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Oncogenic activation of the mTOR signaling pathway occurs frequently in tumor cells and contributes to the devastating features of cancer, including breast cancer. mTOR inhibitors rapalogs are promising anticancer agents in clinical trials; however, rapalogs resistance remains an unresolved clinical challenge. Therefore, understanding the mechanisms by which cells become resistant to rapalogs may guide the development of successful mTOR-targeted cancer therapy. In this study, we found that eEF-2K, which is overexpressed in cancer cells and is required for survival of stressed cells, was involved in the negative-feedback activation of Akt and cytoprotective autophagy induction in breast cancer cells in response to mTOR inhibitors. Therefore, disruption of eEF-2K simultaneously abrogates the two critical resistance signaling pathways, sensitizing breast cancer cells to rapalogs. Importantly, we identified mitoxantrone, an admitted anticancer drug for a wide range of tumors, as a potential inhibitor of eEF-2K via a structure-based virtual screening strategy. We further demonstrated that mitoxantrone binds to eEF-2K and inhibits its activity, and the combination treatment of mitoxantrone and mTOR inhibitor resulted in significant synergistic cytotoxicity in breast cancer. In conclusion, we report that eEF-2K contributes to the activation of resistance signaling pathways of mTOR inhibitor, suggesting a novel strategy to enhance mTOR-targeted cancer therapy through combining mitoxantrone, an eEF-2K inhibitor.

Heart failure (HF) has become an important social problem that seriously threatens human health due to its high morbidity and mortality. G Protein-Coupled Receptors Kinase 2 (GRK2), as a novel target of heart failure, is overexpressed in the pathogenesis and progression of HF. In this study, we conducted a multistep virtual screening process from the ZINC database to discover potential GRK2 inhibitors. First, by using 6 known GRK2 inhibitors with high bioactivity and diverse structures as the template molecules, 7499 compounds were obtained from ligand-based similarity screening by 4 types of fingerprints. Subsequently, these hits were further screened by three quantitative structure-activity relationship (QSAR) models, which were built by 197 known GRK2 compounds and represented by two-dimensional MOE descriptors (MOE2D), molecular fragments (MACCS) and 2- dimensional pharmacophore (CATS) based on random forest algorithm, respectively. These hit compounds were then filtered by three molecular docking packages (MOE, GOLD and GLIDE) to improve the docking accuracy for avoid losing candidate compounds. The compounds with high docking scores were selected for the evaluation of absorption, distribution, metabolism, excretion, and toxicity (ADMET). Finally, 17 compounds were identified the binding affinity to GRK2 by using SPR assay, three of which were considered as novel GRK2 inhibitors, and their binding modes with GRK2 active sites were analyzed. As new potential GRK2 inhibitors, COM5(KD = 0.172 μM) and COM17(KD = 0.595 μM) with the KD value less than 1 μM could be directly used in the next study, while the remaining hit COM4 (KD = 4.72 μM) whose KD over 1 μM can be optimized into the required low nanomole range for further study.

Eukaryotic elongation factor-2 kinase (eEF2K), a calcium/calmodulin-dependent protein kinase, is a potential target for treating cancer. eEF2K can reduce the binding ability of eEF2 to ribosomes by phosphorylating eEF2, thereby inhibiting peptide chain extension and negatively regulating protein synthesis, which provides a cytoprotective mechanism for the development of cancer. eEF2K has been reported to be overexpressed in various types of cancers. Therefore, it is of high importance to discover new eEF2K inhibitors for cancer therapy. Here, we proposed a three-step screening strategy to seek eEF2K inhibitors from the FDA approved drug library: homology modeling of eEF2K protein, SAR-based virtual screening, and docking-based virtual screening. Based on the screening strategy, 13 drugs with high evaluation scores were purchased to measure their binding affinity (KD) to eEF2K by using the SPR assay. The assay results suggested that 4 drugs may be potential eEF2K inhibitors, which are Pemetrexed (KD = 0.104 μM), Entecavir (KD = 2.16 μM), calcium levofolinate (KD = 11.7 μM), and Fosbretabulin (KD = 34.5 μM). To further validate whether these drugs act on eEF2K, Western blot (WB) analysis was performed, which showed that Pemetrexed and Entecavir could inhibit the eEF2K activity. Furthermore, molecular dynamics (MD) simulation was carried out to further demonstrate the interaction of eEF2K with Pemetrexed, the most bioactive drug. The specific in vitro/in vivo interaction mechanisms between these drugs and eEF2K are under investigation.

Virtual Screening (VS) based on molecular docking is an efficient method used for retrieving novel hit compounds in drug discovery. However, the accuracy of the current docking scoring function (SF) is usually insufficient. In this study, in order to improve the screening power of SF, a novel approach named EAT-Score was proposed by directly utilizing the energy auxiliary terms (EAT) provided by molecular docking scoring through eXtreme Gradient Boosting (XGBoost). Here, EAT specifically refers to the output of the Molecular Operating Environment (MOE) scoring, including the energy scores of five different classical SFs and the Protein-Ligand Interaction Fingerprint (PLIF) terms. The performance of EAT-Score to discriminate actives from decoys was strictly validated on the DUD-E diverse subset by using different performance metrics. The results showed that EAT-Score performed much better than classical SFs in VS, with its AUC values exhibiting an improvement of around 0.3. Meanwhile, EAT-Score could achieve comparable even better prediction performance compared with other state-of-the-art VS methods, such as some machine learning (ML)-based SFs and classical SFs implemented in docking programs, in terms of AUC, LogAUC, or BEDROC. Furthermore, the EAT-Score model can capture important binding pattern information from protein-ligand complexes by Shapley additive explanations (SHAP) analysis, which may be very helpful in interpreting the ligand binding mechanism for a certain target and thereby guiding drug design.

Scoring functions (SFs) based on complex machine learning (ML) algorithms have gradually emerged as a promising alternative to overcome the weaknesses of classical SFs. However, extensive efforts have been devoted to the development of SFs based on new protein–ligand interaction representations and advanced alternative ML algorithms instead of the energy components obtained by the decomposition of existing SFs. Here, we propose a new method named energy auxiliary terms learning (EATL), in which the scoring components are extracted and used as the input for the development of three levels of ML SFs including EATL SFs, docking-EATL SFs and comprehensive SFs with ascending VS performance. The EATL approach not only outperforms classical SFs for the absolute performance (ROC) and initial enrichment (BEDROC) but also yields comparable performance compared with other advanced ML-based methods on the diverse subset of Directory of Useful Decoys: Enhanced (DUD-E). The test on the relatively unbiased actives as decoys (AD) dataset also proved the effectiveness of EATL. Furthermore, the idea of learning from SF components to yield improved screening power can also be extended to other docking programs and SFs available.

Hepatotoxicity is a major cause of drug withdrawal from the market. To reduce the drug attrition induced by hepatotoxicity, an accurate and efficient hepatotoxicity prediction system must be constructed. In the present study, we constructed a three-level hepatotoxicity prediction system based on different levels of adverse hepatic effects (AHEs) combined with machine learning, using (1) an end point, hepatotoxicity; (2) four hepatotoxicity severity degrees; and (3) specific AHEs. After collecting and curing 15 873 compound-AHE pairs associated with 2017 compounds and 403 AHEs, we constructed 27 models with three end point levels with the random forest algorithm, and obtained accuracies ranging from 67.0 to 78.2% and the area under receiver operating characteristic curves (AUCs) of 0.715-0.875. The 27 models were fully integrated into a tiered hepatotoxicity prediction system. The existence of hepatotoxicity existence, severity degree, and potential AHEs for a given compound could be inferred simultaneously and systematically. Thus, the tiered hepatotoxicity prediction system allows researchers to have significant confidence in confirming compound hepatotoxicity, analyzing hepatotoxicity from multiple perspectives, obtaining warnings for the potential hepatotoxicity severity, and even rapidly selecting the proper in vitro experiments for hepatotoxicity verification. We also applied three external sets (11 drugs or candidates that failed in clinical trials or were withdrawn from the market, the PharmGKB (offsides) database, and an herbal hepatotoxicity data set) to test and validate the prediction ability of our system. Furthermore, the hepatotoxicity prediction system was adapted into a flow framework based on the Konstanz Information Miner, which was made available for researchers.

In the present study, we examined the morphological features of the adrenal gland in Bactrian camel by means of digital anatomy, light and electron microscopy. Our findings testified that the gland was divided into three parts, capsule, cortex and medulla from outside to inside as other mammals, and the cortex itself was further distinguished into four zones: zona glomerulosa, zona intermedia, zona fasciculate and zona reticularis. Notably, the zona intermedia could be seen clearly in the glands from females and castrated males, whereas it was not morphologically clear in male. There was a great deal of lipid droplets in the zona fasciculate, while it was fewer in the zona glomerulosa and zona reticularis. The cytoplasm of adrenocortical cell contained rich mitochondria and endoplasmic reticulum. The adrenal medulla was well-developed with two separations of external and internal zones. The most obvious histological property of adrenal medulla cells were that they contained a huge number of electron-dense granules enveloped by the membrane, and so medulla cells could be divided into norepinephrine cells and epinephrine cells. Moreover, the cortical cuffs were frequently present in adrenal gland. Results of this study provides a theoretical basis necessary for ongoing investigations on Bactrian camels and their good adaptability in arid and semi-arid circumstances.

Arsenic (As) accumulation in food crops such as rice is of major concern. To investigate whether phytoremediation can reduce As uptake by rice, the As hyperaccumulator Pteris vittata was grown in five contaminated paddy soils in a pot experiment. Over a 9-month period P. vittata removed 3.5–11.4% of the total soil As, and decreased phosphate-extractable As and soil pore water As by 11–38% and 18–77%, respectively. Rice grown following P. vittata had significantly lower As concentrations in straw and grain, being 17–82% and 22–58% of those in the control, respectively. Phytoremediation also resulted in significant changes in As speciation in rice grain by greatly decreasing the concentration of dimethylarsinic acid (DMA). In two soils the concentration of inorganic As in rice grain was decreased by 50–58%. The results demonstrate an effective stripping of bioavailable As from contaminated paddy soils thus reducing As uptake by rice.

How arsenic (As) is transported in phloem remains unknown. To help answer this question, we quantified the chemical species of As in phloem and xylem exudates of castor bean (Ricinus communis) exposed to arsenate [As(V)], arsenite [As(III)], monomethylarsonic acid [MMA(V)], or dimethylarsinic acid. In the As(V)- and As(III)-exposed plants, As(V) was the main species in xylem exudate (55%–83%) whereas As(III) predominated in phloem exudate (70%–94%). The ratio of As concentrations in phloem to xylem exudate varied from 0.7 to 3.9. Analyses of phloem exudate using high-resolution inductively coupled plasma-mass spectrometry and accurate mass electrospray mass spectrometry coupled to high-performance liquid chromatography identified high concentrations of reduced and oxidized glutathione and some oxidized phytochelatin, but no As(III)-thiol complexes. It is thought that As(III)-thiol complexes would not be stable in the alkaline conditions of phloem sap. Small concentrations of oxidized glutathione and oxidized phytochelatin were found in xylem exudate, where there was also no evidence of As(III)-thiol complexes. MMA(V) was partially reduced to MMA(III) in roots, but only MMA(V) was found in xylem and phloem exudate. Despite the smallest uptake among the four As species supplied to plants, dimethylarsinic acid was most efficiently transported in both xylem and phloem, and its phloem concentration was 3.2 times that in xylem. Our results show that free inorganic As, mainly As(III), was transported in the phloem of castor bean exposed to either As(V) or As(III), and that methylated As species were more mobile than inorganic As in the phloem.

To evaluate the long-term effects of arteriovenous fistula (AVF) on heamodynamic changes and cardiac structure and function in non-diabetic hemodialysis patients.Data were collected from 50 non-diabetic hemodialysis patients (aged 18 to 60 years) who had used AVF as the vascular access. AVF flow (Qa), stoke volume (SV), cardiac output (CO), cardiac index (CI), central blood volume (CBV) and peripheral vascular resistance (PR) were measured using the ultrasound dilution technique. Echocardiography was performed in the second day after hemodialysis sessions to evaluate the influence of AVF on the cardiac structure and function.The cubic polynomial regression model best fit the relationships of Qa with SV, CO, and CI. CO and CI significantly increased and PR reduced when the Qa of AVF was more than 2.0 L/min(all P0.05), and no statistical difference of CO, CI and PR in groups of Qa between 0.6-2.0 L/min and less than 0.6 L/min(all P0.05). In different Qa groups, the grades of cardiac function (based on New York Heart Association classification) showed significant difference, among which the cardiac failure was significantly common when Qa2.0 L/min(both P0.05). Echocardiography showed the left atrium dimension, thickness of posterior wall and interventricular seprum of left ventricle, left ventricular end-systolic dimension (LVESD) and end-diastolic dimension (LVEDD), venae cava inferior, and pulmonary artery systolic pressure gradually increased when Qa increased, while the ejection fraction and fractional shortening reduced(all P0.05). Notably, the changes of LVESD, LVEDD, and venae cava inferior with different Qa were statistically significant(all P0.05).Long-term AVF remarkably affects the cardiovascular dynamics of non-diabetic hemodialysis patients. A cubic polynomial regression model best fits the relationship of AVF Qa with SV, CO, and CI. The cardiac adaptic changes after long-term AVF include the enlargement of left ventricle and the thickening of ventricular wall. The risk of cardiac failure significantly increases when the Qa of AVF is more than 2.0 L/min with much higher CO and lower PR.

To prospectively determinate the association of left ventricular systolic and diastolic function with intradialytic hypotension (IDH) in patients on maintenance hemodialysis.Totally 115 patients with sinus rhythm were included in this study and divided into IDH group (n=29) and control group (n=86). The cardiac function was assessed by New York Heart Association (NYHA) classification before hemodialysis. Echocardiograms were performed in the next day after hemodialysis and the software of GE EchoPAC was used to estimate parameters of cardiac systolic and diastolic functions including ejection fraction, fractional shortening of left ventricular diameter, stroke volume (SV), cardiac output (CO), left ventricular mass index, Tei index, isovolumetric relaxation time, E-deceleration time, mitral inflow peak early diastolic velocity (E) to late diastolic velocities (A) ratio, and E to peak mitral annulus velocity (E/Em) ratio.The mean age (p=0.045) and the ratio of heart failure evaluated by the NYHA classification (p0.01) were significantly higher in IDH group than those in control group. No difference was noted for gender, body mass index, duration of dialysis, body weight elevated rate and blood pressure between these two groups (all p0.05). Left ventricular diameters at the end of diastolic phase, SV, and CO in IDH group were significantly lower than those in control group (all P0.05), whereas no significant difference was found for EF, FS, Tei index, IVRT, EDT, E/A and E/Em ratio (all p0.05). Multivariate logistic regression analysis showed that NYHA cardiac function was an independent predictor of IDH, and the risk of IDH increased by 1.134 times with incremental one grade of NYHA classification.IDH is a common complication of hemodialysis. Neither systolic dysfunction nor diastolic dysfunction of left ventricle evaluated in second day after hemodialysis affects its frequency, whereas the clinical cardiac function is an independent predictor of IDH.

To investigate the clinical and pathologic characteristics of anti-glomerular basement membrane(GBM) disease with normal renal function.The clinical and pathologic data of 6 patients with anti-GBM disease and normal renal function in Peking Union Medical College Hospital were reviewed retrospectively. Furthermore, 29 patients with anti-GBM disease and impaired renal function in the same period in the same hospital were enrolled as the control group. Factors that may influence the prognosis were analyzed.Six (17.1%) of all 35 patients maintained normal renal function for 12-133 months during follow-up. Five patients had microhematuria and proteinuria, one had pulmonary hemorrhage only, and three manifested as Goodpasture syndrome. Renal biopsies from 4 patients revealed linear deposition of IgG 2+-3+ along the glomerular capillary walls by immunofluorescence. As shown by normal light microscopy, mild mesangial proliferation and crescentic glomerulonephritis with a large amount of fibrinoid necrosis of glomerular capillary walls were observed in different patients; however, most pathological changes were mild. Five of these six patients were treated with immunosuppressive drugs and/or plasma exchange. Compared with the control group, the 6 patients with normal renal function had significantly higher hemoglobin[(77.97±20.62 vs.(99.67±19.80 g/L P=0.024], lower titers of anti-GBM antibody[(224.34 ± 145.79 vs.(80.23 ± 85.73 EU/ml P=0.027], and lower ratio of glomeruli with crescents[(0.58±0.29 vs.(0.17±0.27 ,P=0.005]. These 6 patients with normal renal function were followed up for 12-133 months, among whom 4 patients achieved complete remission and 2 had mild proteinuria and microhematuria.Anti-GBM disease with normal renal function is not uncommon. Most patients have mild pathologic changes and good prognosis.

To analysis the clinical and pathological features, results of laboratory tests and prognosis of nephrotic syndrome (NS) in patients with non-haematological malignancy.The data were collected from 25 patients who presented with NS around the diagnosis of non-haematological malignancy.Twenty-five cases were investigated (age: (56.6 +/- 17.7) years; male/female ratio: 20/5). Malignancy and NS occurred within one year in 92% patients. There was a wide distribution of malignancy with involvement of 36% in digestive system and 20% in respiratory system. Ten patients (40%) presented with NS as their initial manifestation. Haematuria appeared in 67% patients and acute renal insufficiency was complicated in 12% cases before treatments. Some other non-specific laboratory tests were found including elevated serum gamma-globulin in 50% and anemia not related with renal failure in 28% cases. Membranous nephropathy was the most common pathological changes in 67% cases. Although NS still continued for several weeks in 8 of 9 cases after surgery and/or chemical therapy, glucocorticoids was helpful to achieve the remission in these patients. However, no remission was achieved in patients without the treatment for malignancy.Malignancy may present with NS as its initial manifestation. It should be inspected routinely and regularly in elder patients with NS, especially in those with membranous nephropathy, as well as gamma-globulinemia and anemia.

To evaluate the risk factors of post-renal biopsy bleeding (PBB).We retrospectively analyzed the clinical data of 1 262 patients who received ultrasound-guided biopsy of native kidney at our hospital from January 2005 to December 2006.The overall incidence of PBB was 30.3% (383/1,262), among which the incidence of hematoma was 29.4% (371/1,262) (the percentages of less and more than 5cm were 73.9% and 26.1%, respectively) while that of gross hematuria was only 1.3% (17/1,262). The incidences of minor, intermediate, and major bleeding complications were 21.4% (270/1,262), 8.4% (106/1,262), and 0.6% (7/1,262), respectively. In seven patients with major bleeding complications, six had renal disease secondary to rheumatic disease (lupus nephritis, n = 5; scleroderma crisis, n = 1), while the other one had IgA nephropathy (Lee's classification V). The risk of PBB was relatively higher in women and younger patients.Patients with chronic connective tissue diseases are vulnerable to severe PBB complications. A close monitoring of these patients is necessary.

To explore the regulational effect of oxidized low-density lipoprotein (Ox-LDL) on expression of type A scavenger receptor (SR-A) in human mesangial cells (HMC).HMC line (HMCL) with high expression of SR-A (HMCL-SRA) was established after stable transfection of expressive vector with cDNA encoding SR-A. Uptake of Ox-LDL by HMCL was evaluated using Oil Red "O" staining. SR-A mRNA expression was examined using reverse transcription-polymerase chain reaction (RT-PCR).More uptake of Ox-LDL was observed in the HMCL-SRA than that in the untransfected HMCL. Ox-LDL could induce SR-A mRNA expression in HMC in a dose-dependent manner, and reached a peak level after 24 h of stimulation. After 24 h of stimulation with Ox-LDL at the dose of 10, 50 and 100 micrograms/ml, SR-A mRNA expression was up-regulated to 1.35, 1.83 and 2.30-fold of controls, respectively. However, LDL had no effect on the expression of SR-A.It suggests that SR-A be a major binding receptor to uptake Ox-LDL in HMC. Ox-LDL may promote the progression of chronic renal diseases through up-regulation of SR-A.