Search

MS-222 (tricaine methanesulfonate) and eugenol are the two frequently used fish anesthetics. This study intends to analyze the regulation of these anesthetics in Chinese sea bass, Lateolabrax maculatus, through transcriptomic analysis. L. maculatus were exposed to MS-222 or eugenol, and those without any treatments were regarded as controls. Gills and livers were extracted for transcriptomic analysis after recovery in fresh water for 6 h. Identified genes were assigned to NR, COG, SWISS, GO, and KEGG database for predicting gene functions. A FDR ≤ 0.05 and |log2(FC)| ≥ 1 were applied to determined differentially expressed gene (DEG). A total of 45,626 unigenes were annotated using at least one database. The eugenol-treated liver group presented less DEGs compared with that treated by MS-222. Both the MS-222- and eugenol-treated liver groups presented notable DEGs that participated in human disease and metabolism pathways. The eugenol group showed more pathways related to detoxification activity and xenobiotics biodegradation, and those from the MS-222 group were related to organismal system such as reproduction. By comparing gill and liver samples using the same drug, the enriched pathways were generally consistent among the three comparisons. In conclusion, eugenol and MS-222 could change the pathways related to metabolism and immunity in L. maculatus. MS-222 may trigger more damages on the fish liver and reproduction.

Harmful effects of water pollutants are myriad. Sulfide from water bodies affects the aquatic animals. Intestine barrier function serves as the front-line of animals defense. Our previous study confirmed the toxic effect of sulfide on intestine immune response of Litopenaeus vannamei, but the underlying mechanisms remained elusive. Therefore, in this study, we investigated the transcriptomic and microbiota responses of the L. vannamei intestine subjected to acute sulfide exposure. Sulfide decreased bacterial richness and altered the intestine microbiota composition. Specifically, sulfide increased the abundances of Bacteroidetes and Actinobacteria, but decreased the abundance of Proteobacteria. At the genus level, sulfide increased typical cellulolytic characteristics bacteria, such as Formosa, Sphingomonas, and Demequina. RNA-seq analysis identified differential expression of 1799 genes (701 up-regulated and 1098 down-regulated) were grouped into 267 pathways. The most enriched pathway 'amoebiasis' was related to the intestine mucus homeostasis. A number of immune-related genes associated with antimicrobial, antioxidant, pathogen attachment and recognition, and apoptosis processes in contrasting accessions; they were correlated with the abundance of intestine bacterial at the phylum level. This study provides an insight into the mechanisms associated with molecular and microbiota response and processes involved in adaptation strategies towards sulfide stress.

The purpose of this study was to evaluate the anaesthesia effects of eugenol and MS‐222 sedatives applied on juvenile Lateolabrax maculatus during simulated transport. In experiment 1, the juveniles were divided into two groups, with seven concentrations tested on each group (eugenol [4, 6, 8, 10, 12, 14 and 16 mg/L] and MS‐222 [20, 30, 40, 50, 60, 70 and 80 mg/L]). Induction and recovery times were recorded. The time for anaesthesia was shortened, and the time for complete recovery was prolonged as the anaesthetic concentration increased. The optimal transport concentration for each anaesthetic tested was 6 mg/L of eugenol and 30 mg/L for MS‐222. In experiment 2, the 5‐hr simulated transport test showed that the survival rate of L. maculatus juveniles with anaesthesia was 100%, and without anaesthesia, survival was 60%. After 24 hr of recovery following transport, the fish showed 100% survival for the group with added anaesthetic and 40% for the group without added anaesthetic. Compared to the non‐anaesthetized groups, the anaesthetized transport groups showed significant increases in the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (p

The shrimp intestine barrier serves as the first line of the host defense against pathogen infection. Lipopolysaccharide (LPS) is the cell wall component of gram-negative bacteria, which known as endotoxin and induce the intestine inflammation. In this study, the acute toxicity effects of LPS injection on the morphology, digestive enzymes and immunological responses of intestine from Litopenaeus vannamei was investigated. HE stain showed that LPS injection damaged the intestine connective and epithelium tissue. Specifically, a decrease in the activities of digestive enzymes including of amylase, lipase, trypsin and pepsin was observed. Moreover, LPS injection increased the content of oxidative stress parameters (O2- generation capacity, LPO, MDA and PC), and the expression of HIF-1α gene. Alternatively, the antibacterial activities (PO and T-NOS), and the expression of the antibacterial genes (proPO, ALF, Toll and Imd) and pathogen pattern recognition genes (LGBP and Lec) increased at first and then decreased. Significant variations in anitioxidant enzyme activity of T-AOC, and the expression of the anitioxidant-related genes (SOD, HSP70 and Trx). These results revealed that LPS injection harmed the intestine barrier of L. vannamei by damaging intestine mucosal structure, increasing oxidative stress, and suppressing the digestive and immune status.

The acute toxicity of nitrite on the mucosal morphology and oxidative stress response was investigated in the intestines of Litopenaeus vannamei after the exposure of 20 mg/L nitrite for 72 hr. The duration of nitrite stress influenced the shrimp survival, and the cumulative mortality rate was 36.7% at 72 hr. Hematoxylin and eosin (HE) stain showed that nitrite stress damaged the intestine connective and epithelium tissue. Specifically, nitrite stress increased the content of reactive oxygen species (ROS) production (O2- generation capacity), lipid peroxidation (LPO), and malondialdehyde (MDA). Meanwhile, the activities of antioxidant enzymes including superoxide dismutase (SOD) increased first, then catalase (CAT) and glutathione peroxidase (GPx) increased to the highest at 24 and 12 hr, respectively. The relative expression level of antioxidant genes including heat shock protein 70 (HSP70), ferritin, and thioredoxin (Trx) increased to the highest at 6, 12, and 48 hr respectively. After exposure to nitrite stress for 72 hr, the levels of antioxidant enzymes activity and genes expression all decreased. These results revealed that nitrite stress harmed the intestine barrier of L. vannamei by damaging the mucosal structure, inducing the oxidative stress, and confusing the antioxidant status.

A special denitrifying capsule that was designed according to the theory of maldistribution of DO (dissolved oxygen) for simultaneous nitrification and denitrification (SND) in an aerobic reactor was prepared by an encapsulation technique. The morphology of the denitrifying capsule and its SND performance in an aerobic reactor used for treating synthetic low C/N wastewater were systematically investigated. The spherical capsule comprised a porous membrane, denitrifying active sludge and semen litchi powder, which were cohered together by a physical method. Well-shaped pores were distributed in the membrane and the average pore diameter was approximately 0.25 µm. Circular pores were present in the cross section of the membrane, and the thickness of the membrane ranged from 95.0 µm to 120.0 µm. After 7 days of acclimation, the denitrifying capsule showed good SND performance in an aerobic reactor with a DO concentration of 5.09 mg/L. The NH4+-N concentration of 51.09 mg/L was removed completely after 8 h, the effluent dissolved inorganic nitrogen (DIN) concentration decreased to 6.11 mg/L, the SND ratio reached to 91.6% at 6 h, and finally decreased to 2.09 mg/L at 15 h and could also reach 98.91%. The influent of NH4+-N loading had little effect on the nitrogen removal performance. And decreased of air input induced lower hydraulic shearing force compared to reactors with higher DO had a little effect on the diffusion and mass transfer of NO3--N. With this core shell structure denitrifying capsule, it was easy to achieve SND without controlling the DO concentrations at low levels or using intermittent aeration.

The effects of sulfide stress on oxidative stress and immune response in intestine of Pacific white shrimp Litopenaeus vannamei were evaluated in the present study. Oxidative stress parameters, immune enzymes activity and immune gene mRNA expression level were detected in intestine of L. vannamei after the exposure of 5.0 mg/L sulfide stress 72 h. The duration of sulfide stress influenced the shrimp survival, and the cumulative mortality rate was 30.0% and 33.3% at 48 h and 72 h respectively. HE staining showed that sulfide stress caused the intestine tissue damage symptoms. Compared with the control group, after exposed to sulfide stress, the content of lipid peroxidation (LPO), malondialdehyde (MDA) and ROS production (·O2− generation capacity) increased. Total antioxidant capacity (T-AOC) activity increased at 6 h and decreased at 48 h. Superoxide dismutase (SOD) activity increased in the entire experiment. Inducible nitric oxide synthase (iNOS) activity and nitric oxide (NO) content increased to the highest at 6 h and 12 h respectively, and both decreased at 48 h. The relative mRNA expression level of heat shock protein 70 (HSP70) gene decreased at 6 h and increased to the highest at 48 h. The relative mRNA expression level of hypoxia inducible factor 1α (HIF-1α) gene increased at 12 h and decreased to a lower level at 72 h. The relative mRNA expression level of Toll and immune deficiency (Imd) gene increased to the highest at 12 h and 24 h respectively, and both decreased at 48 h. These results revealed that sulfide stress could induce oxidative stress and immune response via confusion of immune enzymes activity and gene expression level in intestine of L. vannamei.

A green porous composite was prepared using two kinds of natural polysaccharides, konjac glucomannan (KGM) and semen litchi (SL) in aqueous medium under mild conditions. The effect of mass ratios of KGM : SL on structures and denitrification was systematically investigated. The intermolecular hydrogen bonds and other synergistic interactions between KGM and SL gave the composite an amorphous phase and honeycomb network structure with a wide range of macropores. Its high porosity and biocompatibility meant the composite had short acclimation time. Nitrate removal rate could reach up to 98.8% on the first day and denitrification rate could reach up to 320.2 ± 5.82 mg N (L−1 d−1). In particular, the KGM : SL = 3 : 3 composite exhibited better mechanical property and much longer lifetime during the denitrification reaction. The porous composite can be used as an economical and effective carbon source for denitrification in low C/N wastewater.

A porous solid carbon source was prepared by semen litchi (SL), poly(vinyl alcohol) (PVA) and sodium alginate (SA) in aqueous. The effect of SL content on the structures and denitrification performance of the porous solid carbon source in simulated mariculture wastewater was investigated. The SL/PVA/SA beads showed a network structure with a wide range of macropores. Compared with blank beads, the SL/PVA/SA beads showed an increased rough surface and whole distribution on the surface with the increase of SL. In addition, SL/PVA/SA beads have more uniform pore size, but the porosity of SL/PVA/SA beads was decreased with the increase of SL. The porosity of the beads was 83.24%, 74.24%, 71.48% and 71.29% for blank beads and SL/PVA/SA beads contained 30%, 40% and 50% SL, when it was used as a solid carbon source for denitrification. Owing to their good porosity and biocompatibility, SL/PVA/SA beads had shorter acclimation time. Nitrate removal rate could reach up to 100% after two days of adaptation. After the exhaustion of carbon sources, nitrate removal rate less than 50% occurred at the 9th, 10th and 11th day for SL/PVA/SA beads that contained 30%, 40% and 50% SL, respectively. The beads that contained 50% SL exhibited longer lifetime during the denitrification reaction and denitrification rate could reach 243.5 ± 7.08 mg N (L d)

In the present study, the oxidative stress response in hepatopancreas of Marsupenaeus japonicus to desiccation stress and resubmersed in seawater were studied, such as respiratory burst, ROS production ( [Formula: see text] ), activities of antioxidant enzymes (CAT, GPx, SOD, POD and GST) and oxidative damage to lipid and protein (indexed by contents of MDA). The duration of desiccation significantly influenced shrimp survival, and the mortality rates were 37.5% and 87.5% after desiccation 5 h and 10 h, respectively. After desiccation stress 3 h, the respiratory burst, ROS production, and the activity of SOD and CAT were up-regulated significantly. The activity of GPx and POD, and the content of MDA decreased significantly at 0.5 h and 1 h, and then increased significantly at 3 h. But GST activity was no significant change after desiccation. During the resubmersion period, most of the antioxidant enzymes activities could recover to the control level at 24 h, but a small quantity of the oxidative stress still existed in tissues. HE staining showed that desiccation stress induced damage symptoms in hepatopancreas of M. japonicus. These results revealed that desiccation influenced the antioxidative status and caused oxidative stress and tissue damage via confusion of antioxidant enzymes in M. japonicus, but the oxidative stress could be eliminated within a certain range after the shrimps were resubmersed in seawater.

Vibrio parahaemolyticus is a virulent pathogen that affects shrimp aquaculture. Reactive oxygen species are produced by the immune system that defends the host against foreign microorganisms. In the present study, the oxidative stress response in hepatopancreas and gills of Penaeus monodon to V. parahaemolyticus challenge were studied, such as respiratory burst, ROS production (·O2(-) and ·OH), activities of antioxidant enzymes (CAT, GPx, SOD, POD and GST) and oxidative damage to lipid and protein (indexed by contents of MDA). Compared with the control group, after V. parahaemolyticus challenge, respiratory burst and ROS production were up-regulated significantly. GPx and POD activity increased significantly in hepatopancreas and gills of the shrimps at 12 h, but CAT activity decreased markedly at 12 h and 24 h. SOD and GST activity in hepatopancreas of the shrimps increased significantly at 1.5 h, but decreased markedly at 12 h-48 h. MDA content increased significantly after 6 h-24 h challenge. HE staining showed that V. parahaemolyticus challenge induced damage symptoms in hepatopancreas of P. monodon. Our study revealed that V. parahaemolyticus influenced the antioxidative status and caused oxidative stress and tissue damage via confusion of antioxidant enzymes in P. monodon.

The intestine barrier serves as the front-line defense in shrimp. Clostridium butyricum (CB) can produce butyric acid that provides energy for the intestine epithelial cells of the host. However, the effects of dietary CB on the intestine microbiome and the digestion and immunity of the host is not clear. In this study, we therefore investigated the composition and metabolic activity of the intestine microbiome, and digestive and immune-related gene expression in Litopenaeus vannamei fed with diets containing different levels of CB: basal diet (control), 2.5 × 109 CFU kg-1 diet (CB1), 5.0 × 109 CFU kg-1 diet (CB2), and 1.0 × 1010 CFU kg-1 diet (CB3) for 56 days. Dietary CB altered the composition of the intestine microbiome. Specifically, the dominant bacterial phylum Proteobacteria was enriched in the CB3 group and weakened in the CB1 and CB2 groups. The Bacteroidetes was enriched in the CB1 and CB2 groups and weakened in the CB3 group. The Firmicutes was enriched in all three CB groups. At the genus level, the potential pathogen (Desulfovibrio and Desulfobulbus) were weakened, and beneficial bacteria (Bacillus, Clostridium, Lachmoclostridium, Lachnospiraceae, and Lactobacillus) were enriched in response to dietary CB; these might contribute to the expression of the host digestive genes (α-amylase, lipase, trypsin, fatty acid-binding protein, and fatty acid synthase) and immune-related genes (prophenoloxidase, lipopolysaccharide and β-1,3-glucan binding protein, lysozyme, crustin, and superoxide dismutase). Additionally, CB enhanced the bacterial metabolism, especially that of carbohydrates, polymers, amino acids, carboxylic acids, and amines. These results revealed that dietary CB had a beneficial effect on the intestine health of L. vannamei by modulating the composition of the intestine microbiome, enhancing the microbial metabolism activity, and promoting the digestion and immunity of the host. The optimal dietary supplementation dosage was found to be 5.0 × 109 CFU kg-1 in the diet.

Sulfide is a harmful environmental pollutant that affects the survival and immunity of shrimps. The gill is important for shrimp respiratory and osmotic adjustment, the physiological and immune homeostasis of the organ can be influenced by sulfide. In this study, we investigated the acute toxicity of sulfide (5 mg/L) on the morphology, physiological and immune response in the gills of Litopenaeus vannamei. HE the contents of free amino acid including of Gly, Pro, Ser, Thr and Ala were decreased at 72 h. Respiratory metabolic enzymes, such as cytochrome c oxidase and succcinate dehydrogenase activity was decreased at 12 h-72 h, while fumarate reductase and lactate dehydrogenase activity kept a higher level at 12 h-72 h. Significant variations in the activities of immune enzymes (acid phosphatase, alkaline phosphatase, total antioxidant capacity and lysozyme). The expression of immune-related genes (heat shock protein 70, thioredoxin and caspase-3) was increased at first and then decreased, while hypoxia inducible factor 1α kept a higher level at 6 h-72 h. These results revealed that sulfide stress influenced the L. vannamei gills physiological and immune function by damaging histological structure, and confusing osmoregulation, respiratory metabolic and immune capacity.

Intestine barrier serves as the front-line of shrimp defense, which rely on its structural integrity, microbial composition, and mucus immune compounds. Mucins are the major organic components of the intestine mucus layer that contribute to the immunity of intestine mucus. In this study, we examined the histological structure, microbial composition, and mucin genes expression in the intestines of Litopenaeus vanmei under three different conditions: control, ammonia stress, and nitrite stress for 72 h. HE stain showed that ammonia and nitrite stress exposure both damaged the intestine mucosal tissue. High-throughput 16S rDNA sequencing revealed that two stresses exposure decreased the bacterial diversity, and altered the composition of intestine microbial. Specifically, the dominant bacterial phyla Bacteroidetes abundance was increased, while Proteobacteria and Planctomycetes were decreased; at the genus level, Formosa abundance was increased and Photobacterium was decreased, opportunistic pathogens including Nautella and Pseudoalteromonas was also increased. Intestine mucus immune genes including mucin-2 and mucin-19 were up-regulated, while mucin-1, mucin-5AC, and mucin-5B were down-regulated in two stress exposure groups. These results revealed that ammonia and nitrite stress harmed the intestine barrier function of L. vannamei by damaging the mucosal tissue, disrupting the composition of intestine microbial, and suppressing the immune function.

A new agricultural waste semen litchi was used as the sole carbon source to remove nitrate from high salinity wastewater in laboratory reactors. The main nutrient components, the content of heavy metals and the morphology of semen litchi were first studied. The results showed that semen litchi contained about 60% organic carbon and low levels of heavy metals. The milled semen litchi had lots of gap structures and abundant starch granules. Then the release velocity of the carbon source and the denitrification rate in high salinity wastewater were investigated. It was found that semen litchi could supply continuous organic carbon for denitrification. And the maximum TOC concentration could reach 137.29 mg l−1 on the 46th day. The nitrate removal rate and denitrification rate could reach 98.8–99.5% and 192 mg N (l−1 d−1), respectively. During the whole denitrification reaction, the nitrite concentration was lower than 0.01 mg l−1. A microbial community profile by Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) indicated that the denitrifying bacteria (Sphingomonas family and Rhodospirillum family) became enriched in the semen litchi sludge. Furthermore, salinity didn't have a negative effect on denitrification. Semen litchi could be used as an economical and effective carbon source for denitrification in high salinity wastewater.

In the present study, the effect of dietary supplementation of poly-β-hydroxybutyrate (PHB) on the growth performance, intestinal digestive and immune function, intestinal short-chain fatty acids (SCFA) content and body composition of Pacific white shrimp Litopenaeus vannamei (Boone, 1931) was evaluated. The shrimp was fed for 35 days with four different diets: 0%, 1%, 3% and 5% PHB supplemented feed. The results indicated that supplementation of PHB significantly increased the growth performance of the shrimp, and the feed conversion rate (FCR) in 3%PHB treatment group was significantly lower than the control (P 0.05). The intestinal amylase, lipase and trypsin activity in the three PHB treatment groups were all significantly higher than that of the control (P 0.05), but the pepsin activity were only significantly affected by 3%PHB treatment (P 0.05). The activities of intestinal immune enzymes such as total antioxidant capacity (T-AOC) and inducible nitric oxide synthase (iNOS) was significantly induced by 3%PHB treatment (P 0.05), while lysozyme (LSZ) activity was significantly affected by 5%PHB treatment and nitric oxide (NO) content was significantly induced in three PHB treatments. Meanwhile, PHB induced significantly the expression level of intestinal heat shock protein 70 (HSP70), Toll and immune deficiency (Imd) gene. HE staining showed that PHB induced the intestinal health status of L. vannamei. Intestinal SCFA content analysis revealed that the content of propionic and butyric acid of 3%PHB treatment were significantly higher than that of the control (P 0.05). Body composition analysis showed that the crude protein in 3% and 5%PHB treatments, and the crude lipid in 1% and 5%PHB treatments were all significantly higher than the control (P 0.05). These results revealed that PHB could improve the growth performance, modulated intestinal digestive and immune function, increased intestinal SCFA content and body composition in L. vannamei, and the optimum dietary PHB requirement by L. vannamei was estimated at 3% (w/w) diet.

Hematite and goethite are the two most abundant iron oxides in natural environments. Their formation is controlled by multiple environmental factors; therefore, their relative concentration has been used widely to indicate climatic variations. In this study, we aimed to test whether hematite and goethite growth is influenced by ambient magnetic fields of Earth-like values. Ferrihydrite was aged at 95 °C in magnetic fields ranging from ~0 to ~100 μT. Our results indicate a large influence of the applied magnetic field on hematite and goethite growth from ferrihydrite. The synthesized products are a mixture of hematite and goethite for field intensities

Magnetite is an important magnetic remanence carrier in natural samples and therefore is of great interest in paleo-, rock-, and environmental magnetism. The magnetic properties of magnetite depend on many factors, e.g., concentration and grain size distribution (GSD). In this study, we theoretically investigated the temperature-dependent susceptibility (TDS) of magnetite nanoparticles with a lognormal GSD. Results show that the TDS is affected highly by the GSD mainly in three aspects. Firstly, the unblocking process becomes smoother with the increase of distribution width, characterizing as a wider Hopkinson peak on the TDS curve. Secondly, the blocking temperature increases with the increase of the median diameter or/and the distribution width. Thirdly, the maximum susceptibility decreases with the increase of distribution width, and has a logarithmic function relation with the standard deviation of the distribution. As a case study, this model was further applied to the thermal products of the Chinese loess/paleosol samples to determine the granulometry of newly-formed magnetite upon heating based on TDS curves. The results demonstrate the fidelity and feasibility of this method to determine the GSD of nano-sized magnetic particles.

S U M M A R Y Clay minerals and Al-substituted haematite (Al-hm) usually coexist in soils and sediments. However, effects of clay minerals on Al-hm during thermal magnetic measurements in argon environment have not been well studied. In order to quantify such effects, a series of Al-hm samples were synthesized, and were then mixed with clay minerals (illite, chlorite, kaolinite and Ca-montmorillonite). The temperature dependence of magnetic susceptibility curves in an argon environment showed that Al-substituted magnetite was produced during the thermal treatment via the reduction of Al-hm by the clay mineral, which leads to a significant magnetic enhancement of the thermal products. In addition, the reductive capacity varies among different types of clay minerals, that is, illite > chlorite > kaolinite > Ca-montmorillonite. Furthermore, the iron content in the clay minerals and Al content of Al-hm are two predominant factors controlling the reduced haematite content. The iron is released from the clay minerals and provides the reducing agent, while Al decreases the crystallinity of haematite and thus facilitates the chemical reaction. Therefore, the thermal magnetic measurements can be used to quantify the Al content of Al-hm in natural samples. Our study provides significant information for palaeomagnetism and environmental magnetism studies, such as thermal magnetic analysis and palaeomagnetic intensity reconstruction using ancient pottery and kilns.

RAS mutations, especially in NRAS, are common in AML, identified in over 10% of the patients. The mutations lead to constitutive RAS activation and activation of multiple signaling pathways, including RAF/MAPK, PI3K-AKT/mTOR, and others. Many efforts to directly target RAS itself with small molecules have been unsuccessful. Thus, efforts have been directed at targeting canonical downstream effectors of RAS, such as RAF, MEK, and others. The complexity of RAS signaling, including redundancy and activation of compensatory pathways, makes it difficult to predict clinical effects. For example, RAF inhibitors increase proliferation of RAS-transformed cells by paradoxically increasing activation of MEK. In an effort to identify new therapeutic targets related to RAS, we designed a novel chemical screen to identify agents capable of potentiating the activity of the MEK inhibitor, AZD6244 (A), or the mTOR inhibitor, Torin1 (B). As a cell line to be used in the screen, BaF/3 cells were transformed with G12D mutants of NRAS or KRAS (Ba/F3-NRAS or Ba/F3-KRAS cells, respectively) and were shown to be exquisitely dependent on each oncogene for viability. Screen A: We identified the IGF-1R inhibitor, GSK1904529A, as able to selectively potentiate the effects of AZD6244 against mutant RAS-positive leukemia. GSK1904529A and AZD6244 synergized against mutant NRAS-expressing cell lines, OCI-AML3 and HL60, as well as active KRAS-expressing and -dependent cell lines, NOMO-1, NB4, and SKM-1, but not against wild-type (wt) RAS-expressing HEL or MOLM14 cells, or normal mononuclear cells. This result was confirmed with an additional IGF-1R inhibitor, NVP-AEW541, which exhibits 100-fold more selectivity toward IGF-1R than the insulin receptor (IR), and the specificity of IGF-1R as the target of these inhibitors was validated by knockdown (KD) of IGF-1R by shRNA. Mechanistically, IGF-1R protein expression/activity was substantially increased in mutant RAS-expressing cells, and suppression of RAS led to a decrease in IGF-1R. It is hypothesized that the increased IGF-1R levels observed in mutant RAS-expressing cells may contribute significantly to RAS transformation of hematopoietic cells. The synergy between MEK and IGF-1R inhibitors correlated with induction of apoptosis, inhibition of cell cycle progression, and decreased phospho-S6 and phospho-4E-BP1. In vivo, NSG mice tail vein-injected with OCI-AML3-luc+ cells showed significantly lower tumor burden following one week of daily oral administration of 50 mg/kg NVP-AEW541 combined with 25 mg/kg AZD6244, as compared to mice treated with either agent alone, and the combination was more toxic to mutant NRAS-expressing primary AML patient cells, compared to either agent alone. Screen B: We identified the Wee1 kinase inhibitor, MK-1775, which was unexpectedly found to potentiate mTOR inhibition of mutant RAS- leukemia. Wee1 is a protein kinase and inhibitory regulator of the G2/M checkpoint that prevents cells from going through mitosis by inhibiting the activity of CDK1. In response to DNA damage, Wee1 inactivates CDK1, thus leading to G2 arrest; this allows transformed cells the time needed for repair of damaged DNA and thus confers a survival advantage. The synergy was observed in both mutant NRAS- and mutant KRAS-positive AML cell lines and primary patient samples. The observed synergy enhanced dephosphorylation of AKT, 4E-BP1 and S6K, and correlated with increased apoptosis. The specificity of Wee1 as the target of MK-1775 was validated by Wee1 KD, as well as partial reversal of drug combination-induced apoptosis by inhibition of CDK1 by the CDK1 inhibitor, RO-3306. In a mouse in vivo xenotransplantation model, the combination treatment (10 mg/kg MK-1775 combined with 10 mg/kg Torin2) was more effective than single agents at suppressing the growth of NB4-luc cells that were tail vein-injected to NSG mice. The present studies suggest that combinations of drugs that simultaneously inhibit IGF-1R and MEK, or that inhibit Wee1 and mTOR, represent novel targeted therapeutic strategies for mutant RAS-positive leukemias. Disclosures No relevant conflicts of interest to declare.