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Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality. Artemisinin (ART) and SOMCL-14-221 (221), a spirobicyclic analogue of ART, have been reported to inhibit the proliferation of A549 cells with unclear underlying mechanism. In the present study, we validated that both ART and 221 inhibited the proliferation and migration of NSCLC cells and the growth of A549 xenograft tumors without appreciable toxicity. The proteomic data revealed proteins upregulated in ART and 221 groups were involved in “response to endoplasmic reticulum stress” and “amino acid metabolism”. Asparagine synthetase (ASNS) was identified as a key node protein in these processes. Interestingly, knockdown of ASNS improved the antitumor potency of ART and 221 in vitro and in vivo, and treatments with ART and 221 disordered the amino acid metabolism of A549 cells. Moreover, ART and 221 activated ER stress, and inhibition of ER stress abolished the anti-proliferative effects of ART and 221. In conclusion, this study demonstrates that ART and 221 suppress tumor growth by triggering ER stress, and the inhibition of ASNS enhances the antitumor activity of ART and 221, which provides new strategy for drug combination therapy.

Kappa-opioid receptor (KOR) is a member of G-protein coupled receptors (GPCRs) expressed in serotonergic neurons and neuronal terminals. The involvement of KOR ligands in nociception, diuresis, emotion, cognition, and immune system has been extensively studied. Omics-based methods are preferable to understand the signaling cascade after KOR activation in a systematic manner. In this study, an in-depth quantitative phosphoproteomic analysis resulted in 305 phosphosites, which were significantly changed in three KOR-overexpressed cells upon treatment with two KOR agonists. The subsequent substrate-kinase prediction analysis revealed that 18 potential kinases might be activated under stimulation of the agonists. We found that phosphorylation of PAK1/2 (p21-activated kinase 1/2) was induced by KOR agonists, resulting in reduced actin stress fibers and cytoskeletal reorganization. In summary, this quantitative phosphoproteomics-based research studied the downstream phosphorylation events upon KOR activation, which may shed light on the investigations of KOR signaling pathway and targeted therapy for KOR-related diseases.

Prostate cancer (PCa) is the most common solid organ malignancy among men, outnumbering both lung and colorectal cancer, and it is the second leading cause of male tumor-related death in the United States due to high metastasis. Recently, leukemia inhibitory factor receptor (LIFR) has been found to play roles in multiple types of cancer. However, the roles of LIFR in the progression of PCa remain to be revealed. In this study, we found that LIFR plays an oncogenic role in PCa. The phosphorylation of LIFR at S1044 contributes to subsequent activation of the AKT pathway, inducing the expression of a series of proliferation and metastatic genes. Additionally, LIFR-S1044 is phosphorylated by ERK2 but not ERK1. The signal intensity of pLIFR-S1044 and pAKT S473 in PCa tissue displays a tight positive correlation. The ERK2/LIFR/AKT axis modulates PCa progression and offers a promising therapeutic and diagnostic target for PCa.

Shexiang Baoxin Pill (SBP) and Suxiao Jiuxin Pill (SJP) are traditional Chinese medicines used to treat cardiovascular disease (CVD) in China. However, the mechanism of their therapeutic effect on CVD has not been clearly elucidated yet.The aim of this study is to investigate the cardioprotective effect of SBP and SJP in the treatment of acute myocardial infarction (AMI) model rats by applying serum proteomic approach.The rat model of AMI was generated by ligating the left anterior descending coronary artery. 42 rats were randomly divided into four groups: sham-operating (Sham, n = 10) group, model (Mod, n = 8) group, Shexiang Baoxin pills pretreatment (SBP, n = 12) group and Suxiao Jiuxin pills pretreatment (SJP, n = 12) group. Data Independent Acquisition (DIA) proteomic approach was utilized to investigate the serum proteome from the rat individuals. The differentially expressed proteins were subsequently obtained with bioinformatic analysis.DIA-MS identified 415 proteins within 42 samples, and 84 differentially expressed proteins may contribute to the therapeutic effects of SBP and SJP. GOBP and KEGG pathway analysis of 84 differentially expressed proteins revealed that the proteins were mainly involved in platelet activation and adhesion processes. All 84 differentially expressed proteins presented the same changing tendency in the SBP and SJP groups when compared with the Mod group. Among these 84 proteins, 25 proteins were found to be related to CVD. Among these 25 proteins, ACTB, ACTG1, FGA, FGB, FGG, PF4 and VWF were found to be involved in platelet aggregation and activation. FN1, HSPA5 and YWHAZ were associated with adhesion.The results of our study suggest that the cardioprotective effects of SBP and SJP are achieved through the modulation of focal adhesion, platelet activation pathways.

Thermogenesis is a promising approach to limit weight gain in response to excess nutrition. In contrast to cold-induced thermogenesis, the molecular and cellular mechanisms of diet-induced thermogenesis (DIT) have not been fully characterized. Here, we explored the response of brown adipose tissue (BAT) and subcutaneous white adipose tissue (sWAT) to high fat diet (HFD) using proteome and phosphoproteome analysis. We observed that after HFD, Uncoupling protein 1 (UCP1) and its phosphorylation were only increased in BAT. Furthermore, proteins involved in fatty acid oxidation, tricarboxylic acid cycle, and oxidative phosphorylation were also upregulated in BAT. Nevertheless, most metabolic related proteins were downregulated in sWAT. We found that these metabolic changes accompanied with different variation of mitochondrial proteins between BAT and sWAT. After HFD, most mitochondrial proteins were decreased in sWAT, but not in BAT. This effect was correlated with decreased mitochondrial ribosomal proteins in sWAT. Finally, through phosphoproteomic analysis, we predicted the activities of kinases in HFD mice and observed that there were more kinases inactivated in sWAT. Finally, this dataset provides a valuable resource for molecular researchers in the fields of obesity and obesity-related disease. SIGNIFICANCE: Thermogenesis is a promising approach to combat obesity in response to excess energy. Nevertheless, the molecular and cellular mechanisms of DIT have not been fully characterized. Herein, we employed mass spectrometry (MS)-based proteomics and phosphoproteomics to identify differentially regulated proteins and phosphosites in BAT and sWAT of mice fed with HFD. This study unveils the differential regulatory networks of HFD in BAT and sWAT, which provides reference omics data to future researchers.

Background: Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality. Artemisinin (ART) and SOMCL-14-221 (221), a spirobicyclic analogue of ART, have been reported to inhibit the proliferation of A549 cells; yet, the underlying mechanism remains unclear. Methods: Both in vitro and in vivo experiments were performed to examine the anti-proliferative effects of ART and 221. The TMT-based quantitative proteomics and NMR-based metabolomics were conducted to analyze the global proteomic and metabolomic changes in ART and 221 groups, respectively. The potential mechanism of ART and 221 in suppressing tumor growth was studied by knockdown of asparagine synthetase (ASNS) and inhibition of ER stress. Findings: Both ART and 221 inhibited the proliferation of NSCLC cells in vitro and in vivo. The proteomic data revealed 114 upregulated proteins in ART and 221 groups, in which biological process of “response to endoplasmic reticulum stress” were highly represented. ASNS was identified as a key node protein in this process. Interestingly, knockdown of ASNS improved the cytotoxicity of ART and 221, and administration with ART and 221 disordered amino acid metabolism. Moreover, ART and 221 activated ER stress, and inhibition of ER stress abolished the anti-proliferative effects of ART and 221. Interpretation: This study demonstrates that ART and 221 suppress tumor growth by triggering ER stress, and the inhibition of ASNS enhances the antitumor activity of ART and 221, which provides new strategy of combined drug therapy. Funding Statement: This work was supported by the National Key Research and Development Program from the Ministry of Science and Technology of China (No. 2017YFC1700200), the National Science & Technology Major Project "Key New Drug Creation and Manufacturing Program" of China (Grant No. 2018ZX09711002), National Natural Science Foundation of China (No. 31800693, No. 21877120, 81430080) and the ''Personalized Medicines-Molecular Signature-based Drug Discovery and Development" of Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDA12020320). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: All animal studies (including the mice euthanasia procedure) were done in compliance with the regulations and guidelines of Shanghai Institute of Materia Medica (Chinese Academy of Sciences) institutional animal care and conducted according to the AAALAC and the IACUC guidelines.

Background: Hepatic stellate cell (HSC) activation induced by transforming growth factor β1 (TGF-β1) plays a pivotal role in fibrogenesis, while the complex downstream mediators of TGF-β1 in such process are largely unknown. Methods: We performed proteomic profiling of mice liver tissues from control, carbon tetrachloride (CCl4)-induced fibrosis and NPLC0393 administrated groups. Our target Mat2a was overexpressed and knocked down in vivo by tail vein injection of AAV vectors. We examined NF-κB transcriptional and MAT2A promoter activation via luciferase assay. Intracellular SAM contents were analyzed by LC/MS method. Findings: We found that methionine adenosyltransferase 2A (MAT2A) is significantly upregulated in the CCl4-induced fibrosis mice model and application of NPLC0393, a known small molecule inhibitor of TGF-β1 signaling pathway, inhibits the upregulation of MAT2A. Mechanistically, TGF-β1 induces phosphorylation of p65 and activation of NF-κB, thereby promoting mRNA transcription and protein expression of MAT2A and reduces S-adenosylmethionine (SAM) concentration in HSCs. Consistently, in vivo and in vitro knockdown of MAT2A alleviates CCl4- and TGF-β1-induced HSC activation, whereas in vivo overexpression of MAT2A facilitates hepatic fibrosis and abolishes therapeutic effect of NPLC0393. Interpretation: This study identifies TGF-β1/p65/MAT2A pathway that is involved in the regulation of intracellular SAM concentration and liver fibrogenesis, suggesting that this pathway is a potential therapeutic target for hepatic fibrosis. Funding: This work was supported by National Natural Science Foundation of China (No. 81500469, 81573873 and 81774196), Zhejiang Provincial Natural Science Foundation of China (No. Y15H030004), the National Key Research and Development Program from the Ministry of Science and Technology of China (No. 2017YFC1700200) and the Key Program of National Natural Science Foundation of China (No. 8153000502). Declaration of Interest: The authors declare no conflict of interest. Ethical Approval: The animal experiment was approved by Science and Technology Commission of Shanghai Municipality, and all experimental procedures were performed according to the ethical guidelines of Animal care and use committee, Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

Aging of neural stem cell, which can affect brain homeostasis, may be caused by many cellular mechanisms. Autophagy dysfunction was found in aged and neurodegenerative brains. However, little is known about the relationship between autophagy and human neural stem cell (hNSC) aging. The present study used 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) to treat neural precursor cells (NPCs) derived from human embryonic stem cell (hESC) line H9 and investigate related molecular mechanisms involved in this process. MPTP-treated NPCs were found to undergo premature senescence [determined by increased senescence-associated-β-galactosidase (SA-β-gal) activity, elevated intracellular reactive oxygen species level, and decreased proliferation] and were associated with impaired autophagy. Additionally, the cellular senescence phenotypes were manifested at the molecular level by a significant increase in p21 and p53 expression, a decrease in SOD2 expression, and a decrease in expression of some key autophagy-related genes such as Atg5, Atg7, Atg12, and Beclin 1. Furthermore, we found that the senescence-like phenotype of MPTP-treated hNPCs was rejuvenated through treatment with a well-known autophagy enhancer rapamycin, which was blocked by suppression of essential autophagy gene Beclin 1. Taken together, these findings reveal the critical role of autophagy in the process of hNSC aging, and this process can be reversed by activating autophagy.

Summary Hsp90 is a target for anti-cancer drug development. Both the conformational events tuned by ATP/ADP and co-chaperones and the chaperoning cycle timing are required for Hsp90's fully functional display. Interfering with either one of the conformational events or the cycle timing will down-regulate Hsp90's function. In this manuscript, non-covalent allosteric modulators (SOMCL-16-171 and SOMCL-16-175) targeting Hsp90α’s middle domain (Hsp90M) were developed for the first time. Multiple techniques were then applied to characterize the interactions between two active compounds and Hsp90α. Two loops and one α-helix (F349-N360, K443-E451, and D372-G387) in Hsp90M were identified responsible for the recognition of SOMCL-16-171 and SOMCL-16-175. Meanwhile, the binding of SOMCL-16-171 and SOMCL-16-175 to Hsp90M was demonstrated to allosterically modulate the structure and function of Hsp90α’s N-terminal domain. Finally, cellular assays were conducted to evaluate the cellular activity of SOMCL-16-175, and the results indicate that SOMCL-16-175 destabilizes Hsp90's client proteins and reduces cell viability., Graphical Abstract, Highlights • Allosteric modulators targeting Hsp90α's middle domain were developed for the first time • Key elements in Hsp90M for the recognition of allosteric modulators were identified • Compound SOMCL-16-175 promotes Hsp90α’s ATPase activity and reduces cell viability • SOMCL-16-175 destabilizes Hsp90's clients without triggering heat shock response, Molecular Biology; Molecular Structure; Cancer

We performed the first proteogenomic characterization of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) using paired tumor and adjacent liver tissues from 159 patients. Integrated proteogenomic analyses revealed consistency and discordance among multi-omics, activation status of key signaling pathways, and liver-specific metabolic reprogramming in HBV-related HCC. Proteomic profiling identified three subgroups associated with clinical and molecular attributes including patient survival, tumor thrombus, genetic profile, and the liver-specific proteome. These proteomic subgroups have distinct features in metabolic reprogramming, microenvironment dysregulation, cell proliferation, and potential therapeutics. Two prognostic biomarkers, PYCR2 and ADH1A, related to proteomic subgrouping and involved in HCC metabolic reprogramming, were identified. CTNNB1 and TP53 mutation-associated signaling and metabolic profiles were revealed, among which mutated CTNNB1-associated ALDOA phosphorylation was validated to promote glycolysis and cell proliferation. Our study provides a valuable resource that significantly expands the knowledge of HBV-related HCC and may eventually benefit clinical practice.

The structural and magnetic properties of Nd0.5-xPrxSr0.5MnO3 (x = 0, 0.1,0.2,0.3,0.4 and 0.5) system have been investigated. With the substitution of Pr in Nd0.5Sr0.5MnO3, it shows a gradual structure transformation from the Imma orthorhombic symmetry to the tetragonal 14/mcm phase, and the crystallographic transition remains incomplete, even in Pr0.5Sr0.5MnO3. A large bifurcation between zero-field-cooled (ZFC) and field-cooled (FC) susceptibility has been observed below Curie temperature (T-C), which is characteristic of coexistence of ferromagnetism (FM) and antiferromagnetism (AFM) at low temperature region. The magnetization of Pr0.5Sr0.5MnO3 is larger than that of Nd0.5Sr0.5MnO3, while Nd0.5Sr0.5MnO3 with more CE-type AFM shows larger magnetization than Nd0.3Pr0.2Sr0.5MnO3, which mixed with CE-type (majority) and A-type (minority) AFM at low temperature, indicating that the magnetization of Nd0.5-xPrxSr0.5MnO3 system is affected by A-site disorder combined with orbital ordering of A-type AFM and CE-type AFM. (C) 2011 Elsevier B.V. All rights reserved.

Two distinct families of small molecules were discovered as novel alpha7 nicotinic acetylcholine receptor (nAChR) antagonists by pharmacophore-based virtual screening. These novel antagonists exhibited selectivity for the neuronal alpha7 subtype over other nAChRs and good brain penetration. Neuroprotection was demonstrated by representative compounds 7i and 8 in a mouse seizure-like behavior model induced by the nerve agent diisopropylfluorophosphate (DFP). These novel nAChR antagonists have potential use as antidote for organophosphorus nerve agent intoxication.

Organophosphorus (OP) compounds cause toxic symptoms, including convulsions, coma, and death, as the result of irreversible inhibition of acetylcholinesterase (AChE). The development of effective treatments to block these effects and attenuate long-term cognitive and motor disabilities that result from OP intoxication is hampered by a limited understanding of the CNS pathways responsible for these actions. We employed a candidate method (called CNSProfile™) to identify changes in the phosphorylation state of key neuronal phosphoproteins evoked by the OP compound, diisopropyl fluorophosphate (DFP). Focused microwave fixation was used to preserve the phosphorylation state of phosphoproteins in brains of DFP-treated mice; hippocampus and striatum were analyzed by immunoblotting with a panel of phospho-specific antibodies. DFP exposure elicited comparable effects on phosphorylation of brain phosphoproteins in both C57BL/6 and FVB mice. DFP treatment significantly altered phosphorylation at regulatory residues on glutamate receptors, including Serine897 (S897) of the NR1 NMDA receptor. NR1 phosphorylation was bi-directionally regulated after DFP in striatum versus hippocampus. NR1 phosphorylation was reduced in striatum, but elevated in hippocampus, compared with controls. DARPP-32 phosphorylation in striatum was selectively increased at the Cdk5 kinase substrate, Threonine75 (T75). Phencynonate hydrochloride, a muscarinic cholinergic antagonist, prevented seizure-like behaviors and the observed changes in phosphorylation induced by DFP. The data reveal region-specific effects of nerve agent exposure on intracellular signaling pathways that correlate with seizure-like behavior and which are reversed by the muscarinic receptor blockade. This approach identifies specific targets for nerve agents, including substrates for Cdk5 kinase, which may be the basis for new anti-convulsant therapies.

HOXD13, the homeobox-containing gene located at the most 5' end of the HOXD cluster, plays a critical role in limb development. It has been shown that mutations in human HOXD13 can give rise to limb malformations, with variable expressivity and a wide spectrum of clinical manifestations. Polyalanine expansions in HOXD13 cause synpolydactyly, whereas amino acid substitutions in the homeodomain are associated with brachydactyly types D and E. We describe two large Han Chinese families with different limb malformations, one with syndactyly type V and the other with limb features overlapping brachydactyly types A4, D, and E and mild syndactyly of toes 2 and 3. Two-point linkage analysis showed LOD scores3 (theta =0) for markers within and/or flanking the HOXD13 locus in both families. In the family with syndactyly type V, we identified a missense mutation in the HOXD13 homeodomain, c.950A--G (p.Q317R), which leads to substitution of the highly conserved glutamine that is important for DNA-binding specificity and affinity. In the family with complex brachydactyly and syndactyly, we detected a deletion of 21 bp in the imperfect GCN (where N denotes A, C, G, or T) triplet-containing exon 1 of HOXD13, which results in a polyalanine contraction of seven residues. Moreover, we found that the mutant HOXD13 with the p.Q317R substitution was unable to transactivate the human EPHA7 promoter. Molecular modeling data supported these experimental results. The calculated interactions energies were in agreement with the measured changes of the activity. Our data established the link between HOXD13 and two additional limb phenotypes--syndactyly type V and brachydactyly type A4--and demonstrated that a polyalanine contraction in HOXD13, most likely, led to other digital anomalies but not to synpolydactyly. We suggest the term "HOXD13 limb morphopathies" for the spectrum of limb disorders caused by HOXD13 mutations.