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The aim of this study was to explore the effects and mechanisms of different starvation treatments on the compensatory growth of Acipenser dabryanus. A total of 120 fish (60·532 (sem 0·284) g) were randomly assigned to four groups (fasting 0, 3, 7 or 14 d and then refed for 14 d). During fasting, middle body weight decreased significantly with prolonged starvation. The whole-body and muscle composition, serum biochemical indexes, visceral indexes and digestive enzyme activities had been effected with varying degrees of changes. The growth hormone (GH) level in serum was significantly increased in 14D; however, insulin-like growth factor-1 (IGF-1) showed the opposite trend. The neuropeptide Y (npy) mRNA level in brain was significantly improved in 7D; peptide YY (pyy) mRNA level in intestine was significantly decreased during fasting. After refeeding, the final body weight, percentage weight gain, specific growth rate, feed intake, feed efficiency and protein efficiency ratio showed no difference between 0D and 3D. The changes of whole-body and muscle composition, serum biochemical indexes, visceral indexes and digestive enzyme activities had taken place in varying degrees. GH levels in 3D and 7D were significantly higher than those in the 0D; the IGF-1 content decreased significantly during refeeding. There was no significant difference in npy and pyy mRNA levels. These results indicated that short-term fasting followed by refeeding resulted in full compensation and the physiological and biochemical effects on A. dabryanus were the lowest after 3 d of starvation and 14 d of refeeding. Additionally, compensation in A. dabryanus may be mediated by appetite genes and GH, and the degree of compensation is also affected by the duration of starvation.

Atherosclerosis is characterized by the pathological accumulation of cholesterol-laden macrophages in the arterial wall. Atherosclerosis is also the main underlying cause of CVDs, and its development is largely driven by elevated plasma cholesterol. Strong epidemiological data find an inverse association between plasma β-carotene with atherosclerosis, and we recently showed that β-carotene oxygenase 1 (BCO1) activity, responsible for β-carotene cleavage to vitamin A, is associated with reduced plasma cholesterol in humans and mice. In this study, we explore whether intact β-carotene or vitamin A affects atherosclerosis progression in the atheroprone LDLR-deficient mice. Compared with control-fed Ldlr(−/−) mice, β-carotene-supplemented mice showed reduced atherosclerotic lesion size at the level of the aortic root and reduced plasma cholesterol levels. These changes were absent in Ldlr(−/−)/Bco1(−/−) mice despite accumulating β-carotene in plasma and atherosclerotic lesions. We discarded the implication of myeloid BCO1 in the development of atherosclerosis by performing bone marrow transplant experiments. Lipid production assays found that retinoic acid, the active form of vitamin A, reduced the secretion of newly synthetized triglyceride and cholesteryl ester in cell culture and mice. Overall, our findings provide insights into the role of BCO1 activity and vitamin A in atherosclerosis progression through the regulation of hepatic lipid metabolism.

The use of animal serum in culture medium brings safety concerns and batch-to-batch variability, and thus may restrict the clinical use of ex vivo expanded mesenchymal stromal cells (MSCs). Clinically compliant MSCs should be developed in adherence to serum-free, xeno-free and chemically defined medium (S&XFM-CD). In this study, we develop a S&XFM-CD by replacing all serum components with synthetic alternatives for the derivation of clinical-grade umbilical cord-derived MSCs (UCMSCs). The critical aspects including characterization, safety concerns, potency and exogenous factors contamination risk of UCMSCs in S&XFM-CD are compared with serum-containing medium (SCM). UCMSCs in S&XFM-CD retain fibroblastic-like morphology and immunophenotype of MSCs, and exhibit superior clone efficiency, proliferation capacity, and osteogenic and chondrogenic differentiation potential compared with SCM. Moreover, UCMSCs in S&XFM-CD retain similar immunosuppressive potential, and exhibit superior secretion levels of bFGF, PDGF-BB and IGF-1 compared with SCM. In addition, UCMSCs in S&XFM-CD do not undergo transformation, preserve the normal karyotypes and genomic stability, and are less prone to senescence process after long-term in vitro culture, which conforms to the current guidance of international and national evaluation standard. The S&XFM-CD developed here may serve as a GMP-grade production platform of UCMSCs for future clinical application.

Tropomyosin receptor kinase A (Trk A) is a receptor tyrosine kinase activated by nerve growth factor (NGF). TrkA plays an important role in pain sensation, which leads to significant interest in the development of small molecule inhibitors of TrkA. However, TrkA and the other two highly homologous isoforms, TrkB and TrkC, are highly conserved in the ATP binding site, which suggests that achieving TrkA subtype selectivity over TrkB and TrkC in this site may be extremely challenging. Allosteric inhibitors without making any interactions with the conserved ATP binding site may present a more promising approach. Recently, selective TrkA inhibitors

Since China initiated new health-care reforms in early 2009, a variety of measures have been implemented to slow the growth of medical expenses. This study was conducted to investigate the effect of controlling medical expenses. Based on inpatients' medical expenses at the largest tertiary hospital in Shenzhen, China, from 2009 to 2017, this study analyzed the changes in medical expenses and expense structures according to payment sources (insured or self-financed), stratifying the medical expenses according to the ICD-10 classification chapters of the principal diagnoses of the inpatients in two years (2009 and 2017) in order to control for confounding diseases. The results showed that mean inpatient expenses continued to rise from 2009 to 2017, and the expenses of the self-financed group began to exceed those of the insured group after 2011. Drug and consumable expenses were still the main factors that affected inpatient expenses, and consumable expenses remarkably increased, becoming the highest proportion of expenses. New health-care reforms were effective in controlling growing medical expenses for insured patients but did not make a significant difference in the expenses of self-financed patients. The excessive use of consumables has become a new driver of growing medical expenses.

A new geminivirus was identified in common bean (Phaseolus vulgaris) showing severe stunt and leaf curling symptoms in Heilongjiang province of China, via sequencing and assembly of small RNAs. The genome of this geminivirus comprises 2,959 nucleotides (nt) and shares 21.77-54.97% nt sequence identity with other geminiviruses. The coat protein (CP) shares the highest amino acid (aa) sequence identity (23.5%) with that of sesame curly top virus (SeYMV; genus Turncurtovirus), whereas the C1 (Rep) shares the highest aa sequence identity (66.5%) with that of beet severe curly top virus (BSCTV; genus Curtovirus). This geminivirus neighbors the turncurtoviruses in phylogenetic trees based on the full genome sequence or the amino acid sequence of the Rep protein, but it forms a distinct clade in the phylogenetic tree based on the coat protein. Recombination analysis showed that parts of the C1 coding region of this geminivirus were recombined from a curtovirus or turncurtovirus. Based on these results, the name "common bean curly stunt virus" (CBCSV) is proposed for this virus.

DNMDP and related compounds, or velcrins, induce complex formation between the phosphodiesterase PDE3A and the SLFN12 protein, leading to a cytotoxic response in cancer cells that express elevated levels of both proteins. The mechanisms by which velcrins induce complex formation, and how the PDE3A-SLFN12 complex causes cancer cell death, are not fully understood. Here, we show that PDE3A and SLFN12 form a heterotetramer stabilized by binding of DNMDP. Interactions between the C-terminal alpha helix of SLFN12 and residues near the active site of PDE3A are required for complex formation, and are further stabilized by interactions between SLFN12 and DNMDP. Moreover, we demonstrate that SLFN12 is an RNase, that PDE3A binding increases SLFN12 RNase activity, and that SLFN12 RNase activity is required for DNMDP response. This new mechanistic understanding will facilitate development of velcrin compounds into new cancer therapies., The small molecule DNMDP acts as a velcrin by inducing complex formation between phosphodiesterase PDE3A and SLFN12, which kills cancer cells that express sufficient levels of both proteins. Here, the authors present the cryo-EM structure of the DNMDP-stabilized PDE3A-SLFN12 complex and show that SLFN12 is an RNase. PDE3A binding increases SLFN12 RNase activity, and SLFN12 RNase activity is required for DNMDP-mediated cancer cell killing.

Extensive clinical efforts have been made to control the severity of dengue diseases; however, the dengue morbidity and mortality have not declined. Dengue virus (DENV) can infect and cause systemic damage in many organs, resulting in organ failure. Here, we present a novel report showing a tailored stem-cell-based therapy that can aid in viral clearance and rescue liver cells from further damage during dengue infection. We administered a combination of hematopoietic stem cells and endothelial progenitor cells in a DENV-infected BALB/c mouse model and found that delivery of this cell cocktail had improved their liver functions, confirmed by hematology, histopathology, and next-generation sequencing. These stem and progenitor cells can differentiate into target cells and repair the damaged tissues. In addition, the regime can regulate endothelial proliferation and permeability, modulate inflammatory reactions, enhance extracellular matrix production and angiogenesis, and secrete an array of growth factors to create an enhanced milieu for cell reparation. No previous study has been published on the treatment of dengue infection using stem cells combination. In conclusion, dengue-induced liver damage was rescued by administration of stem cell therapy, with less apoptosis and improved repair and regeneration in the dengue mouse model.

Background: Higher serum gamma-glutamyltransferase (GGT) is associated with high risk of hypertension. We aimed to examine the association between sex-specific serum GGT levels and incident of hypertension in a Chinese population without metabolic syndrome.Methods: Participants who were free of hypertension and metabolic syndrome from the First Affiliated Hospital of Wenzhou Medical University between 2009 and 2014 were included. Participants were grouped into sex-specific quartiles of GGT levels (Q1–Q4) defined as: ≤19, 20–26, 27–38, and ≥39 U/L for male; ≤12, 13–15, 16–19, and ≥20 U/L for female. Cox proportional hazards models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for incidence of hypertension according to sex-specific quartiles of GGT levels. Kaplan–Meier analysis and interaction analysis were conducted.Results: Among 38,806 participants included (average age 36.0 years, 54.0% men), 4,505 of them developed hypertension. In the overall study population, using Q1 as the reference group, participants in Q2, Q3, and Q4 showed a higher risk of developing hypertension, with HRs (95% CIs) of 1.126 (1.029–1.232), 1.187 (1.083–1.302), and 1.300 (1.182–1.431), respectively (P < 0.001), after adjusting for known confounders. Sex-specific analysis showed that the adjusted HRs for participants in Q4 (reference: Q1) were greater in females [1.321 (1.100–1.586, P < 0.001)] than in males [1.268 (1.133–1.420, P < 0.001)] (P for interaction = 0.047). Moreover, interaction analysis showed that this association was consistently observed when the participants were stratified by age, body mass index, and fatty liver status.Conclusion: Among Chinese adults without metabolic syndrome, serum GGT level was positively associated with incidence of hypertension, and the association was stronger in females than in males.

The head-like inflorescence in C. × morifolium was considered as a pseudianthia that bears numerous types of floral organs. ABCE-class and CYC2-like genes have been proven to play a key role in the identity of floral organs. To further understand their roles in capitulum development, expression pattern and protein interactions of ABCE-class and CYC2-like genes were detected using quantitative PCR and yeast-two hybrid. A-class genes, DenFUL and ClAP2, particularly accumulated in ray floret corollas and formed heterodimers with CYC2-like proteins. CYC2-like genes are particularly expressed in ray floret corollas, and these proteins form homodimers and heterodimers. The present study indicated that A-class genes in chrysanthemums interacted with CYC2-like genes and might be involved in the identity of reproductive organs and ray florets, especially the differentiation of disc and ray floret corollas. Moreover, the floral organ identity model in Chrysanthemums was proposed based on insights from the gene expression and protein interactions of ABCE-class and CYC2-like genes.

Introduction Endothelin receptor type B (EDNRB) is a potential target gene of miR-124-3p, but the association between miR-124-3p and EDNRB has not yet been reported. The aim of this study was to investigate the role of miR-124-3p in bladder cancer (BC) and to determine whether miR-124-3p regulates cell proliferation by targeting EDNRB. Material and methods Bladder cancer tissues and cell lines were obtained in order to analyze the miR-124-3p and EDNRB expression through quantitative RT-PCR (qRT-PCR) and western blotting analysis. The dual-luciferase reporter assay was employed to confirm the relationship between miR-124-3p and EDNRB. The manipulation of miR-124-3p and EDNRB expression was achieved through cell transfection. Cell proliferation and apoptosis were evaluated by MTS assay, colony forming assay and flow cytometry. A nude mouse tumorigenicity assay was used to detect the effects of miR-124-3p in vivo. Results There was an inverse correlation between the expression of miR-124-3p and EDNRB; miR-124-3p was down-regulated and EDNRB was up-regulated in BC tissues and cell lines. MiR-124-3p was observed to target EDNRB and suppress its expression. Other studies have suggested that the transfection of miR-124-3p mimics and EDNRB siRNA can suppress BC cell proliferation and induce cell apoptosis. Conclusions miR-124-3p regulates the proliferation and apoptosis of BC cells by suppressing EDNRB expression.

Background Within the past years, umbilical cord (UC) and amniotic membrane (AM) expanded in human platelet lysate (PL) have been found to become increasingly candidate of mesenchymal stromal cells (MSCs) in preclinical and clinical studies. Different sources of MSCs have different properties, and lead to different therapeutic applications. However, the similarity and differences between the AMMSCs and UCMSCs in PL remain unclear. Results In this study, we conduct a direct head-to-head comparison with regard to biological characteristics (morphology, immunophenotype, self-renewal capacity, and trilineage differentiation potential) and immunosuppression effects of AMMSCs and UCMSCs expanded in PL. Our results indicated that AMMSCs showed similar morphology, immunophenotype, proliferative capacity and colony efficiency with UCMSCs. Moreover, no significantly differences in osteogenic, chondrogenic and adipogenic differentiation potential were observed between the two types of cells. However, AMMSCs exhibited higher PGE2 expression and IDO activity compared with UCMSCs when primed by IFN-γ and (or) TNF-α induction, and AMMSCs showed a higher inhibitory effect on PBMCs proliferation than UCMSCs. Conclusion The results suggest that AMMSCs expanded in PL showed similar morphology, immunophenotype, self-renewal capacity, and trilineage differentiation potential with UCMSCs. However, AMMSCs possessed superior immunosuppression effects in comparison with UCMSCs. These results suggest that AMMSCs in PL might be more suitable than UCMSCs for treatment of immune diseases. This work provides a novel insight into choosing the appropriate source of MSCs for treatment of immune diseases. Electronic supplementary material The online version of this article (10.1186/s12860-018-0178-8) contains supplementary material, which is available to authorized users.

Flower type is an important quality trait of ornamental plants. Mastering genetic variation in flower types is of practical significance for breeding improved flower types. Chrysanthemum (non-anemone type) flowers consist of several elements; both ray floret shape and the relative number of ray florets (RNRF) on the capitulum are very important factors. However, until recently, few reports have standardized chrysanthemum flower type evaluations, hindering the development of genetic studies of chrysanthemum flower types. In this study, as an important index of ray floret shape, the corolla tube merged degree (CTMD) was defined as the corolla tube length/ray floret length (CTL/RFL). In addition, the number of ray florets/number of florets (NRF/NF) was used to describe the RNRF on the capitulum. Major gene and polygene mixed inheritance analyses were conducted based on 2 F1 populations from 2 pairs of parents that strongly differed in the CTMD and RNRF. ANOVA revealed that all traits strongly significantly differed among the different hybrids, and correlation analysis revealed that the CTMD and RNRF might have evolved independently. Additionally, according to the Q-mode cluster analysis results, we divided the RNRF into 4 levels: single, semi-double I, semi-double II and double. Genetic analysis revealed that both the CTMD and RNRF could be described by a B-2 genetic model via two additive-dominance major genes. In addition, the heritability of the major genes for these traits was greater than 50%, indicating that the CTMD and RNRF were controlled mainly by genetic factors. These results will provide a new theoretical basis for further improvement and breeding of chrysanthemum flower types.

Replication and movement are two critical steps in plant virus infection. Recent advances in the understanding of the architecture and subcellular localization of virus-induced inclusions and the interactions between viral replication complex (VRC) and movement proteins (MPs) allow for the dissection of the intrinsic relationship between replication and movement, which has revealed that recruitment of VRCs to the plasmodesma (PD) via direct or indirect MP-VRC interactions is a common strategy used for cell-to-cell movement by most plant RNA viruses. In this review, we summarize the recent advances in the understanding of virus-induced inclusions and their roles in virus replication and cell-to-cell movement, analyze the advantages of such coreplicational movement from a viral point of view and discuss the possible mechanical force by which MPs drive the movement of virions or viral RNAs through the PD. Finally, we highlight the missing pieces of the puzzle of viral movement that are especially worth investigating in the near future.

The usage of animal serum may ultimately prevent the application of ex vivo cultured mesenchymal stromal cells (MSCs) in a clinical setting due to safety concerns and batch-to-batch variability. Increasing regulatory pressure to limit use of animal serum has been issued and serum-free, xeno-free, and chemically defined media (S&XFM-CD) is encouraged to replace serum-containing media (SCM) in the stem cell preparation process. We previously developed a S&XFM-CD for the expansion of umbilical cord-derived MSCs (UCMSCs). Different culture conditions affect the function of MSCs, which may further affect the therapeutic efficiency and mechanisms of action. In this study, we compared the therapeutic effect and mechanism of UCMSCs in S&XFM-CD (UCMSCS&XFM−CD) in experimental colitis with those in SCM (UCMSCSCM). UCMSCS&XFM−CD exhibited better therapeutic effects than UCMSCSCM by body weight, disease activity index, and histological colitis score. UCMSCS&XFM−CD or UCMSCSCM migrated to the inflammation site of injured colon, but exhibited low levels of recruitment and persistence. Systemic depletion of endogenous macrophages impaired the therapeutic effects of UCMSCSCM and UCMSCS&XFM−CD. Furthermore, UCMSCS&XFM−CD more markedly promoted intestinal macrophage polarisation from M1 to M2 phenotype to produce higher levels of IL-10 and lower levels of TNF-α in colon tissue than UCMSCSCM, while a higher level of IL-4 was produced in UCMSCSCM-treated group. UCMSCS&XFM−CD cocultured with RAW264.7 cells in a transwell system promoted the release of TSG-6 and IL-6, whereas UCMSCSCM increased PGE2 levels. Taken together, we demonstrated that UCMSCs in S&XFM-CD exhibited improved therapeutic effects with altered cytokine secretion in an experimental acute colitis model.

Gastrodin is the main active constituent of Tianma, a famous traditional Chinese herbal medicine. Our previous research has found that gastrodin is absorbed rapidly in the intestine by the sodium-dependent glucose transporter 1 (SGLT1). In the current report, gastrodin is the best glycoside compound absorbed via the glucose transport pathway. This study aimed to investigate the effect of the slight difference in chemical structure on the drug intestinal absorption via the glucose transport pathway. Traditional biopharmaceutical and computer-aided molecular docking methods were used to evaluate the intestinal absorption characteristics of three gastrodin analogues, namely, salicin, arbutin and 4-methoxyphenyl-β-D-glucoside (4-MG). The oil–water partition coefficient (logP) experiments showed that the logP values of the gastrodin analogues followed the order: 4-MG > salicin > arbutin. In vitro Caco-2 cell transport experiments demonstrated that the apparent permeability coefficient (Papp) value of arbutin was higher than those of salicin and 4-MG. In situ single-pass intestinal perfusion experiments showed that the absorption of arbutin and 4-MG was better than that of salicin and that the absorption of the three compounds in the colon was lower than that in the small intestine. Quantitative real-time polymerase chain reaction results confirmed that the SGLT1 mRNA expression in the small intestine of rats was obviously higher than that in the colon of rats. In vivo pharmacokinetic experiments demonstrated that the oral bioavailability of salicin was lower than those of arbutin and 4-MG. In vitro and in vivo experiments showed that glucose or phlorizin (SGLT1 inhibitor) could decrease the intestinal absorption of the three compounds. Contrary to the above biopharmaceutical experiments, the computer-aided molecular docking test showed that the affinity of salicin to the vSGLT receptor was stronger than those of arbutin and 4-MG. In conclusion, the SGLT1 can facilitate the intestinal absorption of salicin, arbutin and 4-MG, and the slight difference in chemical structure can affect absorption.

Neutrophil extracellular traps (NETs) promote inflammation and atherosclerosis progression. NETs are increased in diabetes and impair the resolution of inflammation during wound healing. Atherosclerosis resolution, a process resembling wound healing, is also impaired in diabetes. Thus, we hypothesized that NETs impede atherosclerosis resolution in diabetes by increasing plaque inflammation. Indeed, transcriptomic profiling of plaque macrophages from NET(+) and NET(–) areas in low-density lipoprotein receptor–deficient (Ldlr(–/–)) mice revealed inflammasome and glycolysis pathway upregulation, indicating a heightened inflammatory phenotype. We found that NETs declined during atherosclerosis resolution, which was induced by reducing hyperlipidemia in nondiabetic mice, but they persisted in diabetes, exacerbating macrophage inflammation and impairing resolution. In diabetic mice, deoxyribonuclease 1 treatment reduced plaque NET content and macrophage inflammation, promoting atherosclerosis resolution after lipid lowering. Given that humans with diabetes also exhibit impaired atherosclerosis resolution with lipid lowering, these data suggest that NETs contribute to the increased cardiovascular disease risk in this population and are a potential therapeutic target.

Long non-coding RNAs (lncRNAs) have been extensively studied in recent years. Numerous lncRNAs have been identified in mice, rats, and humans, some of which play important roles in muscle formation and development. However, little is known about lncRNA regulators that affect muscle development in yak (Bos grunniens). LncRNA expression during skeletal muscle development in yak was analyzed by RNA sequencing at three development stages: 3 years (group A), 6 months (group M), and 90-day-old fetuses (group E). A total of 1180 lncRNAs were identified in the three development stages. Compared with group E, 154 were upregulated and 130 were downregulated in group A. Compared with group A, 31 were upregulated and 29 were downregulated in group M. Compared with group E, 147 were upregulated and 149 were downregulated in group M (padj < 0.001, |log2FC| > 1.2). In addition, functional annotation analysis based on gene ontology (GO) and the Kyoto protocol encyclopedia of genes and genomes (KEGG) database showed that differentially expressed lncRNAs (DElncRNAs) were cis–trans target genes. The results showed that DElncRNAs were mainly involved in PI3K-Akt signaling pathway, focal adhesion, MAPK signaling pathway, apoptosis, and p53 signaling pathway. Furthermore, RTL1, IGF2, MEF2C, Pax7, and other well-known muscle development regulators were included in a co-expression network of differentially expressed target genes and lncRNAs. These data will help to further clarify the function of lncRNAs in the different stages of skeletal muscle developmental in yak.

P3N-PIPO, the only dedicated movement protein (MP) of potyviruses, directs cylindrical inclusion (CI) protein from the cytoplasm to the plasmodesma (PD), where CI forms conical structures for intercellular movement. To better understand potyviral cell-to-cell movement, we further characterized P3N-PIPO using Turnip mosaic virus (TuMV) as a model virus. We found that P3N-PIPO interacts with P3 via the shared P3N domain and that TuMV mutants lacking the P3N domain of either P3N-PIPO or P3 are defective in cell-to-cell movement. Moreover, we found that the PIPO domain of P3N-PIPO is sufficient to direct CI to the PD, whereas the P3N domain is necessary for localization of P3N-PIPO to 6K2-labeled vesicles or aggregates. Finally, we discovered that the interaction between P3 and P3N-PIPO is essential for the recruitment of CI to cytoplasmic 6K2-containing structures and the association of 6K2-containing structures with PD-located CI inclusions. These data suggest that both P3N and PIPO domains are indispensable for potyviral cell-to-cell movement and that the 6K2 vesicles in proximity to PDs resulting from multipartite interactions among 6K2, P3, P3N-PIPO, and CI may also play an essential role in this process. IMPORTANCE Potyviruses include numerous economically important viruses that represent approximately 30% of known plant viruses. However, there is still limited information about the mechanism of potyviral cell-to-cell movement. Here, we show that P3N-PIPO interacts with and recruits CI to the PD via the PIPO domain and interacts with P3 via the shared P3N domain. We further report that the interaction of P3N-PIPO and P3 is associated with 6K2 vesicles and brings the 6K2 vesicles into proximity with PD-located CI structures. These results support the notion that the replication and cell-to-cell movement of potyviruses are processes coupled by anchoring viral replication complexes at the entrance of PDs, which greatly increase our knowledge of the intercellular movement of potyviruses.

Aiming to explore novel BRAFV600E and VEGFR-2 dual inhibitors, a series of 1H-pyrazolo[3,4-d]pyrimidine derivatives were designed, synthesized and biologically evaluated in this study. Most of the synthesized 1H-pyrazolo[3,4-d]pyrimidine compounds displayed moderate to high potent activity in both enzymatic and cellular proliferation assays. Among these compounds, 9e, 9g, 9m and 9u showed remarkably high inhibitory activities against both BRAFV600E and VEGFR-2 kinase comparable to positive control Sorafenib. Particularly, compound 9u also showed potent anti-proliferative activity against BRAFV600E-expressing A375 (IC50 = 1.74 μM) and H-29 (IC50 = 6.92 μM) as well as VEGFR-2-expressing HUVEC (IC50 = 5.89 μM), which was also comparable to Sorafenib. Furthermore, kinase selectivity profile showed that 9u had almost poor or no significant inhibitory activity against wild-type BRAF and 15 other tested protein kinases. Flow cytometric analysis showed that compound 9u mainly arrested the A375 and HUVEC cell lines in the G0/G1 stage with a concentration-dependent effect. In addition, the molecular docking and molecular dynamics simulations suggested that 9u adopted a similar binding pattern with Sorafenib at the ATP-binding sites of BRAFV600E and VEGFR-2. Taken together, these results indicated that compound 9u may serve as novel lead compound in research on more effective BRAFV600E and VEGFR-2 dual inhibitors.

The design and synthesis of a novel series of 2,6-disubstituted pyrazine derivatives as CK2 kinase inhibitors is described. Structure-guided optimization of a 5-substituted-3-thiophene carboxylic acid screening hit (3a) led to the development of a lead compound (12b), which shows inhibition in both enzymatic and cellular assays. Subsequent design and hybridization efforts also led to the unexpected identification of analogs with potent PIM kinase activity (14f).

Through sequencing and assembly of small RNAs, an orthotospovirus was identified from a celtuce plant (Lactuca sativa var. augustana) showing vein clearing and chlorotic spots in the Zhejiang province of China. The S, M, and L RNAs of this orthotospovirus were determined to be 3146, 4734, and 8934 nt, respectively, and shared 30.4-72.5%, 43.4-80.8%, and 29.84-82.9% nucleotide sequence identities with that of known orthotospoviruses. The full length nucleoprotein (N) of this orthotospovirus shared highest amino acid sequence identity (90.25%) with that of calla lily chlorotic spot virus isolated from calla lily (CCSV-calla) [China: Taiwan: 2001] and tobacco (CCSV-LJ1) [China: Lijiang: 2014]. Phylogenetic analyses showed that this orthotospovirus is phylogenetically associated with CCSV isolates and clustered with CCSV, tomato zonate spot virus (TZSV), and tomato necrotic spot-associated virus (TNSaV) in a separate sub-branch. These results suggest that this orthotospovirus is a divergent isolate of CCSV and was thus named CCSV-Cel [China: Zhejiang: 2017].

With the aim of discovering potential and selective inhibitors targeting ROS1 kinase, we rationally designed, synthesized and evaluated two series of novel 2-amino-pyridine derivatives with 1-phenylethoxy at C-3 and C-4 position. The enzymic assays results indicated that six of the new compounds 13b-13d and 14a-14c showed remarkably higher inhibitory activities against ROS1 kinase. The most promising compounds, 13d and 14c displayed the most desired ROS1 inhibitory activity with IC50 values of 440 nM and 370 nM respectively. Furthermore, 13d and 14c displayed ROS1 inhibitory selectivity of about 7-fold and 12-fold, relative to that of ALK sharing about 49% amino acid sequence homology in the kinase domains. They also showed good anti-proliferative effects against ROS1-addicted HCC78 cell lines with the IC50 values of 8.1 μM and 65.3 μM, respectively. Moreover, molecular docking and molecular dynamics simulation studies disclosed that compound 14c and 13d shared similar binding poses with Crizotinib except the selective binding site of ROS1. It also gave a probable molecular explanation for their activity and selectivity, which the methoxyl group in benzene ring was the crucial to the selectivity to ROS1 versus ALK.

MuS110 and MT110 are BiTE antibodies bispecific for CD3 and EpCAM, which is the most frequently and highly expressed tumor-associated antigen on breast cancer. And pronounced expression of IDO1 has also been reported in breast cancer. Our study aimed to investigate whether IDO1 inhibitor D-1MT combing with MuS110/MT110 had synergistic antitumor effects on IDO expressing EpCAM-positive breast cancer cells in vitro and in vivo. Data suggested that the expression of IDO1 on Epcam-positive breast cancer 4T1 and MCF-7 decreased MuS110/MT110 antitumor efficacy by the suppression of T cells activation in vitro. Combining D-1MT with MT110 in IDO+MCF-7 cells, or with MuS110 in IDO+4T1 cells, significantly improved the antitumor efficacy of BiTE antibodies via increasing T cell cytotoxicity and contributing to cytokines releasing. In vivo assay, combination of D-1MT with MT110 in NOD/SCID mice bearing IDOhi MCF-7 xenografts or MuS110 in immune competent BALB/c mice bearing IDOhi 4T1 xenografts suggested the similar synergistic effect. Together, IDO inhibition could reverse the suppression of T cells due to IDO expressing on breast cancer, and improve the antitumor efficacy of EpCAM/CD3-bispecific BiTE antibody.

Abscisic acid plays a crucial role in the regulation of fruit development and ripening, however, its role in the floral development and the fruit set is still unclear. In the present study, the ABA accumulation and the expression patterns of genes related to ABA metabolism and signalling in sweet cherry were investigated. The results showed that ABA accumulation increased and peaked at stage V in ovary, at stage VI in stamen, and in young fruit it peaked at 7 days after full bloom. The expression pattern of ABA synthetase PaNCED1 was consistent with the changes of ABA accumulation. Among four ABA degradation enzymes PaCYP707As, PaCYP707A4 was highly expressed in ovary, PaCYP707A1 was mainly in stamen, and PaCYP707A2 was in young fruit, and their expressions were reversed to the trend of PaNCED1. With regard to ABA signalling genes, among three ABA receptors PaPYLs, PaPYL2 and PaPYL3 were high expression genes in ovary and in young fruit with similar expression patterns, while PaPYL3 was the high expression gene in stamen. Within six PaPP2Cs, PaPP2C1/2/3 were highly expressed in ovary and young fruit, while PaPP2C3/4 were mainly in stamen. The six PaSnRK2s showed different expression patterns: PaSnRK2.1/2.2/2.4 were highly expressed in ovary and young fruit, while PaSnRK2.1/2.3 were highly expressed in stamen. In situ hybridization results showed that PaPYL3, PaPP2C3 and PaSnRK2.4 were expressed in seed, pulp and fruit peel during fruit set. In conclusion, ABA and its signaling may play an important role in the regulation of floral development and fruit set.

Acipenser dabryanus, also called Yangtze sturgeon or Dabry's sturgeon, is an endemic species restricted to the upper reaches of the Yangtze River in China. In the present study, we conducted the first full-length transcriptome analysis of A. dabryanus by the use of SMRT sequencing technology. Transcriptome sequencing generated a total of 155,348 unigenes with a mean length of 2996 bp. A total of 131,711 (84.78%) unigenes were annotated in various databases and comparative analysis was performed between A. dabryanus and its the related species. Based on the sequence information, three members in the tripartite motif (TRIM) family, namely, TRIM32, TRIM36 and TRIM71, were identified. The full-length TRIM genes encoded polypeptides with a typical TRIM structure: a RING domain, one or two B-box domains, and variable domains at the C-terminus. Gene expression profile analysis showed that the three TRIM genes were constitutively expressed in A. dabryanus, and the highest expression levels of TRIM32 and TRIM36 were found in the brain, while TRIM71 was predominantly expressed in the muscle. Upon challenge with the bacterial pathogen Edwardsiella tarda, significant upregulation of TRIMs was observed in both mucosal and systemic-associated immune tissues. Our data revealed the possible functional role of TRIMs in both mucosal and systemic defence of A. dabryanus in the immune response against bacterial infection.

BRAFV600E small-molecule inhibitors have recently received increasing attention as very effective agents for the therapy of cancer, especially melanoma. In this current work, a computational investigation was performed to investigate the interaction details of BRAFV600E kinase with inhibitors Vemurafenib (PLX4032) and its analog PLX4720. Binding free energy calculations based on molecular dynamics simulations reveal that ligands PLX4032 and PLX4720 bind to and stabilize the DFG-in conformation of BRAFV600E. Component analysis of binding free energy revealed that vdWaals interactions play a dominating effect on BRAFV600E inhibition. Furthermore, the per-residue binding free energy decomposition revealed that the most favorable contribution came from Ile463, Val471, Ala481, Lys483, Leu514, Ile527, Thr529, Gln530, Trp531, Cys532, Phe583, Gly593, and Asp594. These results agree well with experimental data, which provide valuable resources for understanding the inhibition mechanism of BRAFV600E by PLX4032 and PLX4720 and clues for the design of novel potent BRAFV600E inhibitors.

Anti-cancer uses of non-oncology drugs have been found on occasion, but such discoveries have been serendipitous and rare. We sought to create a public resource containing the growth inhibitory activity of 4,518 drugs tested across 578 human cancer cell lines. To accomplish this, we used PRISM, which involves drug treatment of molecularly barcoded cell lines in pools. Relative barcode abundance following treatment thus reflects cell line viability. We found that an unexpectedly large number of non-oncology drugs selectively inhibited subsets of cancer cell lines. Moreover, the killing activity of the majority of these drugs was predictable based on the molecular features of the cell lines. Follow-up of several of these compounds revealed novel mechanisms. For example, compounds that kill by inducing PDE3A-SLFN12 complex formation; vanadium-containing compounds whose killing is dependent on the sulfate transporter SLC26A2; the alcohol dependence drug disulfiram, which kills cells with low expression of metallothioneins; and the anti-inflammatory drug tepoxalin, whose killing is dependent on high expression of the multi-drug resistance gene ABCB1. These results illustrate the potential of the PRISM drug repurposing resource as a starting point for new oncology therapeutic development. The resource is available at https://depmap.org.

Storage roots are the main sink for photo-assimilate accumulation and reflect cassava yield and productivity. Regulation of sugar partitioning from leaves to storage roots has not been elucidated. Cell wall invertases are involved in the hydrolysis of sugar during phloem unloading of vascular plants to control plant development and sink strength but have rarely been studied in root crops like cassava. MeCWINV3 encodes a typical cell wall invertase in cassava and is mainly expressed in vascular bundles. The gene is highly expressed in leaves, especially mature leaves, in response to diurnal rhythm. When MeCWINV3 was overexpressed in cassava, sugar export from leaves to storage roots was largely inhibited and sucrose hydrolysis in leaves was accelerated, leading to increased transient starch accumulation by blocking starch degradation and reduced overall plant growth. The progress of leaf senescence was promoted in the MeCWINV3 over-expressed cassava plants with increased expression of senescence-related genes. Storage root development was also delayed because of dramatically reduced sugar allocation from leaves. As a result, the transcriptional expression of starch biosynthetic genes such as small subunit ADP-glucose pyrophosphorylase, granule-bound starch synthase I, and starch branching enzyme I was reduced in accordance with insufficient sugar supply in the storage roots of the transgenic plants. These results show that MeCWINV3 regulates sugar allocation from source to sink and maintains sugar balance in cassava, thus affecting yield of cassava storage roots.

ALK and ROS1 kinases have become promising therapeutic targets since Crizotinib was used to treat non-small-cell lung cancer clinically. Aiming to explore new potent inhibitors, a series of 2-amino-4-(1-piperidine) pyridine derivatives that stabilized a novel DFG-shifted conformation in the kinase domain of ALK were designed and synthesized on the base of lead compound A. Biological evaluation highlighted that most of these new compounds could also potently inhibit ROS1 kinase, leading to the promising inhibitors against both ROS1 and ALK. Among them, the representative compound 2e stood out potent anti-proliferative activity against ALK-addicted H3122 and ROS1-addicted HCC78 cell lines (IC50 = 6.27 μM and 10.71 μM, respectively), which were comparable to that of Crizotinib. Moreover, 2e showed impressive enzyme activity against clinically Crizotinib-resistant ALKL1196M with an IC50 value of 41.3 nM, which was about 2-fold more potent than that of Crizotinib. 2e also showed potent inhibitory activity in about 6-fold superior to Crizotinib (IC50: 104.7 nM vs. 643.5 nM) in Ba/F3 cell line harboring ROS1G2032R. Furthermore, molecular modeling disclosed that all the representative inhibitors could dock into the active site of ALK and ROS1, which gave a probable explanation of anti Crizotinib-resistant mutants. These results indicated that our work has established a path forward for the generation of anti Crizotinib-resistant ALK/ROS1 dual inhibitors.

Plant viruses usually encode one or more movement proteins (MP) to accomplish their intercellular movement. A group of positive-strand RNA plant viruses requires three viral proteins (TGBp1, TGBp2, and TGBp3) that are encoded by an evolutionarily conserved genetic module of three partially overlapping open reading frames (ORFs), termed the triple gene block (TGB). However, how these three viral movement proteins function cooperatively in viral intercellular movement is still elusive. Using a novel in vivo double-stranded RNA (dsRNA) labeling system, we showed that the dsRNAs generated by potato virus X (PVX) RNA-dependent RNA polymerase (RdRp) are colocalized with viral RdRp, which are further tightly covered by “chain mail”-like TGBp2 aggregates and localizes alongside TGBp3 aggregates. We also discovered that TGBp2 interacts with the C-terminal domain of PVX RdRp, and this interaction is required for the localization of TGBp3 and itself to the RdRp/dsRNA bodies. Moreover, we reveal that the central and C-terminal hydrophilic domains of TGBp2 are required to interact with viral RdRp. Finally, we demonstrate that knockout of the entire TGBp2 or the domain involved in interacting with viral RdRp attenuates both PVX replication and movement. Collectively, these findings suggest that TGBp2 plays dual functional roles in PVX replication and intercellular movement. IMPORTANCE Many plant viruses contain three partially overlapping open reading frames (ORFs), termed the triple gene block (TGB), for intercellular movement. However, how the corresponding three proteins coordinate their functions remains obscure. In the present study, we provided multiple lines of evidence supporting the notion that PVX TGBp2 functions as the molecular adaptor bridging the interaction between the RdRp/dsRNA body and TGBp3 by forming “chain mail”-like structures in the RdRp/dsRNA body, which can also enhance viral replication. Taken together, our results provide new insights into the replication and movement of PVX and possibly also other TGB-containing plant viruses.

Plants are persistently challenged by various phytopathogens. To protect themselves, plants have evolved multilayered surveillance against all pathogens. For intracellular parasitic viruses, plants have developed innate immunity, RNA silencing, translation repression, ubiquitination-mediated and autophagy-mediated protein degradation, and other dominant resistance gene-mediated defenses. Plant viruses have also acquired diverse strategies to suppress and even exploit host defense machinery to ensure their survival. A better understanding of the defense and counter-defense between plants and viruses will obviously benefit from the development of efficient and broad-spectrum virus resistance for sustainable agriculture. In this review, we summarize the cutting edge of knowledge concerning the defense and counter-defense between plants and viruses, and highlight the unexploited areas that are especially worth investigating in the near future., This study is financially supported by the National Natural Science Foundation of China (grant no: 31671998), the Natural Science Foundation of Heilongjiang Province (grant no: ZD2018002), and the Scientific Research Foundation of Department of Education of Heilongjiang Province for the Returned Overseas Chinese Scholars (grant no: 2018QD0002). J.A.G. and A.V. were supported by grants BIO2016-80572-R and BIO2015-73900-JIN and the FEDER program., We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

Lung cancer is the leading cause of cancer death, and epidermal growth factor receptor (EGFR) kinase domain mutations are a common cause of non-small-cell lung cancer (NSCLC), a major subtype of lung cancers. Patients harboring most of these mutations respond well to the EGFR inhibitors Gefitinib and Erlotinib initially, but soon develop resistance to them due to the emergence of the gatekeeper mutation T790M. The new-generation inhibitors such as AZD9291, HM61713, CO-1686 and WZ4002 can overcome T790M through covalent binding to Cys 797, but ultimately lose their efficacy upon the emergence of the C797S mutation that abolishes the covalent bonding. Allosteric inhibitors EAI001 and EAI045 are a new type of EGFR inhibitors that bind to EGFR away from the ATP-binding site and not relying on Cys 797. In this study, molecular dynamics simulations and free energy calculations were carried out on EAI001 and EAI045 in complex with EGFR, revealing the detailed inhibitory mechanism of EAI001 and EAI045 as EGFR allosteric inhibitor, which was expected to provide a basis for rational drug design of the EGFR allosteric inhibitors. Communicated by Ramaswamy H. Sarma.

Non-alcoholic fatty liver disease (NAFLD) is becoming a severe public health problem that is affecting a large proportion of the world population. Generally, NAFLD in patients is usually accompanied by obesity, hyperglycemia, insulin resistance (IR) and type 2 diabetes (T2D), for which numerous animal models have been generated in order to explore the pathogenesis and therapies of NAFLD. On the contrary, quite a number of NAFLD subjects, especially in Asian regions, are non-obese and non-diabetic; however, few animal models are available for the research of non-obese NAFLD. Here, four approaches (here called approach 1 to 4) corresponding to the variable compositions of diets were used to treat tree shrews (Tupaia belangeri chinensis), which have a closer evolutionary relationship to primates than rodents. Analysis of plasma biochemical parameters, hepatic histology, and the expression of hepatic lipid metabolic genes revealed that all four approaches led to hepatic lipid accumulation, liver injury and hypercholesterolemia, but had no effect on body weight and adipose tissue generation, or glycemia. Hepatic gene expression in tree shrews treated by approach 4 might suggest a different or non-canonical pathway leading to hepatic steatosis. In conclusion, the tree shrew displays hepatic steatosis and dyslipidemia, but remains non-obese and non-diabetic under high energy diets, which suggests that the tree shrew may be useful as a novel animal model for the research of human non-obese NAFLD., Summary: This study suggests that the tree shrew (Tupaia belangeri chinensis) may be useful as a novel animal model for the research of human non-obese non-alcoholic fatty liver disease (NAFLD).

Three series of novel quinazoline and pyrido[2,3-d]pyrimidine derivatives were designed, synthesized and evaluated for their ability to inhibit EGFR tyrosine kinase and a panel of five human cancer cell lines (MCF-7, A549, BT-474, SK-BR-3, and MDA-MB-231). Bioassay results indicated that five of these prepared compounds (12c-12e and 13c-13d) exhibited remarkably higher inhibitory activities against EGFR and SK-BR-3 cell line. Compounds 12c and 12e displayed the most potent EGFR inhibitory activity (IC50 = 2.97 nM and 3.58 nM, respectively) and good anti-proliferative effect against SK-BR-3 cell with the IC50 values of 3.10 μM and 5.87 μM, respectively. Furthermore, molecular docking and molecular dynamics simulation studies verified that compound 12c and 12e shared similar binding pattern with gefitinib in the binding pocket of EGFR. MM-GBSA binding free energy revealed that the compound 12c and 12e have almost the same inhibitory activity against EGFR as gefitinib, and that the dominating effect of van der Waals interactions drives the binding process.

A novel series of covalent inhibitors of EGFR (epidermal growth factor receptor) kinase was discovered through a combination of subset screening and structure-based design. These compounds preferentially inhibit mutant forms of EGFR (activating mutant and T790M mutant) over wild-type EGFR in cellular assays measuring EGFR autophosphorylation and proliferation, suggesting an improved therapeutic index in non-small cell lung cancer patients would be achievable relative to established EGFR inhibitors. We describe our design approaches, resulting in the identification of the lead compound 5, and our efforts to develop an understanding of the structure-activity relationships within this series. In addition, strategies to overcome challenges around metabolic stability and aqueous solubility are discussed. Despite limitations in its physical properties, 5 is orally bioavailable in mice and demonstrates pronounced antitumor activity in in vivo models of mutant EGFR-driven cancers.

Examples of hydroxymethylated analogues of heteroaryl cores such as quinazolin-4-ones, isoquinolin-1(2H)-ones, pyrido[3,4-d]pyrimidin-4(3H)-ones, chromen-4-ones and pyrrolo[2,1-f][1,2,4]triazin-4(3H)-ones are sparse or non-existent in the scientific literature. We have demonstrated that such compounds are accessible by using standard procedures from readily available raw materials.

The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase (RdRp) and responsible for replicating the whole HCV genome with help of viral and cellular proteins. However, how cellular factors influence NS5B and, in turn, regulating HCV replication are still poorly defined. The well known tumor suppressor Fbw7, a component of E3 ubiquitin ligase SCF(Fbw7), targets oncoproteins or cellular regulatory proteins for ubiquitin-mediated degradation through a highly conserved binding site called a Cdc4 phosphodegron (CPD). But little is known about whether Fbw7 plays a role in regulation of viral proteins. In this study, we revealed that the conserved CPD is shared by NS5B of almost all genotype of HCV and our data demonstrated that NS5B is a bona fide substrate of Fbw7. Forced expression of Fbw7 promoted the ubiquination of NS5B and negatively regulated its turnover in the proteasome-dependent manner. We further revealed the interaction between NS5B and Fbw7, which resulted in the relocation of Fbw7 from nucleus to cytoplasm. During HCV replication, ectopic expression of Fbw7 could strongly down-regulate NS5B level and consequently inhibited the virus replication. When endogenous Fbw7 was knocked down, both NS5B protein abundance and HCV replication were remarkably up-regulated. The results provide more insights into the interplay of HCV and cellular factors and shed light on molecular mechanisms of HCV replication and pathogenesis.

A new class of 2-amino-4-(1,2,4-triazol)pyridine derivatives were designed and synthesized as potent epidermal growth factor receptor inhibitors. In particular, compound 10c exhibited significant inhibitory against EGFRL858R/T790M, and also displayed potent anti-proliferative activity against non-small cell lung cancer cell line H1975. Besides, compound 10j showed potent inhibitory activity against glioblastoma cell line U87-EGFRvⅢ, which was at least 3-fold more potent than Osimertinib and 25-fold superior to Lazertinib. Moreover, molecular modeling and molecular dynamics simulations disclosed the binding model of the most active compound to the domain of EGFR. This contribution provides 2-amino-4-(1,2,4-triazol)pyridines as a new scaffold for EGFRT790M and/or EGFRvⅢ inhibitor.

Background: Depression is an independent risk factor for atherosclerosis (AS), which can increase the risk of death and disability from AS. However, the mechanism of AS comorbidity with depression is complex. Methods: ApoE −/− and C57BL/6J mice were fed with a high-fat diet (model group, N=12 ♂) and a normal diet (control group, N=12 ♂). During the 15-week experimental period, the following tests were performed: coat color score, body weight, and sucrose preference tests (every 2 weeks); open-field test (1 st , 7 th , and 15 th weeks); and light/dark and tail suspension tests (15 th week). Oil Red O and hematoxylin and eosin (HE) stainings were used to assess the area of atherosclerotic status. The levels of triglyceride and total and low-density lipoprotein cholesterol in the serum and secretion of pro-inflammatory cytokines were determined using the enzyme-linked immunosorbent assay. The differentially expressed genes (DEGs) in the hippocampus and prefrontal cortex were screened by RNA-sequencing (RNA-seq) and analyzed using the Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations. Results: Our findings showed that compared with C57 mice in the control group, ApoE −/− mice in the model group gradually developed depression-like behavioral changes with elevated blood lipid concentrations, serum inflammatory factor levels, and atherosclerotic plaque formation in the thoracic aorta. Consequently, in the RNA-seq and bioinformatics analysis, the high expression of inflammatory chemokine genes was found in the hippocampus and prefrontal cortex area. The regulation of movement, feeding, and reproduction of the gene expression decreased. Conclusions: These results indicate that when ApoE −/− mice were fed a high-fat diet for 15 weeks, depression-like behavioral changes occurred with the formation of atherosclerotic lesions. The RNA-seq, combined with bioinformatics analysis, showed that this AS comorbidity with depressive behavior was associated with the high expression of inflammation-related genes and pathways in the hippocampus and prefrontal cortex.

The c-ros oncogene 1 (ROS1) has proven to be an important cancer target for the treatment of various human cancers. The anaplastic lymphoma kinase inhibitor crizotinib has been granted approval for the treatment of patients with ROS1 positive metastatic non-small-cell lung cancer by the Food and Drug Administration on 2016. However, serious resistance due to the secondary mutation of glycine 2032 to arginine (G2032R) was developed in clinical studies. Loratinib (PF-06463922), a macrocyclic analog of crizotinib, showed significantly improved inhibitory activity against wild-type (WT) ROS1 and ROS1G2032R mutant. To provide insights into the inhibition mechanism, molecular dynamics simulations and free energy calculations were carried out for the complexes of loratinib with WT and G2032R mutated ROS1. The apo-ROS1WT and apo-ROS1G2032R systems showed similar RMSF distributions, while ROS1G2032R-loratinib showed significantly higher than that of WT ROS1-loratinib, which revealed that the binding of loratinib to ROS1G2032R significantly interfered the fluctuation of protein. Calculations of binding free energies indicate that G2032R mutation significantly reduces the binding affinity of loratinib for ROS1, which arose mostly from the increase of conformation entropy and the decrease of solvation energy. Furthermore, detailed per-residue binding free energies highlighted the increased and decreased contributions of some residues in the G2032R mutated systems. The present study revealed the detailed inhibitory mechanism of loratinib as potent WT and G2032R mutated ROS1 inhibitor, which was expected to provide a basis for rational drug design.

Plasticity of the cell state has been proposed to drive resistance to multiple classes of cancer therapies, thereby limiting their effectiveness. A high-mesenchymal cell state observed in human tumours and cancer cell lines has been associated with resistance to multiple treatment modalities across diverse cancer lineages, but the mechanistic underpinning for this state has remained incompletely understood. Here we molecularly characterize this therapy-resistant high-mesenchymal cell state in human cancer cell lines and organoids and show that it depends on a druggable lipid-peroxidase pathway that protects against ferroptosis, a non-apoptotic form of cell death induced by the build-up of toxic lipid peroxides. We show that this cell state is characterized by activity of enzymes that promote the synthesis of polyunsaturated lipids. These lipids are the substrates for lipid peroxidation by lipoxygenase enzymes. This lipid metabolism creates a dependency on pathways converging on the phospholipid glutathione peroxidase (GPX4), a selenocysteine-containing enzyme that dissipates lipid peroxides and thereby prevents the iron-mediated reactions of peroxides that induce ferroptotic cell death. Dependency on GPX4 was found to exist across diverse therapy-resistant states characterized by high expression of ZEB1, including epithelial-mesenchymal transition in epithelial-derived carcinomas, TGFβ-mediated therapy-resistance in melanoma, treatment-induced neuroendocrine transdifferentiation in prostate cancer, and sarcomas, which are fixed in a mesenchymal state owing to their cells of origin. We identify vulnerability to ferroptic cell death induced by inhibition of a lipid peroxidase pathway as a feature of therapy-resistant cancer cells across diverse mesenchymal cell-state contexts.

Like other positive-strand RNA viruses, plant potyviruses assemble viral replication complexes (VRCs) on modified cellular membranes. Potyviruses encode two membrane proteins, 6K2 and P3. The former is known to play pivotal roles in the formation of membrane-associated VRCs. However, P3 remains to be one of the least characterized potyviral proteins. The P3 cistron codes for P3 as well as P3N-PIPO which results from RNA polymerase slippage. In this study, we show that the P3N-PIPO of Turnip mosaic virus (TuMV) is required for viral cell-to-cell movement but not for viral replication. We demonstrate that the C-terminal region of P3 (P3C) is indispensable for P3 to form cytoplasmic punctate inclusions and target VRCs. We reveal that TuMV mutants that lack P3C are replication-defective. Taken together, these data suggest that the P3 cistron has two distinct functions: P3N-PIPO as a dedicated movement protein and P3 as an essential component of the VRC.

SUMMARYWe previously piloted the concept of a Connectivity Map (CMap), whereby genes, drugs and disease states are connected by virtue of common gene-expression signatures. Here, we report more than a 1,000-fold scale-up of the CMap as part of the NIH LINCS Consortium, made possible by a new, low-cost, high throughput reduced representation expression profiling method that we term L1000. We show that L1000 is highly reproducible, comparable to RNA sequencing, and suitable for computational inference of the expression levels of 81% of non-measured transcripts. We further show that the expanded CMap can be used to discover mechanism of action of small molecules, functionally annotate genetic variants of disease genes, and inform clinical trials. The 1.3 million L1000 profiles described here, as well as tools for their analysis, are available at https://clue.io.HIGHLIGHTSA new gene expression profiling method, L1000, dramatically lowers costThe Connectivity Map database now includes 1.3 million publicly accessible L1000 perturbational profilesThis expanded Connectivity Map facilitates discovery of small molecule mechanism of action and functional annotation of genetic variantsThe work establishes feasibility and utility of a truly comprehensive Connectivity Map

We previously piloted the concept of a Connectivity Map (CMap), whereby genes, drugs, and disease states are connected by virtue of common gene-expression signatures. Here, we report more than a 1,000-fold scale-up of the CMap as part of the NIH LINCS Consortium, made possible by a new, low-cost, high-throughput reduced representation expression profiling method that we term L1000. We show that L1000 is highly reproducible, comparable to RNA sequencing, and suitable for computational inference of the expression levels of 81% of non-measured transcripts. We further show that the expanded CMapcan be used to discover mechanism of action of small molecules, functionally annotate genetic variants ofdisease genes, and inform clinical trials. The 1.3 million L1000 profiles described here, as well as tools for their analysis, are available at https://clue.io.

RAF (Ras activating factor) kinases are important and attractive targets for cancer therapy. With the aim of discovering RAF inhibitors that bind to DFG-out inactive conformation created by the movement of Asp-Phe-Gly (DFG), we conducted structure-based drug design using the X-ray cocrystal structures of BRAF (v-raf murine sarcoma viral oncogene homolog B1), starting from bisarylurea derivative based on 1H-pyrazolo[3,4-d]pyrimidine scaffold 1a. Most of the synthesized compounds showed good to excellent inhibitory activities against BRAFV600E kinase, possessed moderate to potent anti-proliferative activities against four tumor cell lines (A375, HT-29, PC-3 and A549) and good selectivity towards cancer cells rather normal cells (Madin-Darby canine kidney, MDCK). The most promising compound, 1v, exhibited potent inhibitory activity against not only BRAFV600E (half maximal inhibitory concentration, IC50 = 23.6 nM) but also wild-type BRAF (IC50 = 51.5 nM) and C-RAF (IC50 = 8.5 nM), and effective cellular anti-proliferative activities against A375, HT-29, PC-3 and A549 cell lines as well as a very good selectivity profile. Moreover, compound 1v mainly arrested the A375 cell line in the G0/G1 stage, and showed significant suppression of MEK (mitogen-activated protein kinase kinase) phosphorylation in A375 and HT-29 cell lines. Taken together, the optimal compound 1v showed excellent in vitro potency as a pan-RAF inhibitor. In addition, the promise of compound 1v was further confirmed by molecular dynamics simulation and binding free energy calculations.

Amphotericin B (AmpB) nephrotoxicity was used to assess the utility of drug‑induced kidney injury (DIKI) biomarkers in an exploratory study in male cynomolgus monkeys. All animals had quantifiable levels of AmpB in plasma on days 1 and 4. There were no clinical signs of AmpB‑induced toxicity in this study. The gold standard method used to confirm AmpB‑induced DIKI was anatomic pathology which revealed microscopic lesions with varying grades of severity. Immunolocalization of alpha‑1 microglobulin (α‑1M), kidney injury molecule 1 (KIM‑1), osteopontin (OPN) and neutrophil gelatinase‑associated lipocalin (NGAL) proteins was evaluated in formalin‑fixed, paraffin‑embedded monkey kidney tissue sections. AmpB related immunoreactivities were identified in distinct nephron segments of treated monkeys including α‑1M in damaged proximal tubule epithelium, KIM‑1 in damaged medullary tubule epithelium, OPN mostly in the infiltrating cells of cortical tubule interstitium, and NGAL in the granular and cellular cast in dilatated cortical tubules. Variations in α‑1M, KIM‑1, OPN and NGAL immunolocalization appear as promising DIKI protein biomarkers when monitoring for AmpB‑induced corticomedullary tubule injury in male cynomolgus monkeys.

We hypothesized that human genes and disease-associated alleles might be systematically functionally annotated using morphological profiling of cDNA constructs, via a microscopy-based Cell Painting assay. Indeed, 50% of the 220 tested genes yielded detectable morphological profiles, which grouped into biologically meaningful gene clusters consistent with known functional annotation (e.g., the RAS-RAF-MEK-ERK cascade). We used novel subpopulation-based visualization methods to interpret the morphological changes for specific clusters. This unbiased morphologic map of gene function revealed TRAF2/c-REL negative regulation of YAP1/WWTR1-responsive pathways. We confirmed this discovery of functional connectivity between the NF-κB pathway and Hippo pathway effectors at the transcriptional level, thereby expanding knowledge of these two signaling pathways that critically regulate tumor initiation and progression. We make the images and raw data publicly available, providing an initial morphological map of major biological pathways for future study. DOI: http://dx.doi.org/10.7554/eLife.24060.001, eLife digest Many human diseases are caused by particular changes, called mutations, in patients’ DNA. A genome is the complete DNA set of an organism, which contains all the information to build the body and keep it working. This information is stored as a code made up of four chemicals called bases. Humans have about 30,000 genes built from DNA, which contain specific sequences of bases. Genome sequencing can determine the exact order of these bases, and has revealed a long list of mutations in genes that could cause particular diseases. However, over 30% of genes in the human body do not have a known role. Genes can serve multiple roles, some of which are not yet discovered, and even when a gene’s purpose is known, the impact of each particular mutation in a given gene is largely uncatalogued. Therefore, new methods need to be developed to identify the biological roles of both normal and abnormal gene sequences. For hundreds of years, biologists have used microscopy to study how living cells work. Rohban et al. have now asked whether modern software that extracts data from microscopy images could create a fingerprint-like profile of a cell that would reflect how its genes affect its role and appearance. While some genes do not necessarily carry a code with instructions of what a cell should look like, they can indirectly modify the structure of the cell. The resulting changes in the shape of the cell can then be captured in images. The idea was that two cells with matching profiles would indicate that their combinations of genes had matching biological roles too. Rohban et al. tested their approach with human cells grown in the laboratory. In each sample of cells, they ‘turned on’ one of a few hundred relatively well-known human genes, some of which were known to have similar roles. The cells were then stained via a technique called ‘Cell Painting’ to reveal eight specific components of each cell, including its DNA and its surface membrane. The stained cells were imaged under a microscope and the resulting microscopy images analyzed to create a profile of each type of cell. Rohban et al. confirmed that turning on genes known to perform similar biological roles lead to similar-looking cells. The analysis also revealed a previously unknown interaction between two major pathways in the cell that control how cancer starts and develops. In the future, this approach could predict the biological roles of less-understood genes by looking for profiles that match those of well-known genes. Applying this strategy to every human gene, and mutations in genes that are linked to diseases, could help to answer many mysteries about how genes build the human body and keep it working. DOI: http://dx.doi.org/10.7554/eLife.24060.002

ROS1 fusion kinase-highly expressed in a variety of human cancers-has emerged as an important and attractive target for anticancer drug design. Crizotinib, a well-known drug approved by the FDA as an ALK inhibitor to treat advanced NSCLC, also shows potent inhibitoy activity against ROS1. However, the development of serious resistance due to secondary mutations has been observed in clinical studies. To provide insight into the mechanisms of this drug resistance, molecular dynamics simulations and free-energy calculations were carried out for complexes of crizotinib with wild-type (WT) ROS1 as well as the mutated L2026M and G2032R forms. MD simulations indicated that the L2026M and G2032R systems are slightly less flexible than the WT system. Binding free energy calculations showed that the L2026M and G2032R mutations significantly reduce the binding affinity of crizotinib for ROS1, and that the resistance to crizotinib caused by the L2026M and G2032R mutations arises mostly from increases in entropic terms. Furthermore, calculations of per-residue binding free energies highlighted increased and decreased contributions of some residues in the L2026M and G2032R systems relative to those in the WT system. The present study therefore yielded detailed insight into the mechanisms of resistance to crizotinib caused by the L2026M and G2032R mutations, which should provide the basis for rational drug design to combat crizotinib resistance. Graphical Abstract Superposition of the average structures obtained from the last 10 ns of the molecular dynamics simulation trajectoriy for WT (green) and mutated ROS1 (cyan).

Cancer genome characterization efforts now provide an initial view of the somatic alterations in primary tumors. However, most point mutations occur at low frequency, and the function of these alleles remains undefined. We have developed a scalable systematic approach to interrogate the function of cancer-associated gene variants. We subjected 474 mutant alleles curated from 5,338 tumors to pooled in vivo tumor formation assays and gene expression profiling. We identified 12 transforming alleles, including two in genes (PIK3CB, POT1) that have not been shown to be tumorigenic. One rare KRAS allele, D33E, displayed tumorigenicity and constitutive activation of known RAS effector pathways. By comparing gene expression changes induced upon expression of wild-type and mutant alleles, we inferred the activity of specific alleles. Because alleles found to be mutated only once in 5,338 tumors rendered cells tumorigenic, these observations underscore the value of integrating genomic information with functional studies. Significance: Experimentally inferring the functional status of cancer-associated mutations facilitates the interpretation of genomic information in cancer. Pooled in vivo screen and gene expression profiling identified functional variants and demonstrated that expression of rare variants induced tumorigenesis. Variant phenotyping through functional studies will facilitate defining key somatic events in cancer. Cancer Discov; 6(7); 714–26. ©2016 AACR. See related commentary by Cho and Collisson, p. 694. This article is highlighted in the In This Issue feature, p. 681