Cui Zhang1, Yingnan Jiang2, Jin Zhang3, Jian Huang4, Jinhui Wang5. 1. School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China. zhangcui8534@163.com. 2. School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China. yingnanjiang1996@126.com. 3. School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China. jinjinlovemama@163.com. 4. School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China. profhj@163.com. 5. School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China. profwjh@126.com.
Abstract
8-p-Hdroxybenzoyl tovarol (TAW) is a germacrane-type sesquiterpenoid that can be isolated from the roots of Ferula dissecta (Ledeb.) Ledeb. In this study, the growth inhibitory effects induced by TAW were screened on some types of tumor cells, and the mechanism was investigated on TAW-induced growth inhibition, including paraptosis and autophagy in human cervical cancer HeLa cells. TAW-induced paraptosis involved extensive cytoplasmic vacuolization in the absence of caspase activation. Additionally, TAW evoked cell paraptotic death mediated by endoplasmic reticulum (ER) stress and unfolded protein response (UPR). Autophagy induced by TAW was found to antagonize paraptosis in HeLa cells. This effect was enhanced by rapamycin and suppressed by the autophagy inhibitor, 3-methyladenine (3MA). Loss of beclin 1 (an autophagic regulator) function led to promote ER stress. Taken together, these results suggest that TAW induces paraptosis like cell death and protective autophagy in HeLa cells, which would provide a new clue for exploiting TAW as a promising agent for the treatment of cervical cancer.
8-p-Hdroxybenzoyl tovarol (TAW) is a germacrane-type sesquiterpenoid that can be isolated from the roots of Ferula dissecta (Ledeb.) Ledeb. In this study, the growth inhibitory effects induced by TAW were screened on some types of tumor cells, and the mechanism was investigated on TAW-induced growth inhibition, including paraptosis and autophagy in human cervical cancer HeLa cells. TAW-induced paraptosis involved extensive cytoplasmic vacuolization in the absence of caspase activation. Additionally, TAW evoked cell paraptotic death mediated by endoplasmic reticulum (ER) stress and unfolded protein response (UPR). Autophagy induced by TAW was found to antagonize paraptosis in HeLa cells. This effect was enhanced by rapamycin and suppressed by the autophagy inhibitor, 3-methyladenine (3MA). Loss of beclin 1 (an autophagic regulator) function led to promote ER stress. Taken together, these results suggest that TAW induces paraptosis like cell death and protective autophagy in HeLa cells, which would provide a new clue for exploiting TAW as a promising agent for the treatment of cervical cancer.
Natural products have been the main source of traditional medicine since ancient time. Today, numerous compounds from natural plants are reported to possess growth inhibitory effects on tumor cells. The Ferula genus, belonging to the family of Apiaceae, has been reported to be rich in biologically active compounds such as terpenoidcoumarins and sesquiterpene derivatives [1,2,3,4]. Particularly, these compounds have been proven to be cytotoxic on several cancer cell lines and seem to be promising natural products for treatment of humancancers [5,6]. 8-p-Hdroxybenzoyl tovarol (TAW), a germacrane-type sesquiterpenoid, was isolated from the roots of Ferula dissecta (Ledeb.) Ledeb. However, no further investigations have been carried out on its effects on tumor cells, and the mechanisms underlying the growth inhibitory effects of TAW are still unclear so far.Cervical cancer is the most common malignancy of the female reproductive system. Although neoadjuvant chemotherapy, along with concurrent chemo- and radiotherapies have benefited the majority of patients, survival in women with recurrent or metastatic cervical cancer remains poor. Resistance of cancer to chemotherapy is one of the primary causes of treatment failure [7,8]. Thus, novel anticancer drugs to combat cervical cancer are needed.Up to now, cell death could be classified into apoptosis, autophagy, necrosis, cornification and tentative definitions of atypical cell death modalities such as paraptosis, mitotic catastrophe, anoikis, excitotoxicity, wallerian degeneration, pyroptosis, pyronecrosis, entosis etc. [9]. Among these types of cell death, at least three of them, i.e., apoptosis, autophagy and paraptosis, belong to programmed cell death (PCD) based on their morphological features [10]. The PCD observed during development and tissue homeostasis has been classified morphologically into three main types [11]: type 1, also known as nuclear or apoptotic; type 2 or autophagic; and type 3, a non-lysosomal vacuolated degeneration, also being called paraptosis [12]. Apoptosis is a caspase-dependent process and exhibits morphological features including nuclear fragmentation, cell shrinkage, chromatin condensation, membrane blebbing, and the formation of apoptotic bodies [13]. Autophagy, specific to eukaryotic cells, is a dynamic process involving the sequestration of plasmatic portions and intracellular organelles into double-membrane vacuoles called autophagosomes. Autophagy has many crucial physiological functions and can trigger lysosome-dependent protein degradation, organelle turnover, and removal [14]. Paraptosis is a kind of PCD that has not been well characterized. It was originally introduced in 2000 to describe a form of PCD morphologically and biochemically different from apoptosis [12]. The main feature of paraptosis consists of cytoplasmic vacuolization that begins with progressive swelling of mitochondria and/or endoplasmic reticulum (ER). Paraptosis typically does not respond to caspase inhibitors nor does it involve activation of caspases, formation of apoptotic bodies, or other characteristics of apoptotic morphology. In contrast to apoptosis, Bcl-XL is also not involved in paraptosis.In the present study, we demonstrated for the first time that TAW induces paraptosis characterized by cell death in conjunction with cytoplasmic vacuolation, no involvement of caspase activation and DNA fragmentation, dilated ER and mitochondria, and induction of ER stress and unfolded protein response (UPR) signaling markers. In addition, our results showed that TAW also induced autophagy in HeLa cells, and autophagy antagonized paraptosis. Thus, these results suggest that TAW induces paraptosis like cell death and protective autophagy in HeLa cells.
2. Results
2.1. 8-p-Hdroxybenzoyl Tovarol (TAW) Induces Cytoplasmic Vacuolization and Cell Death in HeLa Cells
In HeLa cells, extensive cellular vacuolization could be detected under the light microscope after TAW treatment (Figure 1B). The development of cytoplasmic vacuolization in HeLa cells could be described as follows: in TAW-treated cells, small vacuoles appeared (6 h treatment) and then gradually fused into giant vacuoles (12 h treatment). After 24 h treatment, some of the cells shrunk and became detached from the culture plate with intracellular vacuole surrounding the cell nucleus. The effects of TAW on cell viability of HeLa cells were shown in Figure 1C. TAW inhibited HeLa cell growth in a time- and dose-dependent manner, and the IC50 value of TAW was 18.8 μM in HeLa cells for 24 h, compared with that of 14.8 μM 5-fluorouracil.
Figure 1
8-p-Hdroxybenzoyl tovarol (TAW) induces cytoplasmic vacuole formation in HeLa cells. (A) The chemical structures of TAW; (B) Light micrograph images (×200 magnification) of these cancer cells with or without TAW treatment. Cells were treated with 18 μM TAW (6, 12 h) for HeLa cells. Bar = 20 μm. And the percentage of vacuolated cells was measured using light microscope. Data are expressed as mean ± S.E.M. * p < 0.05 vs. control group; (C) Results of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay of cell viability. Cells were incubated with escalating concentrations (0–50 μM) of TAW for 12, 24 and 36 h. The data are presented as the mean ± S.E.M. of the results from three independent experiments.
8-p-Hdroxybenzoyl tovarol (TAW) induces cytoplasmic vacuole formation in HeLa cells. (A) The chemical structures of TAW; (B) Light micrograph images (×200 magnification) of these cancer cells with or without TAW treatment. Cells were treated with 18 μM TAW (6, 12 h) for HeLa cells. Bar = 20 μm. And the percentage of vacuolated cells was measured using light microscope. Data are expressed as mean ± S.E.M. * p < 0.05 vs. control group; (C) Results of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay of cell viability. Cells were incubated with escalating concentrations (0–50 μM) of TAW for 12, 24 and 36 h. The data are presented as the mean ± S.E.M. of the results from three independent experiments.
2.2. 8-p-Hdroxybenzoyl Tovarol (TAW) Induces Paraptosis Like Cell Death in HeLa Cells
The TAW-induced cytoplasmic vacuolization was further observed by the transmission electron microscope (Figure 2A). The vacuoles appeared clear in HeLa cells treated with 18 μM TAW for 6 h, and no cytoplasmic material was observed in the vacuoles. At 12 h of TAW treatment, fusion among the swollen mitochondria and ER were further progressed. To further characterize the morphological dynamics of the cytoplasmic vacuolization process, experiments were performed in HeLa cells by using Mito-tracker and ER-tracker stains. As shown in Figure 2B, vacuoles could be observed through mitochondria and ER staining in HeLa cells treated with TAW. Cytoplasmic vacuolization and enlarged mitochondria and/or ER have been reported to be the typical features of paraptosis [12]. Paraptosis typically does not respond to caspase inhibitors nor does it involve activation of caspases, the formation of apoptotic bodies, or DNA fragmentation [11]. Next, to examine the involvement of caspase activation, cells were treated with TAW, then caspase-3, 8, 9, 12 and downstream poly-ADP-ribose polymerase (PARP) protein levels were measured. As a result of treatment, intact caspase-3, 8, 9, 12 and PARP protein levels were not changed, and cleaved caspase-3, 8, 9, 12 and cleaved PARP proteins were not detected (Figure 2C). When cells were pretreated with the broad spectrum pan-caspase inhibitor z-VAD-fmk before treatment of TAW, the percentages of dead cells (Figure 2D) and vacuolated cells (Figure 2E) were not altered, regardless of pretreatment with z-VAD-fmk. Moreover, Hoechst 33258 staining assay (Figure 2F) showed that no obvious morphological alterations were caused in the nucleus of TAW-treated HeLa cells at different time points. Taken together, these results demonstrate that TAW induces paraptosis like cell death in HeLa cells.
Figure 2
8-p-Hdroxybenzoyl tovarol (TAW) induces paraptosis like cell death in HeLa cells. (A) Electron microscopy showing ultrastructure of HeLa cells untreated and treated with 18 μM TAW for 6 and 12 h. Control cells with normal mitochondrial and ER. Cells after 6 and 12 h treatment, showing both mitochondrial and ER swelling. Red arrows represent ER, black arrows represent mitochondria; (B) Cells incubated with vehicle (control) or 18 μM TAW for 12 h then stained with ER-tracker and Mito-tracker, and observed by fluorescence microscope (×200 magnification). Bar = 20 μm; (C) HeLa cells were lysed after treatment with 18 μM TAW for 12, 24, and 36 h, and the protein levels of caspase-3, 8, 9, 12 and PARP were detected by western blot analysis. β-Actin was used as an equal loading control; (D,E) Cells were pretreated with z-VAD-fmk at 20 μM for 1 h before treatment of TAW at 18 μM for 12 h, then the inhibitory rate was measured using an MTT assay (D), and the percentages of vacuolated cells were measured (E). Data are expressed as mean ± S.E.M. * p < 0.05 vs. control; (F) The cells were treated with TAW (18 μM) for 12 and 24 h then stained with Hoechst 33258 and observed by fluorescence microscopy (×200 magnification). Bar = 20 μm.
8-p-Hdroxybenzoyl tovarol (TAW) induces paraptosis like cell death in HeLa cells. (A) Electron microscopy showing ultrastructure of HeLa cells untreated and treated with 18 μM TAW for 6 and 12 h. Control cells with normal mitochondrial and ER. Cells after 6 and 12 h treatment, showing both mitochondrial and ER swelling. Red arrows represent ER, black arrows represent mitochondria; (B) Cells incubated with vehicle (control) or 18 μM TAW for 12 h then stained with ER-tracker and Mito-tracker, and observed by fluorescence microscope (×200 magnification). Bar = 20 μm; (C) HeLa cells were lysed after treatment with 18 μM TAW for 12, 24, and 36 h, and the protein levels of caspase-3, 8, 9, 12 and PARP were detected by western blot analysis. β-Actin was used as an equal loading control; (D,E) Cells were pretreated with z-VAD-fmk at 20 μM for 1 h before treatment of TAW at 18 μM for 12 h, then the inhibitory rate was measured using an MTT assay (D), and the percentages of vacuolated cells were measured (E). Data are expressed as mean ± S.E.M. * p < 0.05 vs. control; (F) The cells were treated with TAW (18 μM) for 12 and 24 h then stained with Hoechst 33258 and observed by fluorescence microscopy (×200 magnification). Bar = 20 μm.
To examine the effects of TAW on mitochondrial membrane potential, HeLa cells untreated or treated with TAW for 12, 24 and 36 h were stained with Rhodamine 123 dye and change of fluorescent intensity was assessed by the flow cytometry. It suggests that TAW treatment significantly reduces the MMP of HeLa cells (Figure 3).
Figure 3
Loss of mitochondrial membrane potential induced by 8-p-hdroxybenzoyl tovarol (TAW). HeLa cells untreated or treated with TAW (18 μM) for 12, 24 and 36 h were stained with Rhodamine 123 dye and change in fluorescent intensity was assessed by flow cytometry. Data represent the mean ± S.E.M. of three replicate independent experiments. * p < 0.05 vs. control group, ** p < 0.01 vs. control group.
Loss of mitochondrial membrane potential induced by 8-p-hdroxybenzoyl tovarol (TAW). HeLa cells untreated or treated with TAW (18 μM) for 12, 24 and 36 h were stained with Rhodamine 123 dye and change in fluorescent intensity was assessed by flow cytometry. Data represent the mean ± S.E.M. of three replicate independent experiments. * p < 0.05 vs. control group, ** p < 0.01 vs. control group.
2.4. 8-p-Hdroxybenzoyl Tovarol (TAW) Induced Vacuolation Is Reversed by Treatment with Cycloheximide in HeLa Cells
As shown in Figure 4A, halting of protein synthesis by addition of translation inhibitor cycloheximide (CHX) at 1.25 μM could significantly inhibit the formation of cytoplasmic vacuolization induced by TAW and decreased the number of cells with cytoplasmic vacuolization, suggesting that cytoplasmic vacuolization was interrupted by translation inhibitor, another characteristic of paraptosis [12]. Moreover, the pretreatment of HeLa cells with 1.25 μM CHX effectively decreased the level of TAW-induced cell death (Figure 4B). Taken together, these results demonstrate that TAW induces paraptosis like cell death in HeLa cells, and an active role of ER protein loads in vacuolation process in TAW-induced paraptosis.
Figure 4
The involvement of prevention of vacuolation by cycloheximide, ER-stress and UPR signaling pathway in 8-p-hdroxybenzoyl tovarol (TAW) induced paraptosis in HeLa cells. (A,B) Cells were pretreated with CHX (1.25 μM, 1 h), then treated with TAW at 18 μM for 12 h. The light micrograph images (×400 magnification, Bar = 20 μm) and the percentages of vacuolated cells were measured using a light microscope (A), and the inhibitory rate was measured using an MTT assay (B). Data are expressed as mean ± S.E.M. * p < 0.05 vs. control group. #
p < 0.05 vs. Cells treated with TAW alone; (C) HeLa cells were treated with TAW (18 μM) for indicated time points prior to preparation of lysates, followed by immunoblotting to detect the protein levels of BiP, CHOP, IRE1α, XBP1s, p-PERK, p-eIF2α, and ATF6. β-Actin was used as an equal loading control.
The involvement of prevention of vacuolation by cycloheximide, ER-stress and UPR signaling pathway in 8-p-hdroxybenzoyl tovarol (TAW) induced paraptosis in HeLa cells. (A,B) Cells were pretreated with CHX (1.25 μM, 1 h), then treated with TAW at 18 μM for 12 h. The light micrograph images (×400 magnification, Bar = 20 μm) and the percentages of vacuolated cells were measured using a light microscope (A), and the inhibitory rate was measured using an MTT assay (B). Data are expressed as mean ± S.E.M. * p < 0.05 vs. control group. #
p < 0.05 vs. Cells treated with TAW alone; (C) HeLa cells were treated with TAW (18 μM) for indicated time points prior to preparation of lysates, followed by immunoblotting to detect the protein levels of BiP, CHOP, IRE1α, XBP1s, p-PERK, p-eIF2α, and ATF6. β-Actin was used as an equal loading control.
2.5. 8-p-Hdroxybenzoyl Tovarol (TAW)-Mediated ER-Stress and Unfolded Protein Response (UPR) Lead to Paraptosis Like Cell Death
In view of dilation of the ER-lumen and the percentages of dead cells and vacuolated cells rescued by CHX involved in TAW-induced paraptosis, we reasoned that TAW induced vacuolization may be preceded by induction of ER-stress. We performed a time course experiment to study ER-stress and activation of UPR by evaluating expression of certain ER resident proteins involved in protein folding. Immunoblot analysis of the cells demonstrated that TAW upregulated signature ER-stress markers glucose-regulated protein BiP/GRP78 and C/EBP-homologous protein CHOP (Figure 4C). To further validate the capacity of TAW to induce ER-stress, we asked whether ER stress-induced UPR signaling pathways are evoked upon TAW stimulation. It is clear to note that TAW treatment caused increased expression of endoribonuclease, inositol requiring enzyme 1 (IRE1α), and the splicing of X-box binding protein (XBP1s), indicating the activation of the IRE1 branch of UPR signaling. Additionally, an increase in expression of auto-phosphorylated PKR-like endoplasmic reticulum (ER) resident kinase (p-PERK) and PERK-mediated phosphorylation of eukaryotic initiation factor (p-eIF2α) were also observed, suggesting that TAW engages the PERK branch of UPR signaling. However, there was no change in ATF6 level, indicating that the ATF6 branch of UPR signaling is not involved in (Figure 4C). Altogether, these results indicate that TAW-mediated ER-stress and UPR signaling leads to paraptosis.
2.6. 8-p-Hdroxybenzoyl Tovarol (TAW) Induces Autophagy in HeLa Cells
To determine the effect of TAW on autophagy in HeLa cells, firstly the ultrastructure of cells was examined by transmission electron microscopy. The HeLa cells treated with TAW (18 μM) for 36 h, and ultrastructural analysis revealed an increased number of autophagosomes in TAW-treated HeLa cells (Figure 5A). As shown in Figure 5B, HeLa cells were transfected with a pEGFP-LC3 plasmid, and exogenous EGFP-LC3 puncta were also increased in TAW-treated HeLa cells. The cells were observed with monodansylcadaverine (MDC) staining by fluorescence microscope. Compared with the control group, TAW treatment caused a marked increase in the number of MDC labeled autophagolysosomes in the cells (Figure 5C). Flow cytometric analysis also showed that the MDC-positive cells caused by treatment with TAW were increased in a time-dependent manner (Figure 5D). Western blot analysis revealed that the upregulation of beclin 1, the conversion from LC3 I to LC3 II and the downregulation of p62 were detected in TAW treated cells in a time-dependent manner (Figure 5E). These results demonstrate that TAW induces autophagy in HeLa cells.
Figure 5
8-p-hdroxybenzoyl tovarol (TAW) induces autophagy in HeLa cells. (A) HeLa cells were untreated or treated with 18 μM TAW for 36 h, and formation of autophagic vacuoles was examined by TEM analysis. Red arrows represent double-membrane autophagosome. The data was representative of three independent experiments; (B) HeLa cells were transfected with a pEGFP-LC3 plasmid. After 24 h, cells were untreated or indicated 18 μM of TAW for another 36 h. Formation of EGFP-LC3 puncta was visualized by a fluorescence microscope (×400 magnification, Bar = 20 μm). The data was representative of three independent experiments; (C) Cells were cultured with 18 μM TAW for 36 h and then observed by fluorescence microscopy with MDC staining (×200 magnification, Bar = 20 μm). Arrows indicate cells containing autophagolysosomes; (D) The cells were treated with 18 μM TAW for 12, 24, 36 and 48 h, and quantitative analysis detected a positive ratio of MDC staining by flow cytometric analysis, n = 3, means ± S.E.M. * p < 0.05 vs. control group, ** p < 0.01 vs. control group; (E) HeLa cells were lysed after treatment with 18 μM TAW for 12, 24, 36 and 48 h, and the protein levels of beclin 1, LC3 and p62 were detected by western blot analysis. β-Actin was used as an equal loading control.
8-p-hdroxybenzoyl tovarol (TAW) induces autophagy in HeLa cells. (A) HeLa cells were untreated or treated with 18 μM TAW for 36 h, and formation of autophagic vacuoles was examined by TEM analysis. Red arrows represent double-membrane autophagosome. The data was representative of three independent experiments; (B) HeLa cells were transfected with a pEGFP-LC3 plasmid. After 24 h, cells were untreated or indicated 18 μM of TAW for another 36 h. Formation of EGFP-LC3 puncta was visualized by a fluorescence microscope (×400 magnification, Bar = 20 μm). The data was representative of three independent experiments; (C) Cells were cultured with 18 μM TAW for 36 h and then observed by fluorescence microscopy with MDC staining (×200 magnification, Bar = 20 μm). Arrows indicate cells containing autophagolysosomes; (D) The cells were treated with 18 μM TAW for 12, 24, 36 and 48 h, and quantitative analysis detected a positive ratio of MDC staining by flow cytometric analysis, n = 3, means ± S.E.M. * p < 0.05 vs. control group, ** p < 0.01 vs. control group; (E) HeLa cells were lysed after treatment with 18 μM TAW for 12, 24, 36 and 48 h, and the protein levels of beclin 1, LC3 and p62 were detected by western blot analysis. β-Actin was used as an equal loading control.
2.7. Autophagy Antagonizes Paraptosis in 8-p-Hdroxybenzoyl Tovarol (TAW) Treated HeLa Cells
TAW is therefore able to induce paraptosis and autophagy in HeLa cells. To find out the relationship between paraptosis and autophagy, the specific autophagic inhibitor 3-methyladenine (3MA) and the autophagic agonist rapamycin were applied. The MTT assay showed that TAW inhibited cell growth significantly, but this inhibition was partially augmented by 3MA and was reversed by rapamycin (Figure 6A). Moreover to elucidate the role of autophagy in TAW-induced paraptosis, the expression of the autophagy-associated marker protein beclin 1 in HeLa cells was knocked down by application of short interfering RNA (siRNA). After transfection, the changes at the protein level were evaluated (Figure 6B). TAW-induced up-regulation of beclin 1 was reversed, and the conversion from LC3 I to LC3 II was also suppressed, demonstrating that the autophagy induced by TAW was markedly inhibited (Figure 6C). Next, the loss of beclin 1 further enhanced TAW-induced upregulation of the ER-stress markers such as BiP and CHOP (Figure 6C). These results further demonstrate that autophagy antagonizes paraptosis in HeLa cells treated with TAW.
Figure 6
8-p-Hdroxybenzoyl tovarol (TAW)-induced autophagy antagonizes paraptosis in HeLa cells. (A) Cells were incubated with 18 μM TAW in the presence or absence of 3MA (1 mM) or rapamycin (1 nM) for 24 h, and the inhibitory rate was measured using an MTT assay. Data are expressed as mean ± S.E.M. * p < 0.05 vs. control group, ** p < 0.01 vs. control group; (B) HeLa cells were transfected with either non-specific siRNA (NC-siRNA) or beclin 1-siRNA, and beclin 1 protein was detected 24 h after transfection by western blot analysis. β-Actin was used as an equal loading control; (C) The transfected HeLa cells were lysed after treatment with 18 μM TAW for 24 h; then the expression of beclin 1, conversion from LC3 I to LC3 II, BiP and CHOP were detected by western blot analysis. β-Actin was used as an equal loading control.
8-p-Hdroxybenzoyl tovarol (TAW)-induced autophagy antagonizes paraptosis in HeLa cells. (A) Cells were incubated with 18 μM TAW in the presence or absence of 3MA (1 mM) or rapamycin (1 nM) for 24 h, and the inhibitory rate was measured using an MTT assay. Data are expressed as mean ± S.E.M. * p < 0.05 vs. control group, ** p < 0.01 vs. control group; (B) HeLa cells were transfected with either non-specific siRNA (NC-siRNA) or beclin 1-siRNA, and beclin 1 protein was detected 24 h after transfection by western blot analysis. β-Actin was used as an equal loading control; (C) The transfected HeLa cells were lysed after treatment with 18 μM TAW for 24 h; then the expression of beclin 1, conversion from LC3 I to LC3 II, BiP and CHOP were detected by western blot analysis. β-Actin was used as an equal loading control.
3. Discussion
Natural products isolated from plants or medicinal herbs have been invaluable resources of antineoplastic agents or lead compounds for new drug development [15]. There are at least 250,000 species of plants out of which more than one thousand plants have been found to possess significant anticancer properties [16]. Ferula dissecta, has a long history of application in the treatment of digestive diseases and arthritis in Uygur medicine. Syreiteate A and syreiteate B, two constituents of Ferula dissecta, have been reported to have growth inhibitory activity against cervical cancerHeLa cell line [17]. 8-p-Hdroxybenzoyl tovarol (TAW) is an active germacrane-type sesquiterpenoid which was isolated from the roots of Ferula dissecta. In this study, the cytotoxic effect and the underlying mechanism of TAW are examined. To our best knowledge, this is the first report as to TAW-induced cytotoxicity and the underlying mechanism on human cervical cancer HeLa cells.Classical apoptosis is caspase-dependent programmed cell death (CD-PCD). Accumulating evidence in the literature supports that several types of caspase-independent programmed cell death (CI-PCD) exist in the cells, such as autophagy, cornification, mitotic catastrophe, anoikis, paraptosis, excitotoxicity, Wallerian degeneration and programmed necrosis [18]. Caspase-mediated apoptosis is a major hindrance to tumour growth and metastasis. Moreover, many tumour cells can unexpectedly survive the activation of caspases. As a result, caspase-independent cell death programs are gaining increasing interest among cancer researchers [19]. Therefore, CI-PCD may play an important role in inducing cell death and be the major death style in many tumor cells. Paraptosis, a new type of CI-PCD, is characterized mainly by a process of cytoplasmic vacuolization that begins with the physical enlargement of mitochondria and/or endoplasmic reticulum. This PCD does not involve the apoptotic characteristics of pyknosis, DNA fragmentation or caspase activation, and is known to require new protein synthesis [20]. Studying paraptosis will not only be helpful to clarify the mechanism of cell death but also to improve therapies for cancer treatment. Therapies based on the induction of non-apoptotic cell deaths such as paraptosis might be possible approaches to suppress the multi-drug resistant phenotype often associated with resistance to apoptosis [21]. In our study TAW induced cytoplasmic vacuolation, which was derived from the swelling mitochondria and ER in HeLa cells, with no characteristics of apoptosis, such as DNA fragmentation, apoptotic bodies and activation of caspases. These suggest that this kind of cell death induced by TAW is different from apoptosis. These results indicate that TAW induces paraptosis like cell death in human cervical cancer HeLa cells.Reduction in mitochondrial membrane potential (MMP) can be a checkpoint of apoptosis as it can cause the release of apoptogenic molecules from the intermembrane space into the cytoplasm [22]. In the present study we have observed significant reduction in MMP after exposure to TAW. But lack of caspase activation also was observed suggesting that loss of MMP is not apoptosis in TAW induced cell death. Depletion of MMP was also observed without AIF (apoptosis inducing factor) release in calcium induced paraptotic cell death in Jurkat cells [23]. Summing up these results also suggest that TAW induces paraptosis like cell death in HeLa cells.The endoplasmic reticulum provides a specialized niche for protein maturation that includes posttranslational modification, proper folding, attainment of native state and finally their transport [24]. ER stress occurs when the rate of translation of proteins exceeds their proper rate of folding [25]. This stressful situation induces a transcriptional program called the unfolded protein response (UPR). The UPR causes attenuation of global translation and selectively upregulates chaperones that act to clear unfolded proteins from the stressed ER [26]. The UPR is fundamentally cyto-protective, but the compensatory mechanisms may not be able to fully sustain ER function and the decompensation will activate the cell death pathway if the stress is too severe or lasts for too long [27]. During the UPR, signaling branches are activated by three parallel sensors of stress in the ER membrane: (1) protein kinase RNA-like endoplasmic reticulum kinase (PERK); (2) activation transcription factor 6 (ATF6) and (3) inositol requiring enzyme 1 (IRE1) [28]. The kinase PERK directly inhibits mRNA translation by phosphorylating translation initiation factor 2α (eIF2α) [29]. ATF6 transduces ER stress after its cleavage, which yields a fragment that translocates to the nucleus to regulate gene expression [30]. Active IRE1 lead to an unconventional splicing of the transcription factor XBP1 triggering the removal of 26 nucleotides and the translation of an active transcription factor [31]. ER dilatation and vacuolization are processes that define type III or paraptotic-like cell death and, recently, a relationship has been observed between this type of cell death and the ER stress induced by proteasome inhibitors [32]. Consistently, we show here that TAW-induced paraptosis cell death in HeLa cells, hallmarked by ER-derived vacuolization. Then the TAW induced ER dilatation enhanced the levels of ER-resident chaperone BiP and other markers of ER stress such as CHOP. ER stress trigger UPR signaling pathways via activation of IRE1 and the persistent XBP1 cleavage constitutes the transcription dependent pathway, and global translation inhibition by the elevated eIF2α phosphorylation levels. According to these results, TAW could induce ER stress and UPR in HeLa cells. We pretreated HeLa cells with CHX to suppress ER stress and the percentages of dead cells and vacuolated cells were rescued. These findings suggest that TAW induces paraptotic death in HeLa cells through ER stress and UPR. Moreover, the TAW inhibits caspase-3, caspase-9, caspase-8 and caspase-12 proteins that involve three signaling pathways that trigger apoptosis [27] and triggers a caspase-independent death program. Therefore, the paraptotic-like cell death may be a backup cell death pathway that takes place when a critical amount of unfolded proteins is reached and, concomitantly, the apoptotic machinery is hindered. This hypothesis is consistent with the information presented in other reports [32].Autophagy is a cellular digestive process conserved from yeast to mammals. Depending on the cellular context, autophagy may promote cell survival by preventing the accumulation of deleterious products and organelles [33], or serve as a type of programmed cell death through cell lysis [34]. The self-digestion not only provides cells with nutrients during fasting, but also, it can rid the cell of superfluous or damaged organelles, misfolded proteins and invading microorganisms. Autophagy therefore is important for stress resistance [35]. Dysfunction of autophagy results in accumulation of damages and finally leads to cell death [35]. In addition, autophagy can also be induced in a response to chemotherapy and has been considered as an important mechanism contributing to drug resistance, and the autophagic process may protect cancer cells from toxicity. Therefore, the inhibition of autophagy may result in a greater susceptibility to cell death-inducing mechanisms [36]. In our study, we detected the morphological changes and the expression of autophagy maker genes Beclin 1, LC3 II, and p62 in HeLa cells treatment with TAW. Our detailed results show that TAW induces autophagy in HeLa cells. Then to explore the relationship between paraptosis and autophagy in TAW-induced HeLa cells, the specific autophagic inhibitor 3MA and the autophagic agonist rapamycin were applied. An MTT assay showed that the cytotoxic effect of TAW-treatment HeLa cells was potentiated by an inhibition of autophagy. Furthermore, after transfection Beclin 1-siRNA, the results demonstrate that a blockade of autophagy can increase the expression of the ER stress marker genes BiP and CHOP. These results suggest that ER stress-mediated paraptosis cell death after TAW treatment is potentiated by an inhibition of autophagy.In summary, this study describes HeLa cells growth inhibitory activity of a germacrane-type sesquiterpenoid8-p-hdroxybenzoyl tovarol (TAW), isolated from the roots of Ferula dissecta. The mechanisms are TAW induced ER stress-mediated paraptosis like cell death and protective autophagy in human cervical cancer HeLa cells. These results would provide a new clue for exploiting TAW as a promising agent for the treatment of cervical cancer.
4. Experimental Section
4.1. Plant Materials and Reagents
Biological materials: the roots of F. dissecta were collected in Tuoli, Xinjiang of China in June 2007 and identified by Yong Tan (Shihezi University of China). A voucher specimen (No. 20071018001) is deposited in School of Pharmacy, Shihezi University. Ferula dissecta (Ledeb.) Ledeb are widely distributed along the Junggar Basin in Xinjiang. Ferula dissecta (Ledeb.) Ledeb used in this study were picked in the hills, plains and valleys of Xinjiang Toli County randomly (Toli County located in the west of Junggar Basin). All these locations do not require any specific permission, and none of the field studies involved endangered or protected species.Reagents: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) (Sigma, Tokyo, Japan, M5655), cycloheximide (Sigma, A6185), monodansylcadaverine (MDC) (Sigma, 30432), Rhodamine 123 (Sigma, R8004), Rapamycin (Sigma, R8781), 3-Methyladenine (Sigma, M9281) were purchased from Sigma. Endoplasmic reticulum (ER)-tracker (Invitrogen, Carlsbad, CA, USA, E34250), Mitochondrion (Mito)-tracker (Invitrogen, M7514), Lipofectamine 2000 (Invitrogen, 11668), Lipofectamine RNAiMAX (Invitrogen, 13778) were purchased from Invitrogen. Hankʼs Balanced Salt Solution with calcium and magnesium (HBSS/Ca/Mg, Gibco, Grand Island, NY, USA, #14025-092) was purchased from Gibco.Antibodies used in this study were against: BiP (Cell Signaling, Boston, MA, USA, #3177), CHOP (Cell Signaling, #2895), IRE1α (Cell Signaling, #3294), XBP1s (Cell Signaling, #12782), phospho-PERK (Santa Cruz, Buda, TX, USA, sc-32577), phospho-elF2α (Cell Signaling, #3398), ATF6 (Abcam, Cambridge, UK, ab37149), caspase-3 (Cell Signaling, #9665), caspase-9 (Cell Signaling, #9508), caspase-8 (Cell Signaling, #9746), caspase-12 (Cell Signaling, #2202), PARP (Cell Signaling, #9532), Beclin 1 (Cell Signaling, #3495), LC3 I/II (Cell Signaling, #4108), p62 (Cell Signaling, #8025), β-Actin (Proteintech, Chicago, IL, USA, 66009-1-lg), anti-rabbit lgG (Cell Signaling, #7074), anti-mouse lgG (Cell Signaling, #7076).
4.2. Extraction and Isolation of TAW
The air-dried roots (1.45 kg) of F. dissecta were refluxed with 95% Ethanol (15 L) three times for 2 h. The extract was concentrated under reduced pressure to yield an Ethanol extract (240 g). A part of the Ethanol extract (240 g) was subjected to chromatography over silica gel, eluted with petroleum ether (PE) with an increasing amount of EtOAc to afford fraction E (PE-EtOAc, 100:6, 1.2 g). Fr. E was then separated by silica gel (PE-EtOAc, 100:7.5) to give the crystal block of TAW (30.0 mg). Its chemical structure (Figure 1A) was identified through a comparison of its spectral data (MS, IR, 1H- and 13C-NMR, single crystal X-ray) with those published previously [37], and its purity was above 98% as determined by HPLC-UV.
4.3. Cell Culture
HeLa cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and were cultured in PRMI-1640 (Gibco, Life Technologies, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS) (Biolianshuo, Yuanheng, China) at 37 °C in 5% CO2.
4.4. Cell Viability Assay
The inhibitory effect of TAW on HeLa cells viability was measured by MTT assay. The cells were dispensed in 96-well flat-bottom microtiter plates (BD Falcon, Austin, TX, USA) at a density of 4.0 × 104 cells per well. After 24 h incubation, they were treated with various concentrations of TAW for indicated time points. After that, the cells were incubated with 5.0 mg/mL MTT solution at 37 °C for 4 h. The resulting crystal was dissolved in 100 μL DMSO and the optical density was measured by using a micro-plate reader (Thermo Multiskan MK3, Thermo scientific, Helsinki, Finland). If required, the pan-caspase inhibitor z-VAD-fmk (20 μM), cycloheximide (CHX, 1.25 μM), 3-Methyladenine (3MA, 1 mM) or rapamycin (1 nM) were added to HeLa cells 1 h before TAW treatment.
4.5. Endoplasmic Reticulum (ER)-Tracker and Mito-Tracker
Endoplasmic reticulum or mitochondrion staining was performed according to the instructions of the manufacturer of ER-tracker kit or Mito-tracker kit with slight alteration. Briefly, cells were washed with HBSS or PBS and then incubated in pre-warmed ER-tracker (1 μM) or Mito-tracker (20 nM) dye solution for approximately 30 min at 37 °C. After HBSS or PBS washes, cells were then observed using a fluorescence microscopy (Olympus, Tokyo, Japan).
4.6. Hoechst 33258, MDC and Rhodamine 123 Staining
After incubation with TAW for indicated time points, the cells were stained with Hoechst 33258, MDC or Rhodamin 123 at 37 °C for 30 min, and then the morphology was observed by a fluorescence microscopy (Olympus, Tokyo, Japan). Mitochondrial membrane potential was measured by Rhodamine 123 dye, and the samples were subsequently analysed by a FACSCalibur flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA). The MDC-positive cells were also analysed by a FACSCalibur flow cytometry.
4.7. Transmission Electron Microscopy
The ultra-structure of cytoplasmic vacuolization was observed using a Philips Tecnai-12 Biotwin Transmission Electron Microscope similar to previous reports [38]. Briefly, collected cells were fixed with 2% glutaraldehyde for 2 h, washed with PBS and then post-fixed with 1% OsO4 for 1.5 h at 4 °C. The samples were then washed and dehydrated with graded alcohol. After dehydration, the samples were infiltrated and embedded in 618 epoxy resin. Ultrathin sections were cut, stained with uranyl acetate and lead citrate and then examined under the transmission electron microscope.
4.8. Vacuolated Cell Counting
Vacuolated cells were counted using a method similar to the previous study [39]. Light micrographs were obtained from different fields and the number of vacuolated cells having the total vacuoles occupying area more than 50% of cytoplasmic area was counted for at least 300 cells for each condition.
4.9. Western Blot Analysis
The HeLa cells were harvested, washed twice with cold PBS and then lysed in whole cell lysis buffer, supplemented with the proteinase inhibitors 100 µg/mL at 4 °C for 1 h. After 12,000× g centrifugation at 4 °C for 10 min, the protein concentration was determined by a BCA Protein Assay Kit (CWBIO, Beijing, China). Equal amounts of total proteins were separated by 12% SDS-PAGE, and transferred onto Immobilon-P Transfer Membrane (Millipore Corporation, Billerica, MA, USA). The membranes were blocked with 5% skimmed milk at room temperature for 1 h, incubated with indicated primary antibodies at 4 °C overnight and horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 2 h, then visualized by using ECL reagents.
4.10. Transfection of siRNA
Beclin 1 and negative control (NC) siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cells were transfected with 50 nM Beclin 1-siRNA, or NC-siRNA using Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer’s instructions. The transfected cells were used for subsequent experiments 24 h later.
4.11. Transfection of EGFP-LC3 Plasmid
HeLa cells grown to 80% confluency were transfected with an EGFP-LC3 plasmid. Twenty-four hours post transfection, cells were treated with TAW (18 μM) for another 36 h and analyzed by fluorescence microscopy. The EGFP-LC3 plasmid was kindly provided by Bo Liu (Sichuan University, Chengdu, China).
4.12. Statistical Analysis
Each experiment was repeated for at least three times and results of three independent experiments were used for statistical analysis. The results were shown as mean ± S.E.M. Statistical comparisons were conducted using Student’s t-test in SPSS17.0. p < 0.05 was considered as statistically significant.
Authors: Si Hyoung Kim; Jun Goo Kang; Chul Sik Kim; Sung-Hee Ihm; Moon Gi Choi; Hyung Joon Yoo; Seong Jin Lee Journal: Biochem Biophys Res Commun Date: 2014-10-18 Impact factor: 3.575
Authors: M Bury; A Girault; V Mégalizzi; S Spiegl-Kreinecker; V Mathieu; W Berger; A Evidente; A Kornienko; P Gailly; C Vandier; R Kiss Journal: Cell Death Dis Date: 2013-03-28 Impact factor: 8.469
Authors: Daniel J Klionsky; Amal Kamal Abdel-Aziz; Sara Abdelfatah; Mahmoud Abdellatif; Asghar Abdoli; Steffen Abel; Hagai Abeliovich; Marie H Abildgaard; Yakubu Princely Abudu; Abraham Acevedo-Arozena; Iannis E Adamopoulos; Khosrow Adeli; Timon E Adolph; Annagrazia Adornetto; Elma Aflaki; Galila Agam; Anupam Agarwal; Bharat B Aggarwal; Maria Agnello; Patrizia Agostinis; Javed N Agrewala; Alexander Agrotis; Patricia V Aguilar; S Tariq Ahmad; Zubair M Ahmed; Ulises Ahumada-Castro; Sonja Aits; Shu Aizawa; Yunus Akkoc; Tonia Akoumianaki; Hafize Aysin Akpinar; Ahmed M Al-Abd; Lina Al-Akra; Abeer Al-Gharaibeh; Moulay A Alaoui-Jamali; Simon Alberti; Elísabet Alcocer-Gómez; Cristiano Alessandri; Muhammad Ali; M Abdul Alim Al-Bari; Saeb Aliwaini; Javad Alizadeh; Eugènia Almacellas; Alexandru Almasan; Alicia Alonso; Guillermo D Alonso; Nihal Altan-Bonnet; Dario C Altieri; Élida M C Álvarez; Sara Alves; Cristine Alves da Costa; Mazen M Alzaharna; Marialaura Amadio; Consuelo Amantini; Cristina Amaral; Susanna Ambrosio; Amal O Amer; Veena Ammanathan; Zhenyi An; Stig U Andersen; Shaida A Andrabi; Magaiver Andrade-Silva; Allen M Andres; Sabrina Angelini; David Ann; Uche C Anozie; Mohammad Y Ansari; Pedro Antas; Adam Antebi; Zuriñe Antón; Tahira Anwar; Lionel Apetoh; Nadezda Apostolova; Toshiyuki Araki; Yasuhiro Araki; Kohei Arasaki; Wagner L Araújo; Jun Araya; Catherine Arden; Maria-Angeles Arévalo; Sandro Arguelles; Esperanza Arias; Jyothi Arikkath; Hirokazu Arimoto; Aileen R Ariosa; Darius Armstrong-James; Laetitia Arnauné-Pelloquin; Angeles Aroca; Daniela S Arroyo; Ivica Arsov; Rubén Artero; Dalia Maria Lucia Asaro; Michael Aschner; Milad Ashrafizadeh; Osnat Ashur-Fabian; Atanas G Atanasov; Alicia K Au; Patrick Auberger; Holger W Auner; Laure Aurelian; Riccardo Autelli; Laura Avagliano; Yenniffer Ávalos; Sanja Aveic; Célia Alexandra Aveleira; Tamar Avin-Wittenberg; Yucel Aydin; Scott Ayton; Srinivas Ayyadevara; Maria Azzopardi; Misuzu Baba; Jonathan M Backer; Steven K Backues; Dong-Hun Bae; Ok-Nam Bae; Soo Han Bae; Eric H Baehrecke; Ahruem Baek; Seung-Hoon Baek; Sung Hee Baek; Giacinto Bagetta; Agnieszka Bagniewska-Zadworna; Hua Bai; Jie Bai; Xiyuan Bai; Yidong Bai; Nandadulal Bairagi; Shounak Baksi; Teresa Balbi; Cosima T Baldari; Walter Balduini; Andrea Ballabio; Maria Ballester; Salma Balazadeh; Rena Balzan; Rina Bandopadhyay; Sreeparna Banerjee; Sulagna Banerjee; Ágnes Bánréti; Yan Bao; Mauricio S Baptista; Alessandra Baracca; Cristiana Barbati; Ariadna Bargiela; Daniela Barilà; Peter G Barlow; Sami J Barmada; Esther Barreiro; George E Barreto; Jiri Bartek; Bonnie Bartel; Alberto Bartolome; Gaurav R Barve; Suresh H Basagoudanavar; Diane C Bassham; Robert C Bast; Alakananda Basu; Henri Batoko; Isabella Batten; Etienne E Baulieu; Bradley L Baumgarner; Jagadeesh Bayry; Rupert Beale; Isabelle Beau; Florian Beaumatin; Luiz R G Bechara; George R Beck; Michael F Beers; Jakob Begun; Christian Behrends; Georg M N Behrens; Roberto Bei; Eloy Bejarano; Shai Bel; Christian Behl; Amine Belaid; Naïma Belgareh-Touzé; Cristina Bellarosa; Francesca Belleudi; Melissa Belló Pérez; Raquel Bello-Morales; Jackeline Soares de Oliveira Beltran; Sebastián Beltran; Doris Mangiaracina Benbrook; Mykolas Bendorius; Bruno A Benitez; Irene Benito-Cuesta; Julien Bensalem; Martin W Berchtold; Sabina Berezowska; Daniele Bergamaschi; Matteo Bergami; Andreas Bergmann; Laura Berliocchi; Clarisse Berlioz-Torrent; Amélie Bernard; Lionel Berthoux; Cagri G Besirli; Sebastien Besteiro; Virginie M Betin; Rudi Beyaert; Jelena S Bezbradica; Kiran Bhaskar; Ingrid Bhatia-Kissova; Resham Bhattacharya; Sujoy Bhattacharya; Shalmoli Bhattacharyya; Md Shenuarin Bhuiyan; Sujit Kumar Bhutia; Lanrong Bi; Xiaolin Bi; Trevor J Biden; Krikor Bijian; Viktor A Billes; Nadine Binart; Claudia Bincoletto; Asa B Birgisdottir; Geir Bjorkoy; Gonzalo Blanco; Ana Blas-Garcia; Janusz Blasiak; Robert Blomgran; Klas Blomgren; Janice S Blum; Emilio Boada-Romero; Mirta Boban; Kathleen Boesze-Battaglia; Philippe Boeuf; Barry Boland; Pascale Bomont; Paolo Bonaldo; Srinivasa Reddy Bonam; Laura Bonfili; Juan S Bonifacino; Brian A Boone; Martin D Bootman; Matteo Bordi; Christoph Borner; Beat C Bornhauser; Gautam Borthakur; Jürgen Bosch; Santanu Bose; Luis M Botana; Juan Botas; Chantal M Boulanger; Michael E Boulton; Mathieu Bourdenx; Benjamin Bourgeois; Nollaig M Bourke; Guilhem Bousquet; Patricia Boya; Peter V Bozhkov; Luiz H M Bozi; Tolga O Bozkurt; Doug E Brackney; Christian H Brandts; Ralf J Braun; Gerhard H Braus; Roberto Bravo-Sagua; José M Bravo-San Pedro; Patrick Brest; Marie-Agnès Bringer; Alfredo Briones-Herrera; V Courtney Broaddus; Peter Brodersen; Jeffrey L Brodsky; Steven L Brody; Paola G Bronson; Jeff M Bronstein; Carolyn N Brown; Rhoderick E Brown; Patricia C Brum; John H Brumell; Nicola Brunetti-Pierri; Daniele Bruno; Robert J Bryson-Richardson; Cecilia Bucci; Carmen Buchrieser; Marta Bueno; Laura Elisa Buitrago-Molina; Simone Buraschi; Shilpa Buch; J Ross Buchan; Erin M Buckingham; Hikmet Budak; Mauricio Budini; Geert Bultynck; Florin Burada; Joseph R Burgoyne; M Isabel Burón; Victor Bustos; Sabrina Büttner; Elena Butturini; Aaron Byrd; Isabel Cabas; Sandra Cabrera-Benitez; Ken Cadwell; Jingjing Cai; Lu Cai; Qian Cai; Montserrat Cairó; Jose A Calbet; Guy A Caldwell; Kim A Caldwell; Jarrod A Call; Riccardo Calvani; Ana C Calvo; Miguel Calvo-Rubio Barrera; Niels Os Camara; Jacques H Camonis; Nadine Camougrand; Michelangelo Campanella; Edward M Campbell; François-Xavier Campbell-Valois; Silvia Campello; Ilaria Campesi; Juliane C Campos; Olivier Camuzard; Jorge Cancino; Danilo Candido de Almeida; Laura Canesi; Isabella Caniggia; Barbara Canonico; Carles Cantí; Bin Cao; Michele Caraglia; Beatriz Caramés; Evie H Carchman; Elena Cardenal-Muñoz; Cesar Cardenas; Luis Cardenas; Sandra M Cardoso; Jennifer S Carew; Georges F Carle; Gillian Carleton; Silvia Carloni; Didac Carmona-Gutierrez; Leticia A Carneiro; Oliana Carnevali; Julian M Carosi; Serena Carra; Alice Carrier; Lucie Carrier; Bernadette Carroll; A Brent Carter; Andreia Neves Carvalho; Magali Casanova; Caty Casas; Josefina Casas; Chiara Cassioli; Eliseo F Castillo; Karen Castillo; Sonia Castillo-Lluva; Francesca Castoldi; Marco Castori; Ariel F Castro; Margarida Castro-Caldas; Javier Castro-Hernandez; Susana Castro-Obregon; Sergio D Catz; Claudia Cavadas; Federica Cavaliere; Gabriella Cavallini; Maria Cavinato; Maria L Cayuela; Paula Cebollada Rica; Valentina Cecarini; Francesco Cecconi; Marzanna Cechowska-Pasko; Simone Cenci; Victòria Ceperuelo-Mallafré; João J Cerqueira; Janete M Cerutti; Davide Cervia; Vildan Bozok Cetintas; Silvia Cetrullo; Han-Jung Chae; Andrei S Chagin; Chee-Yin Chai; Gopal Chakrabarti; Oishee Chakrabarti; Tapas Chakraborty; Trinad Chakraborty; Mounia Chami; Georgios Chamilos; David W Chan; Edmond Y W Chan; Edward D Chan; H Y Edwin Chan; Helen H Chan; Hung Chan; Matthew T V Chan; Yau Sang Chan; Partha K Chandra; Chih-Peng Chang; Chunmei Chang; Hao-Chun Chang; Kai Chang; Jie Chao; Tracey Chapman; Nicolas Charlet-Berguerand; Samrat Chatterjee; Shail K Chaube; Anu Chaudhary; Santosh Chauhan; Edward Chaum; Frédéric Checler; Michael E Cheetham; Chang-Shi Chen; Guang-Chao Chen; Jian-Fu Chen; Liam L Chen; Leilei Chen; Lin Chen; Mingliang Chen; Mu-Kuan Chen; Ning Chen; Quan Chen; Ruey-Hwa Chen; Shi Chen; Wei Chen; Weiqiang Chen; Xin-Ming Chen; Xiong-Wen Chen; Xu Chen; Yan Chen; Ye-Guang Chen; Yingyu Chen; Yongqiang Chen; Yu-Jen Chen; Yue-Qin Chen; Zhefan Stephen Chen; Zhi Chen; Zhi-Hua Chen; Zhijian J Chen; Zhixiang Chen; Hanhua Cheng; Jun Cheng; Shi-Yuan Cheng; Wei Cheng; Xiaodong Cheng; Xiu-Tang Cheng; Yiyun Cheng; Zhiyong Cheng; Zhong Chen; Heesun Cheong; Jit Kong Cheong; Boris V Chernyak; Sara Cherry; Chi Fai Randy Cheung; Chun Hei Antonio Cheung; King-Ho Cheung; Eric Chevet; Richard J Chi; Alan Kwok Shing Chiang; Ferdinando Chiaradonna; Roberto Chiarelli; Mario Chiariello; Nathalia Chica; Susanna Chiocca; Mario Chiong; Shih-Hwa Chiou; Abhilash I Chiramel; Valerio Chiurchiù; Dong-Hyung Cho; Seong-Kyu Choe; Augustine M K Choi; Mary E Choi; Kamalika Roy Choudhury; Norman S Chow; Charleen T Chu; Jason P Chua; John Jia En Chua; Hyewon Chung; Kin Pan Chung; Seockhoon Chung; So-Hyang Chung; Yuen-Li Chung; Valentina Cianfanelli; Iwona A Ciechomska; Mariana Cifuentes; Laura Cinque; Sebahattin Cirak; Mara Cirone; Michael J Clague; Robert Clarke; Emilio Clementi; Eliana M Coccia; Patrice Codogno; Ehud Cohen; Mickael M Cohen; Tania Colasanti; Fiorella Colasuonno; Robert A Colbert; Anna Colell; Miodrag Čolić; Nuria S Coll; Mark O Collins; María I Colombo; Daniel A Colón-Ramos; Lydie Combaret; Sergio Comincini; Márcia R Cominetti; Antonella Consiglio; Andrea Conte; Fabrizio Conti; Viorica Raluca Contu; Mark R Cookson; Kevin M Coombs; Isabelle Coppens; Maria Tiziana Corasaniti; Dale P Corkery; Nils Cordes; Katia Cortese; Maria do Carmo Costa; Sarah Costantino; Paola Costelli; Ana Coto-Montes; Peter J Crack; Jose L Crespo; Alfredo Criollo; Valeria Crippa; Riccardo Cristofani; Tamas Csizmadia; Antonio Cuadrado; Bing Cui; Jun Cui; Yixian Cui; Yong Cui; Emmanuel Culetto; Andrea C Cumino; Andrey V Cybulsky; Mark J Czaja; Stanislaw J Czuczwar; Stefania D'Adamo; Marcello D'Amelio; Daniela D'Arcangelo; Andrew C D'Lugos; Gabriella D'Orazi; James A da Silva; Hormos Salimi Dafsari; Ruben K Dagda; Yasin Dagdas; Maria Daglia; Xiaoxia Dai; Yun Dai; Yuyuan Dai; Jessica Dal Col; Paul Dalhaimer; Luisa Dalla Valle; Tobias Dallenga; Guillaume Dalmasso; Markus Damme; Ilaria Dando; Nico P Dantuma; April L Darling; Hiranmoy Das; Srinivasan Dasarathy; Santosh K Dasari; Srikanta Dash; Oliver Daumke; Adrian N Dauphinee; Jeffrey S Davies; Valeria A Dávila; Roger J Davis; Tanja Davis; Sharadha Dayalan Naidu; Francesca De Amicis; Karolien De Bosscher; Francesca De Felice; Lucia De Franceschi; Chiara De Leonibus; Mayara G de Mattos Barbosa; Guido R Y De Meyer; Angelo De Milito; Cosimo De Nunzio; Clara De Palma; Mauro De Santi; Claudio De Virgilio; Daniela De Zio; Jayanta Debnath; Brian J DeBosch; Jean-Paul Decuypere; Mark A Deehan; Gianluca Deflorian; James DeGregori; Benjamin Dehay; Gabriel Del Rio; Joe R Delaney; Lea M D Delbridge; Elizabeth Delorme-Axford; M Victoria Delpino; Francesca Demarchi; Vilma Dembitz; Nicholas D Demers; Hongbin Deng; Zhiqiang Deng; Joern Dengjel; Paul Dent; Donna Denton; Melvin L DePamphilis; Channing J Der; Vojo Deretic; Albert Descoteaux; Laura Devis; Sushil Devkota; Olivier Devuyst; Grant Dewson; Mahendiran Dharmasivam; Rohan Dhiman; Diego di Bernardo; Manlio Di Cristina; Fabio Di Domenico; Pietro Di Fazio; Alessio Di Fonzo; Giovanni Di Guardo; Gianni M Di Guglielmo; Luca Di Leo; Chiara Di Malta; Alessia Di Nardo; Martina Di Rienzo; Federica Di Sano; George Diallinas; Jiajie Diao; Guillermo Diaz-Araya; Inés Díaz-Laviada; Jared M Dickinson; Marc Diederich; Mélanie Dieudé; Ivan Dikic; Shiping Ding; Wen-Xing Ding; Luciana Dini; Jelena Dinić; Miroslav Dinic; Albena T Dinkova-Kostova; Marc S Dionne; Jörg H W Distler; Abhinav Diwan; Ian M C Dixon; Mojgan Djavaheri-Mergny; Ina Dobrinski; Oxana Dobrovinskaya; Radek Dobrowolski; Renwick C J Dobson; Jelena Đokić; Serap Dokmeci Emre; Massimo Donadelli; Bo Dong; Xiaonan Dong; Zhiwu Dong; Gerald W Dorn Ii; Volker Dotsch; Huan Dou; Juan Dou; Moataz Dowaidar; Sami Dridi; Liat Drucker; Ailian Du; Caigan Du; Guangwei Du; Hai-Ning Du; Li-Lin Du; André du Toit; Shao-Bin Duan; Xiaoqiong Duan; Sónia P Duarte; Anna Dubrovska; Elaine A Dunlop; Nicolas Dupont; Raúl V Durán; Bilikere S Dwarakanath; Sergey A Dyshlovoy; Darius Ebrahimi-Fakhari; Leopold Eckhart; Charles L Edelstein; Thomas Efferth; Eftekhar Eftekharpour; Ludwig Eichinger; Nabil Eid; Tobias Eisenberg; N Tony Eissa; Sanaa Eissa; Miriam Ejarque; Abdeljabar El Andaloussi; Nazira El-Hage; Shahenda El-Naggar; Anna Maria Eleuteri; Eman S El-Shafey; Mohamed Elgendy; Aristides G Eliopoulos; María M Elizalde; Philip M Elks; Hans-Peter Elsasser; Eslam S Elsherbiny; Brooke M Emerling; N C Tolga Emre; Christina H Eng; Nikolai Engedal; Anna-Mart Engelbrecht; Agnete S T Engelsen; Jorrit M Enserink; Ricardo Escalante; Audrey Esclatine; Mafalda Escobar-Henriques; Eeva-Liisa Eskelinen; Lucile Espert; Makandjou-Ola Eusebio; Gemma Fabrias; Cinzia Fabrizi; Antonio Facchiano; Francesco Facchiano; Bengt Fadeel; Claudio Fader; Alex C Faesen; W Douglas Fairlie; Alberto Falcó; Bjorn H Falkenburger; Daping Fan; Jie Fan; Yanbo Fan; Evandro F Fang; Yanshan Fang; Yognqi Fang; Manolis Fanto; Tamar Farfel-Becker; Mathias Faure; Gholamreza Fazeli; Anthony O Fedele; Arthur M Feldman; Du Feng; Jiachun Feng; Lifeng Feng; Yibin Feng; Yuchen Feng; Wei Feng; Thais Fenz Araujo; Thomas A Ferguson; Álvaro F Fernández; Jose C Fernandez-Checa; Sonia Fernández-Veledo; Alisdair R Fernie; Anthony W Ferrante; Alessandra Ferraresi; Merari F Ferrari; Julio C B Ferreira; Susan Ferro-Novick; Antonio Figueras; Riccardo Filadi; Nicoletta Filigheddu; Eduardo Filippi-Chiela; Giuseppe Filomeni; Gian Maria Fimia; Vittorio Fineschi; Francesca Finetti; Steven Finkbeiner; Edward A Fisher; Paul B Fisher; Flavio Flamigni; Steven J Fliesler; Trude H Flo; Ida Florance; Oliver Florey; Tullio Florio; Erika Fodor; Carlo Follo; Edward A Fon; Antonella Forlino; Francesco Fornai; Paola Fortini; Anna Fracassi; Alessandro Fraldi; Brunella Franco; Rodrigo Franco; Flavia Franconi; Lisa B Frankel; Scott L Friedman; Leopold F Fröhlich; Gema Frühbeck; Jose M Fuentes; Yukio Fujiki; Naonobu Fujita; Yuuki Fujiwara; Mitsunori Fukuda; Simone Fulda; Luc Furic; Norihiko Furuya; Carmela Fusco; Michaela U Gack; Lidia Gaffke; Sehamuddin Galadari; Alessia Galasso; Maria F Galindo; Sachith Gallolu Kankanamalage; Lorenzo Galluzzi; Vincent Galy; Noor Gammoh; Boyi Gan; Ian G Ganley; Feng Gao; Hui Gao; Minghui Gao; Ping Gao; Shou-Jiang Gao; Wentao Gao; Xiaobo Gao; Ana Garcera; Maria Noé Garcia; Verónica E Garcia; Francisco García-Del Portillo; Vega Garcia-Escudero; Aracely Garcia-Garcia; Marina Garcia-Macia; Diana García-Moreno; Carmen Garcia-Ruiz; Patricia García-Sanz; Abhishek D Garg; Ricardo Gargini; Tina Garofalo; Robert F Garry; Nils C Gassen; Damian Gatica; Liang Ge; Wanzhong Ge; Ruth Geiss-Friedlander; Cecilia Gelfi; Pascal Genschik; Ian E Gentle; Valeria Gerbino; Christoph Gerhardt; Kyla Germain; Marc Germain; David A Gewirtz; Elham Ghasemipour Afshar; Saeid Ghavami; Alessandra Ghigo; Manosij Ghosh; Georgios Giamas; Claudia Giampietri; Alexandra Giatromanolaki; Gary E Gibson; Spencer B Gibson; Vanessa Ginet; Edward Giniger; Carlotta Giorgi; Henrique Girao; Stephen E Girardin; Mridhula Giridharan; Sandy Giuliano; Cecilia Giulivi; Sylvie Giuriato; Julien Giustiniani; Alexander Gluschko; Veit Goder; Alexander Goginashvili; Jakub Golab; David C Goldstone; Anna Golebiewska; Luciana R Gomes; Rodrigo Gomez; Rubén Gómez-Sánchez; Maria Catalina Gomez-Puerto; Raquel Gomez-Sintes; Qingqiu Gong; Felix M Goni; Javier González-Gallego; Tomas Gonzalez-Hernandez; Rosa A Gonzalez-Polo; Jose A Gonzalez-Reyes; Patricia González-Rodríguez; Ing Swie Goping; Marina S Gorbatyuk; Nikolai V Gorbunov; Kıvanç Görgülü; Roxana M Gorojod; Sharon M Gorski; Sandro Goruppi; Cecilia Gotor; Roberta A Gottlieb; Illana Gozes; Devrim Gozuacik; Martin Graef; Markus H Gräler; Veronica Granatiero; Daniel Grasso; Joshua P Gray; Douglas R Green; Alexander Greenhough; Stephen L Gregory; Edward F Griffin; Mark W Grinstaff; Frederic Gros; Charles Grose; Angelina S Gross; Florian Gruber; Paolo Grumati; Tilman Grune; Xueyan Gu; Jun-Lin Guan; Carlos M Guardia; Kishore Guda; Flora Guerra; Consuelo Guerri; Prasun Guha; Carlos Guillén; Shashi Gujar; Anna Gukovskaya; Ilya Gukovsky; Jan Gunst; Andreas Günther; Anyonya R Guntur; Chuanyong Guo; Chun Guo; Hongqing Guo; Lian-Wang Guo; Ming Guo; Pawan Gupta; Shashi Kumar Gupta; Swapnil Gupta; Veer Bala Gupta; Vivek Gupta; Asa B Gustafsson; David D Gutterman; Ranjitha H B; Annakaisa Haapasalo; James E Haber; Aleksandra Hać; Shinji Hadano; Anders J Hafrén; Mansour Haidar; Belinda S Hall; Gunnel Halldén; Anne Hamacher-Brady; Andrea Hamann; Maho Hamasaki; Weidong Han; Malene Hansen; Phyllis I Hanson; Zijian Hao; Masaru Harada; Ljubica Harhaji-Trajkovic; Nirmala Hariharan; Nigil Haroon; James Harris; Takafumi Hasegawa; Noor Hasima Nagoor; Jeffrey A Haspel; Volker Haucke; Wayne D Hawkins; Bruce A Hay; Cole M Haynes; Soren B Hayrabedyan; Thomas S Hays; Congcong He; Qin He; Rong-Rong He; You-Wen He; Yu-Ying He; Yasser Heakal; Alexander M Heberle; J Fielding Hejtmancik; Gudmundur Vignir Helgason; Vanessa Henkel; Marc Herb; Alexander Hergovich; Anna Herman-Antosiewicz; Agustín Hernández; Carlos Hernandez; Sergio Hernandez-Diaz; Virginia Hernandez-Gea; Amaury Herpin; Judit Herreros; Javier H Hervás; Daniel Hesselson; Claudio Hetz; Volker T Heussler; Yujiro Higuchi; Sabine Hilfiker; Joseph A Hill; William S Hlavacek; Emmanuel A Ho; Idy H T Ho; Philip Wing-Lok Ho; Shu-Leong Ho; Wan Yun Ho; G Aaron Hobbs; Mark Hochstrasser; Peter H M Hoet; Daniel Hofius; Paul Hofman; Annika Höhn; Carina I Holmberg; Jose R Hombrebueno; Chang-Won Hong Yi-Ren Hong; Lora V Hooper; Thorsten Hoppe; Rastislav Horos; Yujin Hoshida; I-Lun Hsin; Hsin-Yun Hsu; Bing Hu; Dong Hu; Li-Fang Hu; Ming Chang Hu; Ronggui Hu; Wei Hu; Yu-Chen Hu; Zhuo-Wei Hu; Fang Hua; Jinlian Hua; Yingqi Hua; Chongmin Huan; Canhua Huang; Chuanshu Huang; Chuanxin Huang; Chunling Huang; Haishan Huang; Kun Huang; Michael L H Huang; Rui Huang; Shan Huang; Tianzhi Huang; Xing Huang; Yuxiang Jack Huang; Tobias B Huber; Virginie Hubert; Christian A Hubner; Stephanie M Hughes; William E Hughes; Magali Humbert; Gerhard Hummer; James H Hurley; Sabah Hussain; Salik Hussain; Patrick J Hussey; Martina Hutabarat; Hui-Yun Hwang; Seungmin Hwang; Antonio Ieni; Fumiyo Ikeda; Yusuke Imagawa; Yuzuru Imai; Carol Imbriano; Masaya Imoto; Denise M Inman; Ken Inoki; Juan Iovanna; Renato V Iozzo; Giuseppe Ippolito; Javier E Irazoqui; Pablo Iribarren; Mohd Ishaq; Makoto Ishikawa; Nestor Ishimwe; Ciro Isidoro; Nahed Ismail; Shohreh Issazadeh-Navikas; Eisuke Itakura; Daisuke Ito; Davor Ivankovic; Saška Ivanova; Anand Krishnan V Iyer; José M Izquierdo; Masanori Izumi; Marja Jäättelä; Majid Sakhi Jabir; William T Jackson; Nadia Jacobo-Herrera; Anne-Claire Jacomin; Elise Jacquin; Pooja Jadiya; Hartmut Jaeschke; Chinnaswamy Jagannath; Arjen J Jakobi; Johan Jakobsson; Bassam Janji; Pidder Jansen-Dürr; Patric J Jansson; Jonathan Jantsch; Sławomir Januszewski; Alagie Jassey; Steve Jean; Hélène Jeltsch-David; Pavla Jendelova; Andreas Jenny; Thomas E Jensen; Niels Jessen; Jenna L Jewell; Jing Ji; Lijun Jia; Rui Jia; Liwen Jiang; Qing Jiang; Richeng Jiang; Teng Jiang; Xuejun Jiang; Yu Jiang; Maria Jimenez-Sanchez; Eun-Jung Jin; Fengyan Jin; Hongchuan Jin; Li Jin; Luqi Jin; Meiyan Jin; Si Jin; Eun-Kyeong Jo; Carine Joffre; Terje Johansen; Gail V W Johnson; Simon A Johnston; Eija Jokitalo; Mohit Kumar Jolly; Leo A B Joosten; Joaquin Jordan; Bertrand Joseph; Dianwen Ju; Jeong-Sun Ju; Jingfang Ju; Esmeralda Juárez; Delphine Judith; Gábor Juhász; Youngsoo Jun; Chang Hwa Jung; Sung-Chul Jung; Yong Keun Jung; Heinz Jungbluth; Johannes Jungverdorben; Steffen Just; Kai Kaarniranta; Allen Kaasik; Tomohiro Kabuta; Daniel Kaganovich; Alon Kahana; Renate Kain; Shinjo Kajimura; Maria Kalamvoki; Manjula Kalia; Danuta S Kalinowski; Nina Kaludercic; Ioanna Kalvari; Joanna Kaminska; Vitaliy O Kaminskyy; Hiromitsu Kanamori; Keizo Kanasaki; Chanhee Kang; Rui Kang; Sang Sun Kang; Senthilvelrajan Kaniyappan; Tomotake Kanki; Thirumala-Devi Kanneganti; Anumantha G Kanthasamy; Arthi Kanthasamy; Marc Kantorow; Orsolya Kapuy; Michalis V Karamouzis; Md Razaul Karim; Parimal Karmakar; Rajesh G Katare; Masaru Kato; Stefan H E Kaufmann; Anu Kauppinen; Gur P Kaushal; Susmita Kaushik; Kiyoshi Kawasaki; Kemal Kazan; Po-Yuan Ke; Damien J Keating; Ursula Keber; John H Kehrl; Kate E Keller; Christian W Keller; Jongsook Kim Kemper; Candia M Kenific; Oliver Kepp; Stephanie Kermorgant; Andreas Kern; Robin Ketteler; Tom G Keulers; Boris Khalfin; Hany Khalil; Bilon Khambu; Shahid Y Khan; Vinoth Kumar Megraj Khandelwal; Rekha Khandia; Widuri Kho; Noopur V Khobrekar; Sataree Khuansuwan; Mukhran Khundadze; Samuel A Killackey; Dasol Kim; Deok Ryong Kim; Do-Hyung Kim; Dong-Eun Kim; Eun Young Kim; Eun-Kyoung Kim; Hak-Rim Kim; Hee-Sik Kim; Jeong Hun Kim; Jin Kyung Kim; Jin-Hoi Kim; Joungmok Kim; Ju Hwan Kim; Keun Il Kim; Peter K Kim; Seong-Jun Kim; Scot R Kimball; Adi Kimchi; Alec C Kimmelman; Tomonori Kimura; Matthew A King; Kerri J Kinghorn; Conan G Kinsey; Vladimir Kirkin; Lorrie A Kirshenbaum; Sergey L Kiselev; Shuji Kishi; Katsuhiko Kitamoto; Yasushi Kitaoka; Kaio Kitazato; Richard N Kitsis; Josef T Kittler; Ole Kjaerulff; Peter S Klein; Thomas Klopstock; Jochen Klucken; Helene Knævelsrud; Roland L Knorr; Ben C B Ko; Fred Ko; Jiunn-Liang Ko; Hotaka Kobayashi; Satoru Kobayashi; Ina Koch; Jan C Koch; Ulrich Koenig; Donat Kögel; Young Ho Koh; Masato Koike; Sepp D Kohlwein; Nur M Kocaturk; Masaaki Komatsu; Jeannette König; Toru Kono; Benjamin T Kopp; Tamas Korcsmaros; Gözde Korkmaz; Viktor I Korolchuk; Mónica Suárez Korsnes; Ali Koskela; Janaiah Kota; Yaichiro Kotake; Monica L Kotler; Yanjun Kou; Michael I Koukourakis; Evangelos Koustas; Attila L Kovacs; Tibor Kovács; Daisuke Koya; Tomohiro Kozako; Claudine Kraft; Dimitri Krainc; Helmut Krämer; Anna D Krasnodembskaya; Carole Kretz-Remy; Guido Kroemer; Nicholas T Ktistakis; Kazuyuki Kuchitsu; Sabine Kuenen; Lars Kuerschner; Thomas Kukar; Ajay Kumar; Ashok Kumar; Deepak Kumar; Dhiraj Kumar; Sharad Kumar; Shinji Kume; Caroline Kumsta; Chanakya N Kundu; Mondira Kundu; Ajaikumar B Kunnumakkara; Lukasz Kurgan; Tatiana G Kutateladze; Ozlem Kutlu; SeongAe Kwak; Ho Jeong Kwon; Taeg Kyu Kwon; Yong Tae Kwon; Irene Kyrmizi; Albert La Spada; Patrick Labonté; Sylvain Ladoire; Ilaria Laface; Frank Lafont; Diane C Lagace; Vikramjit Lahiri; Zhibing Lai; Angela S Laird; Aparna Lakkaraju; Trond Lamark; Sheng-Hui Lan; Ane Landajuela; Darius J R Lane; Jon D Lane; Charles H Lang; Carsten Lange; Ülo Langel; Rupert Langer; Pierre Lapaquette; Jocelyn Laporte; Nicholas F LaRusso; Isabel Lastres-Becker; Wilson Chun Yu Lau; Gordon W Laurie; Sergio Lavandero; Betty Yuen Kwan Law; Helen Ka-Wai Law; Rob Layfield; Weidong Le; Herve Le Stunff; Alexandre Y Leary; Jean-Jacques Lebrun; Lionel Y W Leck; Jean-Philippe Leduc-Gaudet; Changwook Lee; Chung-Pei Lee; Da-Hye Lee; Edward B Lee; Erinna F Lee; Gyun Min Lee; He-Jin Lee; Heung Kyu Lee; Jae Man Lee; Jason S Lee; Jin-A Lee; Joo-Yong Lee; Jun Hee Lee; Michael Lee; Min Goo Lee; Min Jae Lee; Myung-Shik Lee; Sang Yoon Lee; Seung-Jae Lee; Stella Y Lee; Sung Bae Lee; Won Hee Lee; Ying-Ray Lee; Yong-Ho Lee; Youngil Lee; Christophe Lefebvre; Renaud Legouis; Yu L Lei; Yuchen Lei; Sergey Leikin; Gerd Leitinger; Leticia Lemus; Shuilong Leng; Olivia Lenoir; Guido Lenz; Heinz Josef Lenz; Paola Lenzi; Yolanda León; Andréia M Leopoldino; Christoph Leschczyk; Stina Leskelä; Elisabeth Letellier; Chi-Ting Leung; Po Sing Leung; Jeremy S Leventhal; Beth Levine; Patrick A Lewis; Klaus Ley; Bin Li; Da-Qiang Li; Jianming Li; Jing Li; Jiong Li; Ke Li; Liwu Li; Mei Li; Min Li; Min Li; Ming Li; Mingchuan Li; Pin-Lan Li; Ming-Qing Li; Qing Li; Sheng Li; Tiangang Li; Wei Li; Wenming Li; Xue Li; Yi-Ping Li; Yuan Li; Zhiqiang Li; Zhiyong Li; Zhiyuan Li; Jiqin Lian; Chengyu Liang; Qiangrong Liang; Weicheng Liang; Yongheng Liang; YongTian Liang; Guanghong Liao; Lujian Liao; Mingzhi Liao; Yung-Feng Liao; Mariangela Librizzi; Pearl P Y Lie; Mary A Lilly; Hyunjung J Lim; Thania R R Lima; Federica Limana; Chao Lin; Chih-Wen Lin; Dar-Shong Lin; Fu-Cheng Lin; Jiandie D Lin; Kurt M Lin; Kwang-Huei Lin; Liang-Tzung Lin; Pei-Hui Lin; Qiong Lin; Shaofeng Lin; Su-Ju Lin; Wenyu Lin; Xueying Lin; Yao-Xin Lin; Yee-Shin Lin; Rafael Linden; Paula Lindner; Shuo-Chien Ling; Paul Lingor; Amelia K Linnemann; Yih-Cherng Liou; Marta M Lipinski; Saška Lipovšek; Vitor A Lira; Natalia Lisiak; Paloma B Liton; Chao Liu; Ching-Hsuan Liu; Chun-Feng Liu; Cui Hua Liu; Fang Liu; Hao Liu; Hsiao-Sheng Liu; Hua-Feng Liu; Huifang Liu; Jia Liu; Jing Liu; Julia Liu; Leyuan Liu; Longhua Liu; Meilian Liu; Qin Liu; Wei Liu; Wende Liu; Xiao-Hong Liu; Xiaodong Liu; Xingguo Liu; Xu Liu; Xuedong Liu; Yanfen Liu; Yang Liu; Yang Liu; Yueyang Liu; Yule Liu; J Andrew Livingston; Gerard Lizard; Jose M Lizcano; Senka Ljubojevic-Holzer; Matilde E LLeonart; David Llobet-Navàs; Alicia Llorente; Chih Hung Lo; Damián Lobato-Márquez; Qi Long; Yun Chau Long; Ben Loos; Julia A Loos; Manuela G López; Guillermo López-Doménech; José Antonio López-Guerrero; Ana T López-Jiménez; Óscar López-Pérez; Israel López-Valero; Magdalena J Lorenowicz; Mar Lorente; Peter Lorincz; Laura Lossi; Sophie Lotersztajn; Penny E Lovat; Jonathan F Lovell; Alenka Lovy; Péter Lőw; Guang Lu; Haocheng Lu; Jia-Hong Lu; Jin-Jian Lu; Mengji Lu; Shuyan Lu; Alessandro Luciani; John M Lucocq; Paula Ludovico; Micah A Luftig; Morten Luhr; Diego Luis-Ravelo; Julian J Lum; Liany Luna-Dulcey; Anders H Lund; Viktor K Lund; Jan D Lünemann; Patrick Lüningschrör; Honglin Luo; Rongcan Luo; Shouqing Luo; Zhi Luo; Claudio Luparello; Bernhard Lüscher; Luan Luu; Alex Lyakhovich; Konstantin G Lyamzaev; Alf Håkon Lystad; Lyubomyr Lytvynchuk; Alvin C Ma; Changle Ma; Mengxiao Ma; Ning-Fang Ma; Quan-Hong Ma; Xinliang Ma; Yueyun Ma; Zhenyi Ma; Ormond A MacDougald; Fernando Macian; Gustavo C MacIntosh; Jeffrey P MacKeigan; Kay F Macleod; Sandra Maday; Frank Madeo; Muniswamy Madesh; Tobias Madl; Julio Madrigal-Matute; Akiko Maeda; Yasuhiro Maejima; Marta Magarinos; Poornima Mahavadi; Emiliano Maiani; Kenneth Maiese; Panchanan Maiti; Maria Chiara Maiuri; Barbara Majello; Michael B Major; Elena Makareeva; Fayaz Malik; Karthik Mallilankaraman; Walter Malorni; Alina Maloyan; Najiba Mammadova; Gene Chi Wai Man; Federico Manai; Joseph D Mancias; Eva-Maria Mandelkow; Michael A Mandell; Angelo A Manfredi; Masoud H Manjili; Ravi Manjithaya; Patricio Manque; Bella B Manshian; Raquel Manzano; Claudia Manzoni; Kai Mao; Cinzia Marchese; Sandrine Marchetti; Anna Maria Marconi; Fabrizio Marcucci; Stefania Mardente; Olga A Mareninova; Marta Margeta; Muriel Mari; Sara Marinelli; Oliviero Marinelli; Guillermo Mariño; Sofia Mariotto; Richard S Marshall; Mark R Marten; Sascha Martens; Alexandre P J Martin; Katie R Martin; Sara Martin; Shaun Martin; Adrián Martín-Segura; Miguel A Martín-Acebes; Inmaculada Martin-Burriel; Marcos Martin-Rincon; Paloma Martin-Sanz; José A Martina; Wim Martinet; Aitor Martinez; Ana Martinez; Jennifer Martinez; Moises Martinez Velazquez; Nuria Martinez-Lopez; Marta Martinez-Vicente; Daniel O Martins; Joilson O Martins; Waleska K Martins; Tania Martins-Marques; Emanuele Marzetti; Shashank Masaldan; Celine Masclaux-Daubresse; Douglas G Mashek; Valentina Massa; Lourdes Massieu; Glenn R Masson; Laura Masuelli; Anatoliy I Masyuk; Tetyana V Masyuk; Paola Matarrese; Ander Matheu; Satoaki Matoba; Sachiko Matsuzaki; Pamela Mattar; Alessandro Matte; Domenico Mattoscio; José L Mauriz; Mario Mauthe; Caroline Mauvezin; Emanual Maverakis; Paola Maycotte; Johanna Mayer; Gianluigi Mazzoccoli; Cristina Mazzoni; Joseph R Mazzulli; Nami McCarty; Christine McDonald; Mitchell R McGill; Sharon L McKenna; BethAnn McLaughlin; Fionn McLoughlin; Mark A McNiven; Thomas G McWilliams; Fatima Mechta-Grigoriou; Tania Catarina Medeiros; Diego L Medina; Lynn A Megeney; Klara Megyeri; Maryam Mehrpour; Jawahar L Mehta; Alfred J Meijer; Annemarie H Meijer; Jakob Mejlvang; Alicia Meléndez; Annette Melk; Gonen Memisoglu; Alexandrina F Mendes; Delong Meng; Fei Meng; Tian Meng; Rubem Menna-Barreto; Manoj B Menon; Carol Mercer; Anne E Mercier; Jean-Louis Mergny; Adalberto Merighi; Seth D Merkley; Giuseppe Merla; Volker Meske; Ana Cecilia Mestre; Shree Padma Metur; Christian Meyer; Hemmo Meyer; Wenyi Mi; Jeanne Mialet-Perez; Junying Miao; Lucia Micale; Yasuo Miki; Enrico Milan; Małgorzata Milczarek; Dana L Miller; Samuel I Miller; Silke Miller; Steven W Millward; Ira Milosevic; Elena A Minina; Hamed Mirzaei; Hamid Reza Mirzaei; Mehdi Mirzaei; Amit Mishra; Nandita Mishra; Paras Kumar Mishra; Maja Misirkic Marjanovic; Roberta Misasi; Amit Misra; Gabriella Misso; Claire Mitchell; Geraldine Mitou; Tetsuji Miura; Shigeki Miyamoto; Makoto Miyazaki; Mitsunori Miyazaki; Taiga Miyazaki; Keisuke Miyazawa; Noboru Mizushima; Trine H Mogensen; Baharia Mograbi; Reza Mohammadinejad; Yasir Mohamud; Abhishek Mohanty; Sipra Mohapatra; Torsten Möhlmann; Asif Mohmmed; Anna Moles; Kelle H Moley; Maurizio Molinari; Vincenzo Mollace; Andreas Buch Møller; Bertrand Mollereau; Faustino Mollinedo; Costanza Montagna; Mervyn J Monteiro; Andrea Montella; L Ruth Montes; Barbara Montico; Vinod K Mony; Giacomo Monzio Compagnoni; Michael N Moore; Mohammad A Moosavi; Ana L Mora; Marina Mora; David Morales-Alamo; Rosario Moratalla; Paula I Moreira; Elena Morelli; Sandra Moreno; Daniel Moreno-Blas; Viviana Moresi; Benjamin Morga; Alwena H Morgan; Fabrice Morin; Hideaki Morishita; Orson L Moritz; Mariko Moriyama; Yuji Moriyasu; Manuela Morleo; Eugenia Morselli; Jose F Moruno-Manchon; Jorge Moscat; Serge Mostowy; Elisa Motori; Andrea Felinto Moura; Naima Moustaid-Moussa; Maria Mrakovcic; Gabriel Muciño-Hernández; Anupam Mukherjee; Subhadip Mukhopadhyay; Jean M Mulcahy Levy; Victoriano Mulero; Sylviane Muller; Christian Münch; Ashok Munjal; Pura Munoz-Canoves; Teresa Muñoz-Galdeano; Christian Münz; Tomokazu Murakawa; Claudia Muratori; Brona M Murphy; J Patrick Murphy; Aditya Murthy; Timo T Myöhänen; Indira U Mysorekar; Jennifer Mytych; Seyed Mohammad Nabavi; Massimo Nabissi; Péter Nagy; Jihoon Nah; Aimable Nahimana; Ichiro Nakagawa; Ken Nakamura; Hitoshi Nakatogawa; Shyam S Nandi; Meera Nanjundan; Monica Nanni; Gennaro Napolitano; Roberta Nardacci; Masashi Narita; Melissa Nassif; Ilana Nathan; Manabu Natsumeda; Ryno J Naude; Christin Naumann; Olaia Naveiras; Fatemeh Navid; Steffan T Nawrocki; Taras Y Nazarko; Francesca Nazio; Florentina Negoita; Thomas Neill; Amanda L Neisch; Luca M Neri; Mihai G Netea; Patrick Neubert; Thomas P Neufeld; Dietbert Neumann; Albert Neutzner; Phillip T Newton; Paul A Ney; Ioannis P Nezis; Charlene C W Ng; Tzi Bun Ng; Hang T T Nguyen; Long T Nguyen; Hong-Min Ni; Clíona Ní Cheallaigh; Zhenhong Ni; M Celeste Nicolao; Francesco Nicoli; Manuel Nieto-Diaz; Per Nilsson; Shunbin Ning; Rituraj Niranjan; Hiroshi Nishimune; Mireia Niso-Santano; Ralph A Nixon; Annalisa Nobili; Clevio Nobrega; Takeshi Noda; Uxía Nogueira-Recalde; Trevor M Nolan; Ivan Nombela; Ivana Novak; Beatriz Novoa; Takashi Nozawa; Nobuyuki Nukina; Carmen Nussbaum-Krammer; Jesper Nylandsted; Tracey R O'Donovan; Seónadh M O'Leary; Eyleen J O'Rourke; Mary P O'Sullivan; Timothy E O'Sullivan; Salvatore Oddo; Ina Oehme; Michinaga Ogawa; Eric Ogier-Denis; Margret H Ogmundsdottir; Besim Ogretmen; Goo Taeg Oh; Seon-Hee Oh; Young J Oh; Takashi Ohama; Yohei Ohashi; Masaki Ohmuraya; Vasileios Oikonomou; Rani Ojha; Koji Okamoto; Hitoshi Okazawa; Masahide Oku; Sara Oliván; Jorge M A Oliveira; Michael Ollmann; James A Olzmann; Shakib Omari; M Bishr Omary; Gizem Önal; Martin Ondrej; Sang-Bing Ong; Sang-Ging Ong; Anna Onnis; Juan A Orellana; Sara Orellana-Muñoz; Maria Del Mar Ortega-Villaizan; Xilma R Ortiz-Gonzalez; Elena Ortona; Heinz D Osiewacz; Abdel-Hamid K Osman; Rosario Osta; Marisa S Otegui; Kinya Otsu; Christiane Ott; Luisa Ottobrini; Jing-Hsiung James Ou; Tiago F Outeiro; Inger Oynebraten; Melek Ozturk; Gilles Pagès; Susanta Pahari; Marta Pajares; Utpal B Pajvani; Rituraj Pal; Simona Paladino; Nicolas Pallet; Michela Palmieri; Giuseppe Palmisano; Camilla Palumbo; Francesco Pampaloni; Lifeng Pan; Qingjun Pan; Wenliang Pan; Xin Pan; Ganna Panasyuk; Rahul Pandey; Udai B Pandey; Vrajesh Pandya; Francesco Paneni; Shirley Y Pang; Elisa Panzarini; Daniela L Papademetrio; Elena Papaleo; Daniel Papinski; Diana Papp; Eun Chan Park; Hwan Tae Park; Ji-Man Park; Jong-In Park; Joon Tae Park; Junsoo Park; Sang Chul Park; Sang-Youel Park; Abraham H Parola; Jan B Parys; Adrien Pasquier; Benoit Pasquier; João F Passos; Nunzia Pastore; Hemal H Patel; Daniel Patschan; Sophie Pattingre; Gustavo Pedraza-Alva; Jose Pedraza-Chaverri; Zully Pedrozo; Gang Pei; Jianming Pei; Hadas Peled-Zehavi; Joaquín M Pellegrini; Joffrey Pelletier; Miguel A Peñalva; Di Peng; Ying Peng; Fabio Penna; Maria Pennuto; Francesca Pentimalli; Cláudia Mf Pereira; Gustavo J S Pereira; Lilian C Pereira; Luis Pereira de Almeida; Nirma D Perera; Ángel Pérez-Lara; Ana B Perez-Oliva; María Esther Pérez-Pérez; Palsamy Periyasamy; Andras Perl; Cristiana Perrotta; Ida Perrotta; Richard G Pestell; Morten Petersen; Irina Petrache; Goran Petrovski; Thorsten Pfirrmann; Astrid S Pfister; Jennifer A Philips; Huifeng Pi; Anna Picca; Alicia M Pickrell; Sandy Picot; Giovanna M Pierantoni; Marina Pierdominici; Philippe Pierre; Valérie Pierrefite-Carle; Karolina Pierzynowska; Federico Pietrocola; Miroslawa Pietruczuk; Claudio Pignata; Felipe X Pimentel-Muiños; Mario Pinar; Roberta O Pinheiro; Ronit Pinkas-Kramarski; Paolo Pinton; Karolina Pircs; Sujan Piya; Paola Pizzo; Theo S Plantinga; Harald W Platta; Ainhoa Plaza-Zabala; Markus Plomann; Egor Y Plotnikov; Helene Plun-Favreau; Ryszard Pluta; Roger Pocock; Stefanie Pöggeler; Christian Pohl; Marc Poirot; Angelo Poletti; Marisa Ponpuak; Hana Popelka; Blagovesta Popova; Helena Porta; Soledad Porte Alcon; Eliana Portilla-Fernandez; Martin Post; Malia B Potts; Joanna Poulton; Ted Powers; Veena Prahlad; Tomasz K Prajsnar; Domenico Praticò; Rosaria Prencipe; Muriel Priault; Tassula Proikas-Cezanne; Vasilis J Promponas; Christopher G Proud; Rosa Puertollano; Luigi Puglielli; Thomas Pulinilkunnil; Deepika Puri; Rajat Puri; Julien Puyal; Xiaopeng Qi; Yongmei Qi; Wenbin Qian; Lei Qiang; Yu Qiu; Joe Quadrilatero; Jorge Quarleri; Nina Raben; Hannah Rabinowich; Debora Ragona; Michael J Ragusa; Nader Rahimi; Marveh Rahmati; Valeria Raia; Nuno Raimundo; Namakkal-Soorappan Rajasekaran; Sriganesh Ramachandra Rao; Abdelhaq Rami; Ignacio Ramírez-Pardo; David B Ramsden; Felix Randow; Pundi N Rangarajan; Danilo Ranieri; Hai Rao; Lang Rao; Rekha Rao; Sumit Rathore; J Arjuna Ratnayaka; Edward A Ratovitski; Palaniyandi Ravanan; Gloria Ravegnini; Swapan K Ray; Babak Razani; Vito Rebecca; Fulvio Reggiori; Anne Régnier-Vigouroux; Andreas S Reichert; David Reigada; Jan H Reiling; Theo Rein; Siegfried Reipert; Rokeya Sultana Rekha; Hongmei Ren; Jun Ren; Weichao Ren; Tristan Renault; Giorgia Renga; Karen Reue; Kim Rewitz; Bruna Ribeiro de Andrade Ramos; S Amer Riazuddin; Teresa M Ribeiro-Rodrigues; Jean-Ehrland Ricci; Romeo Ricci; Victoria Riccio; Des R Richardson; Yasuko Rikihisa; Makarand V Risbud; Ruth M Risueño; Konstantinos Ritis; Salvatore Rizza; Rosario Rizzuto; Helen C Roberts; Luke D Roberts; Katherine J Robinson; Maria Carmela Roccheri; Stephane Rocchi; George G Rodney; Tiago Rodrigues; Vagner Ramon Rodrigues Silva; Amaia Rodriguez; Ruth Rodriguez-Barrueco; Nieves Rodriguez-Henche; Humberto Rodriguez-Rocha; Jeroen Roelofs; Robert S Rogers; Vladimir V Rogov; Ana I Rojo; Krzysztof Rolka; Vanina Romanello; Luigina Romani; Alessandra Romano; Patricia S Romano; David Romeo-Guitart; Luis C Romero; Montserrat Romero; Joseph C Roney; Christopher Rongo; Sante Roperto; Mathias T Rosenfeldt; Philip Rosenstiel; Anne G Rosenwald; Kevin A Roth; Lynn Roth; Steven Roth; Kasper M A Rouschop; Benoit D Roussel; Sophie Roux; Patrizia Rovere-Querini; Ajit Roy; Aurore Rozieres; Diego Ruano; David C Rubinsztein; Maria P Rubtsova; Klaus Ruckdeschel; Christoph Ruckenstuhl; Emil Rudolf; Rüdiger Rudolf; Alessandra Ruggieri; Avnika Ashok Ruparelia; Paola Rusmini; Ryan R Russell; Gian Luigi Russo; Maria Russo; Rossella Russo; Oxana O Ryabaya; Kevin M Ryan; Kwon-Yul Ryu; Maria Sabater-Arcis; Ulka Sachdev; Michael Sacher; Carsten Sachse; Abhishek Sadhu; Junichi Sadoshima; Nathaniel Safren; Paul Saftig; Antonia P Sagona; Gaurav Sahay; Amirhossein Sahebkar; Mustafa Sahin; Ozgur Sahin; Sumit Sahni; Nayuta Saito; Shigeru Saito; Tsunenori Saito; Ryohei Sakai; Yasuyoshi Sakai; Jun-Ichi Sakamaki; Kalle Saksela; Gloria Salazar; Anna Salazar-Degracia; Ghasem H Salekdeh; Ashok K Saluja; Belém Sampaio-Marques; Maria Cecilia Sanchez; Jose A Sanchez-Alcazar; Victoria Sanchez-Vera; Vanessa Sancho-Shimizu; J Thomas Sanderson; Marco Sandri; Stefano Santaguida; Laura Santambrogio; Magda M Santana; Giorgio Santoni; Alberto Sanz; Pascual Sanz; Shweta Saran; Marco Sardiello; Timothy J Sargeant; Apurva Sarin; Chinmoy Sarkar; Sovan Sarkar; Maria-Rosa Sarrias; Surajit Sarkar; Dipanka Tanu Sarmah; Jaakko Sarparanta; Aishwarya Sathyanarayan; Ranganayaki Sathyanarayanan; K Matthew Scaglione; Francesca Scatozza; Liliana Schaefer; Zachary T Schafer; Ulrich E Schaible; Anthony H V Schapira; Michael Scharl; Hermann M Schatzl; Catherine H Schein; Wiep Scheper; David Scheuring; Maria Vittoria Schiaffino; Monica Schiappacassi; Rainer Schindl; Uwe Schlattner; Oliver Schmidt; Roland Schmitt; Stephen D Schmidt; Ingo Schmitz; Eran Schmukler; Anja Schneider; Bianca E Schneider; Romana Schober; Alejandra C Schoijet; Micah B Schott; Michael Schramm; Bernd Schröder; Kai Schuh; Christoph Schüller; Ryan J Schulze; Lea Schürmanns; Jens C Schwamborn; Melanie Schwarten; Filippo Scialo; Sebastiano Sciarretta; Melanie J Scott; Kathleen W Scotto; A Ivana Scovassi; Andrea Scrima; Aurora Scrivo; David Sebastian; Salwa Sebti; Simon Sedej; Laura Segatori; Nava Segev; Per O Seglen; Iban Seiliez; Ekihiro Seki; Scott B Selleck; Frank W Sellke; Joshua T Selsby; Michael Sendtner; Serif Senturk; Elena Seranova; Consolato Sergi; Ruth Serra-Moreno; Hiromi Sesaki; Carmine Settembre; Subba Rao Gangi Setty; Gianluca Sgarbi; Ou Sha; John J Shacka; Javeed A Shah; Dantong Shang; Changshun Shao; Feng Shao; Soroush Sharbati; Lisa M Sharkey; Dipali Sharma; Gaurav Sharma; Kulbhushan Sharma; Pawan Sharma; Surendra Sharma; Han-Ming Shen; Hongtao Shen; Jiangang Shen; Ming Shen; Weili Shen; Zheni Shen; Rui Sheng; Zhi Sheng; Zu-Hang Sheng; Jianjian Shi; Xiaobing Shi; Ying-Hong Shi; Kahori Shiba-Fukushima; Jeng-Jer Shieh; Yohta Shimada; Shigeomi Shimizu; Makoto Shimozawa; Takahiro Shintani; Christopher J Shoemaker; Shahla Shojaei; Ikuo Shoji; Bhupendra V Shravage; Viji Shridhar; Chih-Wen Shu; Hong-Bing Shu; Ke Shui; Arvind K Shukla; Timothy E Shutt; Valentina Sica; Aleem Siddiqui; Amanda Sierra; Virginia Sierra-Torre; Santiago Signorelli; Payel Sil; Bruno J de Andrade Silva; Johnatas D Silva; Eduardo Silva-Pavez; Sandrine Silvente-Poirot; Rachel E Simmonds; Anna Katharina Simon; Hans-Uwe Simon; Matias Simons; Anurag Singh; Lalit P Singh; Rajat Singh; Shivendra V Singh; Shrawan K Singh; Sudha B Singh; Sunaina Singh; Surinder Pal Singh; Debasish Sinha; Rohit Anthony Sinha; Sangita Sinha; Agnieszka Sirko; Kapil Sirohi; Efthimios L Sivridis; Panagiotis Skendros; Aleksandra Skirycz; Iva Slaninová; Soraya S Smaili; Andrei Smertenko; Matthew D Smith; Stefaan J Soenen; Eun Jung Sohn; Sophia P M Sok; Giancarlo Solaini; Thierry Soldati; Scott A Soleimanpour; Rosa M Soler; Alexei Solovchenko; Jason A Somarelli; Avinash Sonawane; Fuyong Song; Hyun Kyu Song; Ju-Xian Song; Kunhua Song; Zhiyin Song; Leandro R Soria; Maurizio Sorice; Alexander A Soukas; Sandra-Fausia Soukup; Diana Sousa; Nadia Sousa; Paul A Spagnuolo; Stephen A Spector; M M Srinivas Bharath; Daret St Clair; Venturina Stagni; Leopoldo Staiano; Clint A Stalnecker; Metodi V Stankov; Peter B Stathopulos; Katja Stefan; Sven Marcel Stefan; Leonidas Stefanis; Joan S Steffan; Alexander Steinkasserer; Harald Stenmark; Jared Sterneckert; Craig Stevens; Veronika Stoka; Stephan Storch; Björn Stork; Flavie Strappazzon; Anne Marie Strohecker; Dwayne G Stupack; Huanxing Su; Ling-Yan Su; Longxiang Su; Ana M Suarez-Fontes; Carlos S Subauste; Selvakumar Subbian; Paula V Subirada; Ganapasam Sudhandiran; Carolyn M Sue; Xinbing Sui; Corey Summers; Guangchao Sun; Jun Sun; Kang Sun; Meng-Xiang Sun; Qiming Sun; Yi Sun; Zhongjie Sun; Karen K S Sunahara; Eva Sundberg; Katalin Susztak; Peter Sutovsky; Hidekazu Suzuki; Gary Sweeney; J David Symons; Stephen Cho Wing Sze; Nathaniel J Szewczyk; Anna Tabęcka-Łonczynska; Claudio Tabolacci; Frank Tacke; Heinrich Taegtmeyer; Marco Tafani; Mitsuo Tagaya; Haoran Tai; Stephen W G Tait; Yoshinori Takahashi; Szabolcs Takats; Priti Talwar; Chit Tam; Shing Yau Tam; Davide Tampellini; Atsushi Tamura; Chong Teik Tan; Eng-King Tan; Ya-Qin Tan; Masaki Tanaka; Motomasa Tanaka; Daolin Tang; Jingfeng Tang; Tie-Shan Tang; Isei Tanida; Zhipeng Tao; Mohammed Taouis; Lars Tatenhorst; Nektarios Tavernarakis; Allen Taylor; Gregory A Taylor; Joan M Taylor; Elena Tchetina; Andrew R Tee; Irmgard Tegeder; David Teis; Natercia Teixeira; Fatima Teixeira-Clerc; Kumsal A Tekirdag; Tewin Tencomnao; Sandra Tenreiro; Alexei V Tepikin; Pilar S Testillano; Gianluca Tettamanti; Pierre-Louis Tharaux; Kathrin Thedieck; Arvind A Thekkinghat; Stefano Thellung; Josephine W Thinwa; V P Thirumalaikumar; Sufi Mary Thomas; Paul G Thomes; Andrew Thorburn; Lipi Thukral; Thomas Thum; Michael Thumm; Ling Tian; Ales Tichy; Andreas Till; Vincent Timmerman; Vladimir I Titorenko; Sokol V Todi; Krassimira Todorova; Janne M Toivonen; Luana Tomaipitinca; Dhanendra Tomar; Cristina Tomas-Zapico; Sergej Tomić; Benjamin Chun-Kit Tong; Chao Tong; Xin Tong; Sharon A Tooze; Maria L Torgersen; Satoru Torii; Liliana Torres-López; Alicia Torriglia; Christina G Towers; Roberto Towns; Shinya Toyokuni; Vladimir Trajkovic; Donatella Tramontano; Quynh-Giao Tran; Leonardo H Travassos; Charles B Trelford; Shirley Tremel; Ioannis P Trougakos; Betty P Tsao; Mario P Tschan; Hung-Fat Tse; Tak Fu Tse; Hitoshi Tsugawa; Andrey S Tsvetkov; David A Tumbarello; Yasin Tumtas; María J Tuñón; Sandra Turcotte; Boris Turk; Vito Turk; Bradley J Turner; Richard I Tuxworth; Jessica K Tyler; Elena V Tyutereva; Yasuo Uchiyama; Aslihan Ugun-Klusek; Holm H Uhlig; Marzena Ułamek-Kozioł; Ilya V Ulasov; Midori Umekawa; Christian Ungermann; Rei Unno; Sylvie Urbe; Elisabet Uribe-Carretero; Suayib Üstün; Vladimir N Uversky; Thomas Vaccari; Maria I Vaccaro; Björn F Vahsen; Helin Vakifahmetoglu-Norberg; Rut Valdor; Maria J Valente; Ayelén Valko; Richard B Vallee; Angela M Valverde; Greet Van den Berghe; Stijn van der Veen; Luc Van Kaer; Jorg van Loosdregt; Sjoerd J L van Wijk; Wim Vandenberghe; Ilse Vanhorebeek; Marcos A Vannier-Santos; Nicola Vannini; M Cristina Vanrell; Chiara Vantaggiato; Gabriele Varano; Isabel Varela-Nieto; Máté Varga; M Helena Vasconcelos; Somya Vats; Demetrios G Vavvas; Ignacio Vega-Naredo; Silvia Vega-Rubin-de-Celis; Guillermo Velasco; Ariadna P Velázquez; Tibor Vellai; Edo Vellenga; Francesca Velotti; Mireille Verdier; Panayotis Verginis; Isabelle Vergne; Paul Verkade; Manish Verma; Patrik Verstreken; Tim Vervliet; Jörg Vervoorts; Alexandre T Vessoni; Victor M Victor; Michel Vidal; Chiara Vidoni; Otilia V Vieira; Richard D Vierstra; Sonia Viganó; Helena Vihinen; Vinoy Vijayan; Miquel Vila; Marçal Vilar; José M Villalba; Antonio Villalobo; Beatriz Villarejo-Zori; Francesc Villarroya; Joan Villarroya; Olivier Vincent; Cecile Vindis; Christophe Viret; Maria Teresa Viscomi; Dora Visnjic; Ilio Vitale; David J Vocadlo; Olga V Voitsekhovskaja; Cinzia Volonté; Mattia Volta; Marta Vomero; Clarissa Von Haefen; Marc A Vooijs; Wolfgang Voos; Ljubica Vucicevic; Richard Wade-Martins; Satoshi Waguri; Kenrick A Waite; Shuji Wakatsuki; David W Walker; Mark J Walker; Simon A Walker; Jochen Walter; Francisco G Wandosell; Bo Wang; Chao-Yung Wang; Chen Wang; Chenran Wang; Chenwei Wang; Cun-Yu Wang; Dong Wang; Fangyang Wang; Feng Wang; Fengming Wang; Guansong Wang; Han Wang; Hao Wang; Hexiang Wang; Hong-Gang Wang; Jianrong Wang; Jigang Wang; Jiou Wang; Jundong Wang; Kui Wang; Lianrong Wang; Liming Wang; Maggie Haitian Wang; Meiqing Wang; Nanbu Wang; Pengwei Wang; Peipei Wang; Ping Wang; Ping Wang; Qing Jun Wang; Qing Wang; Qing Kenneth Wang; Qiong A Wang; Wen-Tao Wang; Wuyang Wang; Xinnan Wang; Xuejun Wang; Yan Wang; Yanchang Wang; Yanzhuang Wang; Yen-Yun Wang; Yihua Wang; Yipeng Wang; Yu Wang; Yuqi Wang; Zhe Wang; Zhenyu Wang; Zhouguang Wang; Gary Warnes; Verena Warnsmann; Hirotaka Watada; Eizo Watanabe; Maxinne Watchon; Anna Wawrzyńska; Timothy E Weaver; Grzegorz Wegrzyn; Ann M Wehman; Huafeng Wei; Lei Wei; Taotao Wei; Yongjie Wei; Oliver H Weiergräber; Conrad C Weihl; Günther Weindl; Ralf Weiskirchen; Alan Wells; Runxia H Wen; Xin Wen; Antonia Werner; Beatrice Weykopf; Sally P Wheatley; J Lindsay Whitton; Alexander J Whitworth; Katarzyna Wiktorska; Manon E Wildenberg; Tom Wileman; Simon Wilkinson; Dieter Willbold; Brett Williams; Robin S B Williams; Roger L Williams; Peter R Williamson; Richard A Wilson; Beate Winner; Nathaniel J Winsor; Steven S Witkin; Harald Wodrich; Ute Woehlbier; Thomas Wollert; Esther Wong; Jack Ho Wong; Richard W Wong; Vincent Kam Wai Wong; W Wei-Lynn Wong; An-Guo Wu; Chengbiao Wu; Jian Wu; Junfang Wu; Kenneth K Wu; Min Wu; Shan-Ying Wu; Shengzhou Wu; Shu-Yan Wu; Shufang Wu; William K K Wu; Xiaohong Wu; Xiaoqing Wu; Yao-Wen Wu; Yihua Wu; Ramnik J Xavier; Hongguang Xia; Lixin Xia; Zhengyuan Xia; Ge Xiang; Jin Xiang; Mingliang Xiang; Wei Xiang; Bin Xiao; Guozhi Xiao; Hengyi Xiao; Hong-Tao Xiao; Jian Xiao; Lan Xiao; Shi Xiao; Yin Xiao; Baoming Xie; Chuan-Ming Xie; Min Xie; Yuxiang Xie; Zhiping Xie; Zhonglin Xie; Maria Xilouri; Congfeng Xu; En Xu; Haoxing Xu; Jing Xu; JinRong Xu; Liang Xu; Wen Wen Xu; Xiulong Xu; Yu Xue; Sokhna M S Yakhine-Diop; Masamitsu Yamaguchi; Osamu Yamaguchi; Ai Yamamoto; Shunhei Yamashina; Shengmin Yan; Shian-Jang Yan; Zhen Yan; Yasuo Yanagi; Chuanbin Yang; Dun-Sheng Yang; Huan Yang; Huang-Tian Yang; Hui Yang; Jin-Ming Yang; Jing Yang; Jingyu Yang; Ling Yang; Liu Yang; Ming Yang; Pei-Ming Yang; Qian Yang; Seungwon Yang; Shu Yang; Shun-Fa Yang; Wannian Yang; Wei Yuan Yang; Xiaoyong Yang; Xuesong Yang; Yi Yang; Ying Yang; Honghong Yao; Shenggen Yao; Xiaoqiang Yao; Yong-Gang Yao; Yong-Ming Yao; Takahiro Yasui; Meysam Yazdankhah; Paul M Yen; Cong Yi; Xiao-Ming Yin; Yanhai Yin; Zhangyuan Yin; Ziyi Yin; Meidan Ying; Zheng Ying; Calvin K Yip; Stephanie Pei Tung Yiu; Young H Yoo; Kiyotsugu Yoshida; Saori R Yoshii; Tamotsu Yoshimori; Bahman Yousefi; Boxuan Yu; Haiyang Yu; Jun Yu; Jun Yu; Li Yu; Ming-Lung Yu; Seong-Woon Yu; Victor C Yu; W Haung Yu; Zhengping Yu; Zhou Yu; Junying Yuan; Ling-Qing Yuan; Shilin Yuan; Shyng-Shiou F Yuan; Yanggang Yuan; Zengqiang Yuan; Jianbo Yue; Zhenyu Yue; Jeanho Yun; Raymond L Yung; David N Zacks; Gabriele Zaffagnini; Vanessa O Zambelli; Isabella Zanella; Qun S Zang; Sara Zanivan; Silvia Zappavigna; Pilar Zaragoza; Konstantinos S Zarbalis; Amir Zarebkohan; Amira Zarrouk; Scott O Zeitlin; Jialiu Zeng; Ju-Deng Zeng; Eva Žerovnik; Lixuan Zhan; Bin Zhang; Donna D Zhang; Hanlin Zhang; Hong Zhang; Hong Zhang; Honghe Zhang; Huafeng Zhang; Huaye Zhang; Hui Zhang; Hui-Ling Zhang; Jianbin Zhang; Jianhua Zhang; Jing-Pu Zhang; Kalin Y B Zhang; Leshuai W Zhang; Lin Zhang; Lisheng Zhang; Lu Zhang; Luoying Zhang; Menghuan Zhang; Peng Zhang; Sheng Zhang; Wei Zhang; Xiangnan Zhang; Xiao-Wei Zhang; Xiaolei Zhang; Xiaoyan Zhang; Xin Zhang; Xinxin Zhang; Xu Dong Zhang; Yang Zhang; Yanjin Zhang; Yi Zhang; Ying-Dong Zhang; Yingmei Zhang; Yuan-Yuan Zhang; Yuchen Zhang; Zhe Zhang; Zhengguang Zhang; Zhibing Zhang; Zhihai Zhang; Zhiyong Zhang; Zili Zhang; Haobin Zhao; Lei Zhao; Shuang Zhao; Tongbiao Zhao; Xiao-Fan Zhao; Ying Zhao; Yongchao Zhao; Yongliang Zhao; Yuting Zhao; Guoping Zheng; Kai Zheng; Ling Zheng; Shizhong Zheng; Xi-Long Zheng; Yi Zheng; Zu-Guo Zheng; Boris Zhivotovsky; Qing Zhong; Ao Zhou; Ben Zhou; Cefan Zhou; Gang Zhou; Hao Zhou; Hong Zhou; Hongbo Zhou; Jie Zhou; Jing Zhou; Jing Zhou; Jiyong Zhou; Kailiang Zhou; Rongjia Zhou; Xu-Jie Zhou; Yanshuang Zhou; Yinghong Zhou; Yubin Zhou; Zheng-Yu Zhou; Zhou Zhou; Binglin Zhu; Changlian Zhu; Guo-Qing Zhu; Haining Zhu; Hongxin Zhu; Hua Zhu; Wei-Guo Zhu; Yanping Zhu; Yushan Zhu; Haixia Zhuang; Xiaohong Zhuang; Katarzyna Zientara-Rytter; Christine M Zimmermann; Elena Ziviani; Teresa Zoladek; Wei-Xing Zong; Dmitry B Zorov; Antonio Zorzano; Weiping Zou; Zhen Zou; Zhengzhi Zou; Steven Zuryn; Werner Zwerschke; Beate Brand-Saberi; X Charlie Dong; Chandra Shekar Kenchappa; Zuguo Li; Yong Lin; Shigeru Oshima; Yueguang Rong; Judith C Sluimer; Christina L Stallings; Chun-Kit Tong Journal: Autophagy Date: 2021-02-08 Impact factor: 13.391
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6