite this, there is a noticeable dearth of research on genetic factors that could potentially extend lifespan. The genetic role of TOR/let-363 and its interactome was delved into in the model organism C. elegans. Utilizing RNA interference (RNAi), the impact of predicted interactors within the TOR pathway were explored on aging, with a particular emphasis on healthspan. This was achieved through comprehensive lifespan and pumping rate assays. Of the 20 genes examined, only the knockdown of F56F11.4 resulted in a reduction in both lifespan and early-stage pumping rate. To elucidate the interplay between F56F11.4 and TOR/let-363, double knockdown and RT-qPCR assays were employed to reveal their intricate regulatory roles in the aging process. Additionally, the conserved nature of F56F11.4 and let-363 across five well-studied organisms (C. elegans, H. sapiens, M. musculus, R. norvegicus, and D. melanogaster) was identified using bioinformatics analyses. The discovery of the influence of F56F11.4 on healthspan through the modulation of TOR/let-363 signaling suggests a promising target for the development of novel therapeutic strategies aimed at enhancing healthy aging.

Validation of metaxin-2 deficient C. elegans as a model for MandibuloAcral Dysplasia associated to mtx-2 (MADaM) syndrome

MandibuloAcral Dysplasia associated to MTX2 gene (MADaM) is a recently described progeroid syndrome (accelerated aging disease) whose clinical manifestations include skin abnormalities, growth retardation, and cardiovascular diseases. We previously proposed that mtx-2-deficient C. elegans could be used as a model for MADaM and to support this, we present here our comprehensive phenotypic characterization of these worms using atomic force microscopy (AFM), transcriptomic, and oxygen consumption rate analyses. AFM analysis showed that young mtx-2-less worms had a significantly rougher, less elastic cuticle which becomes significantly rougher and less elastic as they age, and abnormal mitochondrial morphology. mtx-2 C. elegans displayed slightly delayed development, decreased pharyngeal pumping, significantly reduced mitochondrial respiratory capacities, and transcriptomic analysis identified perturbations in the aging, TOR, and WNT-signaling pathways. The phenotypic characteristics of mtx-2 worms shown here are analogous to many of the human clinical presentations of MADaM and we believe this validates their use as a model which will allow us to uncover the molecular details of the disease and develop new therapeutics and treatments.

The Punicalagin Compound Mitigates Bronchial Epithelial Cell Senescence Induced by Cigarette Smoke Extract through the PAR2/mTOR Pathway.

Tobacco smoke is an important inducer of airway epithelial cell aging. Punicalagin(PCG) is a natural anti-aging compound. The effect of PCG on tobacco smoke-induced airway epithelial cell senescence is unknown. Our study investigated whether PCG can treat the human bronchial epithelial cell line (BEAS-2B) aging by inhibiting the protease-activated receptor 2 (PAR2)/m- TOR pathway. Bioinformatics techniques were used to analyze the potential biological functions of PAR2. Molecular dynamics evaluated the binding ability of PCG and PAR2. The CCK8 assay was used to detect the cytotoxicity of CSE and PCG. The activity of the PAR2/mTOR pathway and the expression of the characteristic aging markers p16, p21, and SIRT1 are detected by qRT-PCR and Western blotting. Cell senescence was observed by Senescence-associated β-galactosidase (SA-β-gal) staining. The senescence-associated secretory phenotype (SASP): concentrations of interleukin IL-6, IL-8, and TNF- α were detected by ELISA. The GSE57148 bioinformatics analysis dataset showed that PAR2 regulates lung senescence through the mTOR signaling pathway. Molecular dynamics results found that PCG and PAR2 had a strong and stable binding force. CSE induces BEAS-2B cell senescence and activates the PAR2/mTOR pathway. Inhibition of PAR2 mitigated the senescence changes. In addition, PCG's pretreatment can significantly alleviate CSE-induced BEAS-2B cell senescence while inhibiting the PAR2/mTOR pathway. PCG has a therapeutic effect on the senescence of airway epithelial cells.

AMPK activation by dietary acadesine improves fillet texture in large yellow croaker (Larimichthys crocea) fed a linseed oil-based diet

The impacts of AMP-activated protein kinase (AMPK) activation by dietary acadesine (AICAR) on growth performance and muscle quality of large yellow croaker (Larimichthys crocea) fed a linseed oil-based diet (LO) were investigated by a 56-day feeding trial in this study. Fish (initial weight: 92.24 ± 0.30 g) were fed fish oil-based diet (FO), LO or LA (LO diet supplemented with 150 mg kg−1 AICAR) with three replicates of each treatment. Results indicated that weight gain rate and specific growth rate (SGR) were significantly decreased in the LO and LA groups than those of the fish fed FO, while the opposite was true for the feed conversion ratio. Moreover, the SGR of fish fed LA was significantly lower than that of the LO group. Dietary linseed oil and AICAR significantly lowered muscle eicosapentaenoic acid and docosahexaenoic acid contents. Furthermore, the LO group showed significantly lower fillet hardness, chewiness and pH, while higher lipid loss than those of the FO group. However, the FO and LA groups had similar fillet hardness, lipid loss and pH. Compared with the fish fed FO and LA, the LO group possessed significantly lower p-AMPK protein level and p-AMPK/AMPK ratio in muscle. The phosphorylation of TOR protein and the ratios of p-TOR/TOR and p-S6/S6 were significantly decreased in muscle of the LA group than those of the fish fed FO and LO. Meanwhile, totally replacing fish oil with linseed oil inhibited the AMPK/SIRT1/PGC-1α pathway, changed myofibre characteristics (e.g., density, diameter and type of myofibre) and raised the expression of genes or activity of enzyme related to anaerobic metabolism in fish muscle. Dietary AICAR activated AMPK/SIRT1/PGC-1α pathway, restored myofibre characteristics and promoted aerobic metabolism-related genes expression in muscle to some extent. In conclusion, total substitution of dietary fish oil with linseed oil reduced growth, feed utilization and nutritive value of large yellow croaker. And high level of dietary linseed oil induced softer texture and poorer liquid holding capacity (LHC) of muscle, which may be ascribed to the myofibre characteristics changed by linseed oil through depressing muscle AMPK/SIRT1/PGC-1α pathway. Inclusion of AICAR in a linseed oil-based diet improved fish muscle hardness and LHC, which may be relevant to the activated muscle AMPK/SIRT1/PGC-1α pathway and restored myofibre characteristics by AICAR. Notably, dietary AICAR could inhibit TOR pathway by activating AMPK in muscle, which may be responsible for the further decreased SGR of fish fed a linseed oil-based diet.

Insulin and IGF-1 extend the lifespan of Caenorhabditis elegans by inhibiting insulin/insulin-like signaling and mTOR signaling pathways: C. elegans - Focused cancer research

The mutations in Caenorhabditis elegans (C. elegans) that extend lifespan slow down aging by interfering with several signaling pathways, including the insulin/IGF-1 signaling (IIS) pathway, AMP-activated protein kinase (AMPK), and mechanistic target of rapamycin (mTOR). The tumor suppressor pRb (retinoblastoma protein) is believed to be involved in almost all human cancers. Lin-35, the C. elegans orthologue of the tumor suppressor pRb, was included in the study to explore the effects of insulin and IGF-1 because it has been linked to cancer-related pRb function in mammals and exhibits a tumor suppressor effect by inhibiting mTOR or IIS signaling. According to our results, IGF-1 or insulin increased the lifespan of lin-35 worms compared to N2 worms by increasing fertilization efficiency, also causing a significant increase in body size. It was concluded that the expression of daf-2 and rsks-1 decreased after insulin or IGF-1 administration, thus extending the lifespan of C. elegans lin-35 worms through both IIS and mTOR-dependent mechanisms. This suggests that it was mediated by the combined effect of the TOR and IIS pathways. These results, especially obtained in cancer-associated mutant lin-35 worms, will be useful in elucidating the C. elegans cancer model in the future.

Sirtulin-Ypk1 regulation axis governs the TOR signaling pathway and fungal pathogenicity in Cryptococcus neoformans.

Cryptococcus neoformans is an important opportunistic fungal pathogen in humans. While there are currently few effective antifungal treatments, the absence of novel molecular targets in fungal pathogenicity hinders the development of new drugs. There is increasing evidence that protein post-translational modifications (PTMs) can modulate the pathogenicity of fungi. In this study, we discovered that the pathogenicity of C. neoformans was significantly impacted by the dynamic acetylation changes of Ypk1, the immediate downstream target of the TOR complex. We discovered that Ypk1 is acetylated at lysines 315 and 502, both of which are within kinase functional domains. Deacetylation of Ypk1 is necessary for formation of the capsule structure, the response to the TOR pathway inhibitor rapamycin, nutrient utilization, and host infection. We also demonstrate that the sirtuin protein family is involved in the Ypk1 deacetylation mechanism. We anticipate that the sirtuin-Ypk1 regulation axis could be used as a potential target for the development of antifungal medications.

TIPRL, a Potential Double-edge Molecule to be Targeted and Re-targeted Toward Cancer.

The target of rapamycin (TOR) proteins exhibits phylogenetic conservation across various species, ranging from yeast to humans, and are classified as members of the phosphatidylinositol kinase (PIK)-related kinase family. Multiple serine/threonine (Ser/Thr) protein phosphatases (PP)2A, PP4, and PP6, have been recognized as constituents of the TOR signaling pathway in mammalian cells. The protein known as TOR signaling pathway regulator-like (TIPRL) functions as a regulatory agent by impeding the activity of the catalytic subunits of PP2A. Various cellular contexts have been postulated for TIPRL, encompassing the regulation of mechanistic target of rapamycin (mTOR) signaling, inhibition of apoptosis and biogenesis, and recycling of PP2A. According to reports, there has been an observed increase in TIPRL levels in several types of carcinomas, such as non-small-cell lung carcinoma (NSCLC) and hepatocellular carcinomas (HCC). This review aims to comprehensively examine the significance of the Tor pathway in regulating apoptosis and proliferation of cancer cells, with a specific focus on the role of TOR signaling and TIPRL in cancer.

Dietary fishmeal replacement by Clostridium autoethanogenum protein meal influences the nutritional and sensory quality of turbot (Scophthalmus maximus) via the TOR/AAR/AMPK pathways

Clostridium autoethanogenum protein (CAP) is a promising protein source for aquaculture; however, how CAP influences fish quality is worth extensive research. We randomly allocated 630 turbot with initial body weights of about 180 g into 6 groups, with fishmeal-based control diet or diet with CAP replacing 15% (CAP15), 30% (CAP30), 45% (CAP45), 60% (CAP60), or 75% (CAP75) of fishmeal protein. After a 70-d feeding trial, the fillet yield (P = 0.015) and content of protein (P = 0.017), collagen (P < 0.001), hydroxyproline (P < 0.001), C20:5n-3 (P = 0.007), and ∑n-3/∑n-6 polyunsaturated fatty acids ratio (P < 0.001) in turbot muscle was found to decrease linearly with increasing CAP. However, turbot fed CAP15 diet maintained these parameters (P > 0.05). By contrast, the muscle hardness increased linearly with increasing CAP (P = 0.004), accompanied by linear reduction of muscle fiber area (P = 0.003) and expression of myogenesis-related genes, including cathepsin D (ctsdP < 0.001) and muscle ring finger protein 1 (murf 1, P < 0.001). Phosphorylation of protein kinase B (Akt, P < 0.001), target of rapamycin (TOR, P = 0.001), eukaryotic initiation factor 4E-binding protein 1 (4E-BP1, P < 0.001), and ribosomal protein S6 (S6, P < 0.001) decreased linearly; however, phosphorylation of AMP-activated protein kinase (AMPK, P < 0.001), eukaryotic initiation factor 2α (eIF2α, P < 0.001), and the abundance of activating transcription factor 4 (ATF4, P < 0.001) increased with increasing CAP, suggesting that the TOR signaling pathway was inhibited, and the amino acid response (AAR) and AMPK pathways were activated. Additionally, expression of genes related to protein degradation, including myogenic factor 5 (myf 5, P < 0.001), myogenic differentiation (myod, P < 0.001), paired box 7 (pax 7, P < 0.001), and ctsd (P < 0.001), decreased linearly with increasing CAP. In conclusion, CAP could be used to replace up to 15% of fishmeal without negatively impacting turbot quality. However, higher levels of CAP decreased fillet yield, muscle protein content, and muscle fiber diameter while increasing muscle hardness, which could be attributed to the inhibition of the TOR pathway and activation of the AAR and AMPK pathways.

Effects of supplementing coated methionine in a high plant-protein diet on growth, antioxidant capacity, digestive enzymes activity and expression of TOR signaling pathway associated genes in gibel carp, Carassius auratus gibelio.

This study explored the impacts of supplementation of different levels of coated methionine (Met) in a high-plant protein diet on growth, blood biochemistry, antioxidant capacity, digestive enzymes activity and expression of genes related to TOR signaling pathway in gibel carp (Carassius auratus gibeilo). A high-plant protein diet was formulated and used as a basal diet and supplemented with five different levels of coated Met at 0.15, 0.30, 0.45, 0.60 and 0.75%, corresponding to final analyzed Met levels of 0.34, 0.49, 0.64, 0.76, 0.92 and 1.06%. Three replicate groups of fish (initial mean weight, 11.37 ± 0.02g) (20 fish per replicate) were fed the test diets over a 10-week feeding period. The results indicated that with the increase of coated Met level, the final weight, weight gain (WG) and specific growth rate initially boosted and then suppressed, peaking at 0.76% Met level (P< 0.05). Increasing dietary Met level led to significantly increased muscle crude protein content (P< 0.05) and reduced serum alanine aminotransferase activity (P< 0.05). Using appropriate dietary Met level led to reduced malondialdehyde concentration in hepatopancreas (P< 0.05), improved superoxide dismutase activity (P< 0.05), and enhanced intestinal amylase and protease activities (P< 0.05). The expression levels of genes associated with muscle protein synthesis such as insulin-like growth factor-1, protein kinase B, target of rapamycin and eukaryotic initiation factor 4E binding protein-1 mRNA were significantly regulated, peaking at Met level of 0.76% (P< 0.05). In conclusion, supplementing optimal level of coated Met improved on fish growth, antioxidant capacity, and the expression of TOR pathway related genes in muscle. The optimal dietary Met level was determined to be 0.71% of the diet based on quadratic regression analysis of WG.

Design of PI3K-mTOR Dual Inhibitors for Ovarian Cancer: Are We on the Right Track?

Ovarian cancer is one of the most familiar kinds of gynecological cancer seen in women. Though it is not as familiar as breast cancer, the survival rate for ovarian cancer is very low when compared with breast cancer. Even after being one among the familiar types, to date, there are no proper treatments available for ovarian cancer. All the treatments that are present currently show a high rate of recurrence after the treatment. Therefore, treating this silent killer from the roots is the need of the hour. PI3K/AKT/m- TOR pathway is one of the pathways that get altered during ovarian cancer. Studies are already going on for the inhibition of PI3K and mTOR separately. Efforts have been made to inhibit either PI3K or mTOR separately earlier. However, due to its side effects and resistance to the treatments available, current studies are based on the inhibition of PI3K and mTOR together. Inhibition of PI3K and mTOR simultaneously reduces the chances of negative feedback, thus decreasing the toxicity. This review contains the evolution of PI3K and mTOR drugs that are approved by the FDA and are in the trials for different cancer types, including Ovarian cancer. In this article, how a molecular targeted therapy can be made successful and free from toxicity for treating ovarian cancer is discussed. Therefore, this review paves the way for finding an effective scaffold rather than the clinical part. The scaffold thus selected can be further modified and synthesized in the future as dual PI3K/mTOR inhibitors specifically for OC.

Suitable Cottonseed Protein Concentrate Supplementation in Common Carp (Cyprinus carpio) Serves as an Effective Strategy for Fish Meal Sparing Based on Improvement in Intestinal Antioxidant Capacity, Barrier and Microbiota Composition.

The application of cottonseed protein concentrate (CPC) is an effective strategy to moderate the shortage of fish meal (FM) for the aquafeed industry. However, little attention has been paid to the effects of replacing fishmeal with CPC on cyprinid fish. This study used common carp (Cyprinus carpio) as the biological model and assessed the potential of applying CPC as a substitute for fishmeal in the diet of common carp. The proportion of fish meal substituted with CPC in the six diets was 0% (CPC0), 25% (CPC25), 50% (CPC50), 75% (CPC75), and 100% (CPC100). Each diet was fed to three replicate groups of common carp (4.17 ± 0.02 g) for 56 days. Results revealed that the CPC50 group significantly increased the growth indexes via up-regulating the genes of the GH/IGF axis and the TOR pathway. The intestinal digestive ability was also elevated in the CPC50 group via markedly increasing intestinal villus height, protease and lipase activities in the whole intestine, and the amylase activity of the foregut and midgut. The CPC50 group captured significantly higher activities and gene expressions of antioxidant enzymes and lower malonaldehyde contents via evoking the Nrf2/Keap1 signal pathway. The CPC50 group enhance the intestinal mechanical barrier via up-regulating the gene expressions of tight junction proteins and heighten the intestinal biological barrier by increasing the probiotics (Lactococcus) and decreasing the harmful bacteria (Enterococcus). But excessive substitution levels (75% and 100%) would compromise growth performance, intestinal antioxidant capacity, and immune function. The optimum substitution level was estimated to be 46.47%, 47.72%, and 46.43% using broken-line regression analyses based on mass gain rate, protein efficiency ratio, and feed conversion rate. Overall, the fishmeal in common carp feed could be substituted up to 50% by CPC without negative influence on growth, feed utilization, and or intestinal health.

Evaluation of spray-dried blood meal for application in commercial-like feed for juvenile swimming crab (Portunus trituberculatus)

The objective of the present study was to evaluate the effects of replacing fish meal (FM) with spray-dried blood meal (SDBM) on growth performance, antioxidant capacity, digestive enzyme activity, tissue Fe content, gene expression related to inflammation, and the TOR pathway in juvenile swimming crab (Portunus trituberculatus). Four isonitrogenous (47.0% protein) and isolipidic (9.0% lipid) diets were formulated to contain different levels of SDBM, with FM replaced by SDBM at 0, 10%, 20%, and 30%, respectively. The results showed that crabs fed diet SDBM30 exhibited the lowest survival among all treatments, and crabs fed the SDBM20 diet had the highest final weight, percent weight gain, specific growth rate and feed efficiency among all treatments. In addition, the protein content of the crab hepatopancreas decreased significantly and the lipid content increased significantly as SDBM replaced FM in the diets. Compared to other diets, crabs fed the SDBM30 diet had the lowest activities of antioxidant enzymes and the highest concentration of malondialdehyde in the hepatopancreas and hemolymph. The Fe contents in hepatopancreas and gill increased significantly, and the color of the hepatopancreas got darker as replacement of FM with SDBM increased from 0% to 30%. The expression of genes related to TOR (tor, akt, s6, and s6k1) and antioxidant (gpx) pathways in the hepatopancreas were down-regulated significantly with increased replacement of dietary FM with SDBM. In contrast, expression levels of genes related to protein translation (4ebp1, eif4e1a, eif4e2, and eif4e3) and inflammation pathways (myd88, traf6, relish, and irak4) were up-regulated significantly in crabs fed diets with SDBM supplementation. In summary, replacing FM with less than 20% of SDBM can promote the growth, but can darken the hepatopancreas, negatively affect antioxidant capacity, and induce an inflammatory response.

TOR regulates variability of protein synthesis rates

Cellular processes are subject to inherent variability, but the extent to which cells can regulate this variability has received little investigation. Here, we explore the characteristics of the rate of cellular protein synthesis in single cells of the eukaryote fission yeast. Strikingly, this rate is highly variable despite protein synthesis being dependent on hundreds of reactions which might be expected to average out at the overall cellular level. The rate is variable over short time scales, and exhibits homoeostatic behaviour at the population level. Cells can regulate the level of variability through processes involving the TOR pathway, suggesting there is an optimal level of variability conferring a selective advantage. While this could be an example of bet-hedging, but we propose an alternative explanation: regulated ‘loose’ control of complex processes of overall cellular metabolism such as protein synthesis, may lead to this variability. This could ensure cells are fluid in control and agile in response to changing conditions, and may constitute a novel organisational principle of complex metabolic cellular systems.

Changes in growth performance, gene expressions related to protein absorption and hepatic metabolism of triploid crucian carp (Carassius carassius triploid) caused by dietary protein level

The aim of this study was to investigate the effects of dietary protein inclusion level on growth, protein absorption pathway and liver metabolism-related pathway in triploid crucian carp (Carassius carassius triploid). Six experimental diets with different protein levels (26%, 29%, 32%, 35%, 38%, 41%) were formulated. A total of 540 fish with an initial weight of 11.79 ± 0.09 g were randomly assigned to 18 cages and six treatments with three replicates of 30 fish each for 8 weeks feeding. The results showed that the specific growth rate (SGR) and feed efficiency (FE) of triploid crucian carp were highly positively correlated with protein content (p < 0.05). The broken-line regression analysis showed that the optimal dietary inclusion level was determined to be 39% considering SGR of triploid crucian carp. In addition, genes related to protein absorption were influenced by the protein inclusion level of dietary. The mRNA expressions of pept1, lat2, cdx2, jak2 and sp1 exhibited similar change tendency and promoted to highest expression at 41% groups, 32% followed. Furthermore, the expressions of hepatic metabolism genes tor, s6k1, igf1, ampd1 and adsl reached peak at 38% dietary protein inclusion level, while 4e-bp2 gene showed the highest expression levels at 41% dietary protein inclusion level. Finally, igf1 expression in liver and cdx2 in intestine showed high pearson coefficients with dietary protein level suggesting their expressions were significant positively regulated by dietary protein level. In conclusion, this study evidenced the influence of dietary protein level on growth, protein absorption pathway, TOR and IGF pathways of hepatic metabolism related in triploid crucian carp. 39% dietary protein inclusion level would be optimum level of triploid crucian carp dietary, which will lead to a better growth and high feed efficiency of triploid crucian carp.

LKB1 regulates autophagy through AMPK/TOR signaling pathway to alleviate the damage caused by Vibrio alginolyticus infection

LKB1 (liver kinase B1) is a key upstream kinase of AMPK and plays an important role in various cellular activities. While the function and mechanism of LKB1 have been widely reported in the study of tumor, there are few reports on its role in bacterial infectious diseases, especially in shrimp. In the present study, molecular characterization revealed that LvLKB1 has an open reading frame (ORF) of 1266 bp encoding 421 amino acids with a molecular weight of about 48 KDa, including the kinase region, N-terminal regulatory domain and C-terminal regulatory domain. LvLKB1 in hepatopancreas and hemocytes was significantly upregulated after infection with Vibrio alginolyticus (V. alginolyticus). After silencing LvLKB1 gene in Litopenaeus vannamei (L. vannamei) and artificially infecting V. alginolyticus, the survival rate of L. vannamei was significantly decreased. Subsequently, it was found that the expression of inflammatory factors in hepatopancreas and hemocytes of shrimp was up-regulated, and the expression of lipid oxidation factors was decreased after silencing LKB1, leading to the phenomenon of lipid accumulation in hepatopancreas. In order to explore the mechanism, autophagy levels of shrimp were detected after silencing LKB1, which showed that autophagy levels in hepatopancreas and hemocytes were significantly reduced. Further studies conclusively showed that silencing LvLKB1 inhibited AMPK phosphorylation induced by V. alginolyticus infection, thereby activating TOR pathway and inhibiting autophagy in shrimp. These results indicate that LvLKB1 regulates autophagy through AMPK/TOR signaling pathway to alleviate the damage caused by V. alginolyticus infection.

Replacement of fish meal with cottonseed protein concentrate in Chinese mitten crab (Eriocheir sinensis): Nutrient digestibility, growth performance, free amino acid profile, and expression of genes related to nutrient metabolism

This study aimed to investigate the application of cottonseed protein concentrate (CPC) in Chinese mitten crabs (Eriocheir sinensis). First, the apparent digestibility coefficient (ADC) of CPC, fish meal and soybean meal were compared in crabs (21.72 ± 0.33 g). The protein ADC of CPC was 90.42%, which was significantly higher than that of soybean meal (83.16%) (P < 0.05). The ADC of Phe, Cys and Glu of CPC were significantly higher than those of fish meal, while the ADC of Ile, Leu, Lys, Met, Thr and Ala of CPC were significantly lower (P < 0.05). Second, we investigated the effects of fish meal substitution by CPC on growth performance, free amino acid profile, and expression of genes related to nutrient metabolism in crabs. Six diets were formulated by replacing 0%, 15%, 30%, 45%, 60% and 75% fish meal with CPC, namely FM, CPC15, CPC30, CPC45, CPC60, and CPC75. A total of 630 crabs (1.68 ± 0.00 g) were randomly divided into 18 tanks (3 tanks per group) and fed 3 times daily for 9 weeks. Results showed that CPC75 group significantly reduced growth performance, feed conversion efficiency, and free Ile, Leu, Lys, Met, and Thr contents in muscle (P < 0.05). The contents of free amino acids (Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Val, Ala, Cys, Glu, Gly, Ser and Tyr) in hepatopancreas decreased linearly with the increase of dietary CPC level (P < 0.05). The substitution of more than 45% fish meal with CPC significantly decreased the concentration of delicious amino acids (Ala, Glu and Gly) in hepatopancreas (P < 0.05), which might adversely affect crab flavor. The expression of genes related to antioxidant capacity, protein transport, TOR pathway and lipid metabolism was significantly downregulated by increasing dietary CPC level (P < 0.05). In conclusion, based on the quadratic regression analysis of FCR and PER, the optimal replacement levels of fish meal with CPC in crab diet containing 35% fish meal were 32.36% and 35.38%, respectively. It is recommended that Ile, Leu and Thr be supplemented in addition to Met and Lys in the application of CPC.