Lixuan Zhan1, Siyuan Chen1, Kongping Li1, Donghai Liang2, Xinyong Zhu1, Liu Liu1, Zhiwei Lu1, Weiwen Sun1, En Xu1. 1. Institute of Neurosciences and Department of Neurology of The Second Affiliated Hospital of Guangzhou Medical University, Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and The Ministry of Education of China, Guangzhou 510260, China. 2. Department of Environmental Health, Rollins School of Public Health, Emory University, Atlanta 30322, Georgia.
Abstract
Autophagy disruption leads to neuronal damage in hypoxic-ischemic brain injury. Rab7, a member of the Rab GTPase superfamily, has a unique role in the regulation of autophagy. Hypoxic preconditioning (HPC) provides neuroprotection against transient global cerebral ischemia (tGCI). However, the underlying mechanisms remain poorly understood. Thus, the current study explored the potential molecular mechanism of the neuroprotective effect of HPC by investigating how Rab7 mediates autophagosome (AP) maturation after tGCI in adult rats. We found that HPC attenuated AP accumulation in the hippocampal CA1 region after tGCI via restoration of autophagic flux. We also confirmed that this HPC-induced neuroprotection was not caused by the increase in lysosomes or the improvement of lysosomal function after tGCI. Electron microscopic analysis then revealed an increase in autolysosomes in CA1 neurons of HPC rats. Moreover, the inhibition of autophagosome-lysosome fusion by chloroquine significantly aggravated neuronal death in CA1, indicating that AP maturation contributes to HPC-induced neuroprotection against neuronal injury after tGCI. Furthermore, the activation of Rab7 was found to be involved in the neuroprotective effect of AP maturation after HPC. At last, the knockdown of ultraviolet radiation resistance-associated gene (UVRAG) in vivo disrupted the interaction between Vps16 and Rab7, attenuated the activation of Rab7, interrupted autophagic flux, and ultimately abrogated the HPC-induced neuroprotection against tGCI. Our results indicated that AP maturation was enhanced by the activation of Rab7 via UVRAG-Vps16 interaction, which further demonstrated the potential neuroprotective role of Rab7 in HPC against tGCI-induced neuronal injury in adult rats.
Autophagy disruption leads to neuronal damage in hypoxic-ischemic brain injury. Rab7, a member of the RabGTPase superfamily, has a unique role in the regulation of autophagy. Hypoxic preconditioning (HPC) provides neuroprotection against transient global cerebral ischemia (tGCI). However, the underlying mechanisms remain poorly understood. Thus, the current study explored the potential molecular mechanism of the neuroprotective effect of HPC by investigating how Rab7 mediates autophagosome (AP) maturation after tGCI in adult rats. We found that HPC attenuated AP accumulation in the hippocampal CA1 region after tGCI via restoration of autophagic flux. We also confirmed that this HPC-induced neuroprotection was not caused by the increase in lysosomes or the improvement of lysosomal function after tGCI. Electron microscopic analysis then revealed an increase in autolysosomes in CA1 neurons of HPCrats. Moreover, the inhibition of autophagosome-lysosome fusion by chloroquine significantly aggravated neuronal death in CA1, indicating that AP maturation contributes to HPC-induced neuroprotection against neuronal injury after tGCI. Furthermore, the activation of Rab7 was found to be involved in the neuroprotective effect of AP maturation after HPC. At last, the knockdown of ultraviolet radiation resistance-associated gene (UVRAG) in vivo disrupted the interaction between Vps16 and Rab7, attenuated the activation of Rab7, interrupted autophagic flux, and ultimately abrogated the HPC-induced neuroprotection against tGCI. Our results indicated that AP maturation was enhanced by the activation of Rab7 via UVRAG-Vps16 interaction, which further demonstrated the potential neuroprotective role of Rab7 in HPC against tGCI-induced neuronal injury in adult rats.
Transient global cerebral ischemia (tGCI) can be caused by drowning, cardiac arrest or cardiopulmonary bypass surgery, thus leading to delayed neuronal death in the hippocampal CA1 subregion. Ischemic preconditioning (IPC) confers neuroprotection of CA1 pyramidal neurons against a subsequent severe ischemic injury.[1] We previously reported that hypoxia preconditioning (HPC) with 8% oxygen for 30–120 min applied 1–4 days before ischemia reduced cell death in CA1 after tGCI.[2] However, the molecular mechanisms underlying ischemic tolerance induced by HPC remain incompletely understood.Autophagy is a process that degrades intracellular organelles and long-lived cytosolic proteins to maintain cell homeostasis. It includes four stages: initiation, elongation, maturation and degradation.[3, 4] Autophagy is initiated by forming a double-membraned compartment known as an autophagosome (AP). APs can fuse with lysosomes to produce autolysosomes (ALs), which allows APs to obtain hydrolytic enzymes indispensable for the subsequent autophagic degradation.[5] This process is known as AP maturation. Defects in AP maturation or AP clearance can cause accumulation of APs within a cell, which can bring about serious disorders.[6, 7, 8] Particularly, autophagic dysfunction in neurons is associated with various neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease and Huntinton’s disease.[9, 10] However, it is still unclear whether AP maturation participated in HPC-induced neuroprotection against tGCI.AP maturation is modulated by multiple factors, such as E1 ubiquitin activating enzyme, Cathepsin D and small GTP-binding protein Rab7 (Ref. 6, 11), among which only Rab7 is crucial for complete autophagic flux.[12] Rab7, a small 208-amino acid protein in the family of GTPases, is predominantly detected in late endosomes.[13] It can promote lysosome biogenesis and maintain lysosome’s function.[14] Rab7 has a key role in catalyzing the fusion of vacuoles with lysosomes,[15] and in AP maturation. Mutations in Rab7, as well as its abnormal expression and activity, might be associated with neurodegenerative diseases, lipid storage disorders and heart diseases.[16, 17, 18] In addition, the downregulation of Rab7 induced by myocardial ischemia would lead to disturbance of AP maturation.[19] However, little is known about the roles and the molecular mechanisms of Rab7 in AP maturation after cerebral ischemia.The activity of Rab7 depends on its active (GTP-bound) and inactive (GDP-bound) states.[20] The GDP/GTP cycle of Rab7 is regulated by two interrelated proteins, the guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins.[21, 22] GEFs mediate the activation of Rab7 by switching from GDP to GTP form. Collins et al.[23] reported that the homotypic fusion and vacuole protein sorting (HOPS) complex interacts with Ypt-RabGTPase and activates Ypt7p (the yeastRab7 orthologue) during membrane fusion.The HOPS complex consists of six subunits, including the Class B vacuolar protein sorting (Vps) complex and class C Vps (C-Vps) complex. The latter contains Vps11, Vps16, Vps18 and Vps33. C-Vps complex acts as a GEF of Rab7 and promotes the activation of Rab7.[24] However, the function of C-Vps complex on autophagy remains less established.Existing studies demonstrate that the C-Vps complex can be positively regulated by the ultraviolet radiation resistance-associated gene (UVRAG).[25] The UVRAG consists of four major regions: the proline-rich domain, the calcium-dependent lipid-binding C2 domain, the coiled-coil domain and a C-terminal domain. C2 domain is required for UVRAG-mediated membrane-lipid association,[25] and the deletion of this domain disrupts UVRAG’s interaction with the C-Vps complex, thereby impairing the ability of UVRAG in promoting AP fusion with lysosomes.[25] Meanwhile, UVRAG-C-Vps interaction potentiates GDP/GTP exchange of Rab7, which in turn promotes endosome maturation.[25, 26] However, whether the UVRAG-C-Vps interaction can activate Rab7 during AP maturation remains to be clarified.In this study, we sought to investigate the impact of AP maturation deficiency on neuronal damage after tGCI and to examine a potential link between the Rab7 activation and AP maturation in HPC-induced ischemic tolerance. Furthermore, we tested a hypothesis that UVRAG-C-Vps interaction enhances the ability of the C-Vps complex to activate Rab7, which ultimately promotes AP maturation to protect neurons from tGCI-induced damage after HPC.
Results
HPC decreases the accumulation of APs in CA1 after tGCI
To examine the activity of autophagy, immunohistochemistry and western blot of microtubule-associated protein 1 light chain 3 (LC3) in CA1 were performed. The number of LC3-positive cells was significantly higher at 0–4 h, peaked at 48 h, and decreased by day 7 after tGCI as compared with Sham. Contrary to tGCIrats, the change of LC3 immunoreactivity was reversed at each time point described above in HPC groups (Figure 1A and B). We further confirmed that the LC3-II/LC3-I ratio in CA1 increased in time-dependent manner after tGCI, and this increase was reversed by HPC (Figure 1C).
Figure 1
Hypoxic preconditioning decreases AP accumulation in hippocampal CA1 region after tGCI. (A) Immunohistochemistry for LC3 in the hippocampus after tGCI with or without HPC. Representative images show Sham-operated group (a and b), 48 h after reperfusion of tGCI group (c and d), 48 h after reperfusion of HPC group (e and f), 7 days after reperfusion of tGCI group (g and h), and 7 days after reperfusion of HPC group (i and j), respectively. Scale bar: a, c, e, g, i: 250 μm; b, d, f, h, j: 25 μm. (B) Quantitative analysis of immunoreactive cell counting of LC3 in CA1. Data are shown as mean±S.D. *P<0.05 versus Sham-operated group and #P<0.05 versus tGCI group at the same time point (n=6 in each group). (C) Western blot analysis of LC3 in CA1 of ischemic and hypoxic preconditioned rats. The histogram presents the quantitative analyses of the ratio of LC3-II/LC3-I. Data are expressed as percentage of value of Sham-operated animals. *P<0.05 versus Sham-operated animals and #P<0.05 versus tGCI group at the same time point (n=5 in each group). (D) TEM micrographs of CA1 neurons in ischemic and hypoxic preconditioned rats. CA1 neurons from Sham-operated rats (a) contained normal polyribosomes (arrowheads), nucleus (N), mitochondria (M), endoplasmic reticulum (ER) and Golgi apparatus (G). CA1 neurons from brains subjected to 10 min of ischemia followed by 48 h reperfusion (b) displayed dissociation of polyribosomes, fragmentation of the neuronal Golgi apparatus and marked accumulation of intracellular vesicles and protein aggregates (arrows), as well as a dramatic increase in APs. Most abnormal morphological changes such as protein aggregates and dissociation of polyribosomes were absent in CA1 neurons from HPC group at 48 h after reperfusion (c), whereas moderate dilated endoplasmic reticulum and swollen mitochondria, and an obviously increase in ALs were observed. Scale bar: 0.5 μm. (E) TEM micrographs of autophagic ultrastructure in CA1 neurons from ischemic and hypoxic preconditioned rats at 48 h after reperfusion. The APs can be identified by its contents (morphologically intact cytoplasm, including ribosomes and ER), and a limiting membrane that is partially visible as two bilayers separated by a narrow elecron-lucent cleft, that is, as a double membrane (asterisk). a: one double-membraned cistern, containing ribosomes and dilated ER; b: an AP containing two mitochondrion and membranous structures; c: an AP containing vesicles and an AP containing dilated or damaged mitochondrion; d: an AP containing dilated or damaged mitochondrion. ALs typically have only one limiting membrane; frequently they contain electon dense cytoplasmic materials and/or organelles at various stages of degradation. e: an AL with partially digested organelles and a lysosome. f–h: ALs partially digested mitochondrion and cytoplasmic materials. Scale bar: 0.1 μm. (F) The cytoplasmic volume fractions of APs and ALs in CA1 neurons from ischemic and hypoxic preconditioned rats. Data expressed as percentage±S.D. show the cytoplasmic volume fractions of APs and ALs. *P<0.05 versus Sham-operated group and #P<0.05 versus tGCI group at the same time point (n=5 in each group)
Next, we examined the brain sections by transmission electron microscopy (TEM). Neurons from CA1 in Sham-operated group displayed normal nucleus, mitochondria, endoplasmic reticulum and Golgi apparatus (Figure 1D, a), whereas neurons in tGCI group demonstrated a remarkable increase in APs, protein aggregate, dilated endoplasmic reticulum and swollen mitochondria after 48 h of reperfusion (Figure 1D, b and Figure 1E, a–d). Seven days after tGCI, neurons exhibited morphologic characteristics of necrotic cell death such as amorphous organelles and cytoplasm disorganization (data not shown). In contrast to tGCI groups, although a dilated endoplasmic reticulum and swollen mitochondria were observed, there seemed to be significantly more ALs in HPC groups (Figure 1D, c and Figure 1E, e–h). The cytoplasmic volume fractions of recognizable APs and ALs within the defined CA1 were counted at 48 h of reperfusion. Relative to Sham-operative rats, the cytoplasmic volume fractions of APs largely increased after tGCI, but this increase was reversed with HPC. Interestingly, the cytoplasmic volume fractions of ALs substantially increased in HPC group compared with tGCI group (Figure 1F).
HPC decreases the accumulation of APs via restoration of autophagic flux after tGCI
To investigate the potential mechanism of autophagy after tGCI with HPC, we examined the expression of autophagy-related protein 5 (Atg5) (Supplementary Fig. I in the Supplementary Material). Atg5-positive cells from Sham animals had round nuclei and spindle cell bodies with elongated axons, showing a typical neuron-like morphology. Alternatively, Atg5 staining appeared in cells with elongated and irregular nuclei at 168 h after tGCI. Quantitative analysis showed no significant difference in the number of Atg5-immunopositive cells in CA1 from ischemic or HPC brains up to 168 h of reperfusion. Similar results were obtained in western blot. These data indicates that decreased AP accumulation induced by HPC was not due to the suppression of autophagy initiation.Then, we investigated whether the restoration of autophagic flux contributed to the decrease of AP accumulation by HPC. SQSTM1/p62 increased markedly through 48 h of reperfusion after tGCI with a time to peak of 24 h. In contrast, HPC eliminated the effects in the expression of SQSTM1/p62 induced by tGCI (Figure 2a). Moreover, both the LC3-II/LC3-I ratio and SQSTM1/p62 in CA1 remarkably increased at 24 h after reperfusion of HPC group with bafilomycin-A1 (BFA) pretreatment. However, these increases induced by BFA pretreatment were relatively less substantial in tGCI group (Figures 2b and c). These results indicate that HPC restored autophagic flux after tGCI.
Figure 2
Hypoxic preconditioning restores autophagic flux after tGCI. (a) Western blot analysis of SQSTM1/p62 in CA1 of ischemic and hypoxic preconditioned rats. The histogram presents the quantitative analyses of SQSTM1/p62 levels. Data are expressed as percentage of value of Sham-operated animals. Each bar represents the mean±S.D.*P<0.05 versus Sham-operated animals and #P<0.05 versus tGCI group at the same time point (n=7 in each group). (b) Effects of BFA on the expression of LC3 in ischemic and hypoxic preconditioned rats analyzed by western blot. Rats were treated with 2.5 μg of BFA or Vehicle intracerebroventricularly at 30 min before tGCI or hypoxia. The same amount of protein lysates from CA1 was subjected to immunoblot analysis using anti-LC3 antibody at 24 h after reperfusion. The histogram presents the quantitative analyses of the ratio of LC3-II/LC3-I. (c) Effects of BFA on the expression of SQSTM1/p62 in ischemic and hypoxic preconditioned rats analyzed by western blot. The histogram presents the quantitative analyses of SQSTM1/p62 levels. Data are expressed as percentage of value of Sham-operated animals. The values are expressed as mean±S.D. *P<0.05 versus Sham-operated animals, #P<0.05 versus tGCI group and &P<0.05 versus HPC+Vehicle group (n=7 in each group). p62, SQSTM1/p62
HPC induces neuroprotection against tGCI via promoting AP maturation
The accumulation of autophagic vacuoles could result from three sources, a selective impairment in autophagosome-lysosome fusion, the dysfunction of lysosomal proteolysis, or a decrease in the number of lysosomes. Accordingly, we measured the levels of lysosomal-associated membrane protein-2 (LAMP2) and Cathepsin D, respectively. Most LAMP2-positive cells from the Sham-operated rats had round nuclei with a granular appearance (Figure 3A), and double-fluorescent immunohistochemistry revealed that these cells were neuronal nuclei (NeuN)-positive. Also, only a minority of LAMP2-positive cells were complement receptor type 3 (OX-42)-positive, which displayed an irregular appearance with polymorphic somata and processes (data not shown). Our results showed no significant differences in the number and distribution of LAMP2-positive cells in CA1 between ischemic and HPC brains until 48 h (Figure 3B). Similar results were observed in western blot (Figure 3D). Alternatively, at 168 h after ischemia, LAMP2 staining mainly existed in cells which exhibited the typical features of activated microglia (Figure 3A, e–h). The results from double-fluorescent immunohistochemistry supported the predominant localization of LAMP2 in microglia (Figure 3C, p–r).
Figure 3
The effect of hypoxic preconditioning on the protein expression of LAMP2 in CA1 after tGCI. (A) Immunohistochemistry for LAMP2 in the hippocampus after tGCI with or without HPC. Representative images show Sham-operated group (a and b), 48 h after reperfusion of HPC group (c and d), 7 days after reperfusion of tGCI group (e and f), and 7 days after reperfusion of HPC group (g and h), respectively. LAMP2-positive neuron-like cells had rounded nuclei, and spindle cell body with elongated axon (arrow) and LAMP2-positive glia-like cells appeared mostly in cells with elaborate array of processes and irregular nuclei (asterisk). Scale bar: a, c, e, g: 250 μm; b, d, f, h: 25 μm. (B) Quantitative analysis of immunoreactive cell counting of LAMP2-positive neuron-like cells and LAMP2-positive glia-like cells in the CA1 subregion. Data are shown as mean±S.D. *P<0.05 versus Sham-operated animals (n=6 in each group). (C) Representative photomicrographs show fluorescent double staining of LAMP2 and NeuN (red), LAMP2 (green) and GFAP (red) and LAMP2 (green) and OX-42 (red) in the rat brains at 48 h and 7 days after tGCI with hypoxia, respectively. The overlapped images show that the vast majority of LAMP2 was colocalized with NeuN (c), and only minority of LAMP2 overlapped with OX-42 at 48 h after reperfusion (i). However, LAMP2 and OX-42 were almost overlapped at 7 days after reperfusion. Scale bar: 75 μm. (D) Western blot analysis of LAMP2 in CA1 of ischemic and hypoxic preconditioned rats. The histogram presents the quantitative analyses of LAMP2 levels. Data are expressed as percentage of value of Sham-operated animals. Each bar represents the mean±S.D. (n=5 in each group)
Unlike LAMP2, the levels of Cathepsin D sharply increased beside a transient decrease at 4 h after tGCI. However, compared with tGCI groups, a persistent increase of Cathepsin D was detected in HPC groups (Figures 4A–C). These data implied that the decreased Cathepsin D in the early stage after ischemia may have led to lysosomal malfunction and thus potentially contributed to the neuronal damage. Accordingly, we evaluated the effects of Pepstatin A (PA), the Cathepsin D inhibitor, on the neuronal death after HPC. Compared with the neuronal morphology in Sham-operated group (Figure 4D, a–d), significant neuronal damage in CA1 was observed in tGCI group (Figure 4D, e–h, and Figure 4E). In HPC group, survival cells and NeuN-positive cells sharply increased, whereas Fluoro-Jade B (FJ-B)-positive cells markedly decreased compared with tGCIrats (Figure 4D, i–l and Figure 4E). Interestingly, after confirming that PA had no neurotoxic effects on the cells of hippocampus in Sham-operated rats (Figure 4D, m–p, and Figure 4E), we further demonstrated that pretreatment with PA did not block the neuroprotection of HPC (Figure 4D, q–t, and Figures 4E and F). Taken together, our results suggest that the neuroprotection induced by HPC may not have resulted from the increase in lysosome number or the improvement of lysosomal function.
Figure 4
Hypoxic preconditioning improves lysosomal function after tGCI. (A) Immunohistochemistry for Cathepsin D in the hippocampus after tGCI with or without HPC. Representative images show Sham-operated group (a and b), 4 h after reperfusion of tGCI group (c and d), 4 h after reperfusion of HPC group (e and f), 7 days after reperfusion of tGCI group (g and h), and 7 days after reperfusion of HPC group (i and j), respectively. Scale bar: a, c, e, g, i: 250 μm; b, d, f, h, j: 25 μm. (B) Quantitative analysis of Cathepsin D-positive fiber density in CA1. Data are shown as mean±S.D. *P<0.05 versus Sham-operated group and #P<0.05 versus tGCI group at the same time point (n=6 in each group). (C) Western blot analysis of Cathepsin D in CA1 of ischemic and hypoxic preconditioned rats. The histogram presents the quantitative analyses of Cathepsin D levels. Data are expressed as percentage of value of Sham-operated animals. Each bar represents the mean±S.D. *P<0.05 versus Sham-operated animals and #P<0.05 versus tGCI group at the same time point (n=5 in each group). (D) Representative microphotographs of cresyl violet staining, immunostaining of NeuN and FJ-B staining in the hippocampus at 7 days after HPC with or without PA treatment. Sham group (a–d); tGCI group (e–h); HPC group (i–l); PA+Sham group (m–p), pretreatment with PA without ischemia or hypoxia; PA+HPC group, pretreatment with PA at 2 h before HPC (q–t). Scale bar: a, e, i, m, q: 250 μm, b–d, f–h, j–l, n–p, r–t: 25 μm. (E) Quantitative analyses of survival neurons, NeuN-positive cells and FJ-B-positive cells in CA1. Each bar represents the mean±S.D. *P<0.05 versus Sham-operated animals and #P<0.05 versus tGCI group (n=6 in each group). (F) Effects of PA on Cathepsin D in HPC rats. Rats were treated with PA intraperitoneally at 2 h before HPC. The same amount of protein lysates from CA1 were subjected to immunoblot analysis using anti-Cathepsin D antibody at 0 h of reperfusion. The values are expressed as mean±S.D. *P<0.05 versus Sham-operated animals and #P<0.05 versus tGCI group and &P<0.05 versus Vehicle group (n=5 in each group)
Similarly, to further ascertain whether the promotion of autophagosome-lysosome fusion have contributed to HPC-induced neuroprotection against tGCI, rats were pretreated with chloroquine (CQ) (Figure 5), which inhibits autophagosome-lysosome fusion.[27] The pretreatment with CQ before HPC completely eliminated the neuroprotective effect of HPC, and increased LC3-II and SQSTM1/p62 in CA1 after tGCI. These results support that HPC restores autophagic flux owing to the promotion of autophagosome-lysosome fusion, which in turn contributes to neuroprotection against neuronal injury after tGCI.
Figure 5
Impaired AP maturation by pretreatment with chloroquine causes neuronal damage in CA1 of hypoxic preconditioned rats. (A) Representative microphotographs of cresyl violet staining, immunostaining of NeuN, and FJ-B staining in the hippocampus at 7 days after HPC with or without CQ treatment. Sham+CQ group (a–d), pretreatment with CQ without ischemia or hypoxia; HPC+Vehicle group (e–h), pretreatment with normal saline at 2 h before HPC; HPC+CQ group (i–l), pretreatment with CQ at 2 h before HPC. Scale bar: a, e, i: 250 μm, b–d, f–h, j–l: 25 μm. (B–D) Quantitative analyses of survival neurons, NeuN-positive cells and FJ-B-positive cells in CA1. Each bar represents the mean±S.D. *P<0.05 versus Sham-operated animals, #P<0.05 versus tGCI group and &P<0.05 versus HPC+Vehicle group (n=6 in each group). (E) Effects of CQ on LC3 in hypoxic preconditioned rats. Rats were treated with 60 mg/kg of CQ or vehicle intracerebroventricularly at 2 h before hypoxia. The same amount of protein lysates from CA1 was subjected to immunoblot analysis using anti-LC3 antibody at 48 h after reperfusion. The histogram presents the quantitative analyses of the ratio of LC3-II/LC3-I. The values are expressed as mean±S.D. *P<0.05 versus Sham-operated animals, #P<0.05 versus tGCI group and &P<0.05 versus HPC+Vehicle group (n=5 in each group). (F) Western blot analysis of SQSTM1/p62 in CA1 of hypoxic preconditioned rats after pretreatment with CQ. The histogram presents the quantitative analyses of SQSTM1/p62 levels. Data are expressed as percentage of value of Sham-operated animals. The values are expressed as mean±S.D. *P<0.05 versus Sham-operated animals, #P<0.05 versus tGCI group and &P<0.05 versus HPC+Vehicle group (n=6 in each group). CQ: chloroquine
HPC induces AP maturation via activating Rab7 after tGCI
To explore the molecular mechanisms underlying AP maturation in HPCrats, we measured the expression of Rab7 in CA1. Rab7-positive cells from Sham-operated rats had large spindle-shaped soma with single apical axon (Figure 6 A, a–b). Double-fluorescent immunohistochemistry revealed that they surrounded NeuN-positive cells (Figure 6E, a–c). No colocalization of Rab7 with glial fibrillary acidic protein (GFAP) was found (Figure 6E, d–f). Notably, at 168 h of reperfusion after tGCI, Rab7 located mainly in cells with elongated and irregular nuclei (Figure 6A, e–f), and double-fluorescent immunohistochemistry revealed that they were positive for ionized calcium binding adaptor molecule 1 (Iba-1) (Figure 6E, g–i) and GFAP (Figure 6E, j–l). Alternatively, most Rab7-positive cells in HPCrats had large spindle-shaped soma with single apical axon (Figure 6A, g–h). Double-fluorescent immunohistochemistry revealed that they were microtubule-associated protein-2 (MAP-2)-positive (Figure 6E, m–o). There were no significant differences in the number of Rab7-positive cells between ischemic and HPC brains up to 168 h of reperfusion (Figures 6A and B). Similar results were observed in western blot (Figure 6C). These results indicate that the alteration of Rab7 protein may not have been involved in the effect of AP maturation after HPC. We further examined the activity of Rab7 using a Rab7 effector’s protein co-immunoprecipitation assay.[28] The results showed that Rab7-interacting lysosomal protein (RILP) interacting with Rab7 in HPCrats was substantially higher than that in tGCIrats, especially at 0–4 h after reperfusion (Figure 6D). These data indicates that the activation of Rab7 after HPC may have facilitated AP maturation.
Figure 6
Hypoxic preconditioning increases the activation of Rab7 in hippocampal CA1 region after tGCI. (A) Immunohistochemistry for Rab7 in the hippocampus after tGCI with or without HPC. Representative images show Sham-operated group (a and b), 48 h after reperfusion of HPC group (c and d), 7 days after reperfusion of tGCI group (e and f), and 7 days after reperfusion of HPC group (g and h), respectively. Scale bar: a, c, e, g: 250 μm; b, d, f, h : 25 μm. (B) Quantitative analysis of immunoreactive cell counting of Rab7 in CA1. Data are shown as mean±S.D. (n=6 in each group). (C) Western blot analysis of Rab7 in CA1 of ischemic and hypoxic preconditioned rats. The histogram presents the quantitative analyses of Rab7 levels. Data are expressed as percentage of value of Sham-operated animals. Each bar represents the mean±S.D.*P<0.05 versus Sham-operated animals (n=6 in each group). (D) Immunoprecipitation blots showing the Rab7-RILP complex in CA1 of ischemic and hypoxic preconditioned rats. RILP was immunoprecipitated (IP) using anti-RILP antibody. Rab7 was detected by western blot (WB). The experiments were repeated twice (n=3 in each group). (E) Representative photomicrographs show fluorescent double staining of Rab7 (green) and NeuN (red), Rab7 (green) and GFAP (red), Rab7 (green) and Iba-I (red) and Rab7 (green) and MAP-2 (green) in the rat brains, respectively. The overlapped images show that Rab7 surrounded NeuN in Sham (c). However, Rab7 located mainly in Iba-1 and GFAP-positive cells in CA1 at 168 h after tGCI (i, l). Alternative, Rab7 located mainly in MAP-2 and GFAP-positive cells in CA1 at 168 h after HPC (i, l) Scale bar: 50 μm
HPC activates Rab7 via mediating UVRAG-Vps16 interaction after tGCI
We then hypothesized that UVRAG and its modulation by Vps16 were required for the activation of Rab7 after HPC. Our results showed that UVRAG in tGCI groups was remarkably downregulated after reperfusion compared with the Sham-operated group. However, in HPC groups, it increased immediately after reperfusion, and continued to increase up to 4 h and then returned to the Sham level at 24 h post-ischemia (Figure 7A–C). To investigate the involvement of UVRAG in the activation of Rab7, we performed small-interfering RNA (siRNA)-mediated knockdown of UVRAG. The silencing efficacy of siRNAs in vitro is shown in Figure 7D. UVRAG-rat-371 and UVRAG-rat-425 had higher silencing efficacy in in vitro study with retinal ganglion cells (RGC-5), which was determined by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Then, UVRAG-rat-371 and UVRAG-rat-425 were selected to combine with HVJ-Envelope (HVJ-E) to transfer into the dorsal CA1 pyramidal layer of rats. The expression of UVRAG in CA1 detected by western blot significantly decreased after the administration of UVRAG siRNAs either in Sham-operated or in HPCrats (Figure 7E). Moreover, the expression of UVRAG with the vector of UVRAG-rat-425 was lower than that of UVRAG-rat-371.
Figure 7
Hypoxic preconditioning increases the expression of UVRAG and enhances the ability of the C-Vps complex to activate Rab7 in hippocampal CA1 region after tGCI. (A) Immunohistochemistry for UVRAG in the hippocampus after tGCI with or without HPC. Representative images show Sham-operated group (a and b), 4 h after reperfusion of tGCI group (c and d), 4 h after reperfusion of HPC group (e and f), 7 days after reperfusion of tGCI group (g and h), and 7 days after reperfusion of HPC group (i and j), respectively. Scale bar: a, c, e, g, i: 250 μm; b, d, f, h, j: 25 μm. (B) Quantitative analysis of immunoreactive cell counting of UVRAG in CA1. Data are shown as mean±S.D. *P<0.05 versus Sham-operated group and #P<0.05 versus tGCI group at the same time point (n=6 in each group). (C) Western blot analysis of UVRAG in CA1 of ischemic and hypoxic preconditioned rats. The histogram presents the quantitative analyses of UVRAG levels. Data are expressed as percentage of value of Sham-operated animals. Each bar represents the mean±S.D. *P<0.05 versus Sham-operated animals and #P<0.05 versus tGCI group at the same time point (n=6 in each group). (D) RT-qPCR showed that UVRAG mRNA expression in RGC-5 was significantly downregulated after transfection with siRNAs, and siRNA-371 and siRNA-425 at 50 nM presented the higher efficacy. *P<0.05 versus negative control (NC, n=3 in each group). (E) Representative picture from western blot showed that protein expressions of UVRAG in CA1 of Sham-operated and hypoxic preconditioned rats were remarkably decreased after transfection with siRNAs into the dorsal CA1 pyramidal layer of rat brains. Data are expressed as percentage of value of Sham+NC animals. Each bar represents the mean±S.D. *P<0.05 versus Sham+NC animals and #P<0.05 versus HPC+NC group (n=6 in each group). (F) Immunoprecipitation blots showing the interaction of Vps16 with Rab7 in CA1 of ischemic and hypoxic preconditioned rats. Vps16 was immunoprecipitated using anti-Vps16 antibody. Rab7 was detected by WB. (G–H) The effects of UVRAG knockdown on the interaction of Vps16 with Rab7 and the activation of Rab7. The same amount of protein lysates from CA1 were obtained at 4 h after reperfusion in HPC or 24 h after injection of vectors in Sham-operated group. RILP was immunoprecipitated using anti-RILP antibody. Rab7 was detected by WB. The experiments were repeated twice (n=3 in each group)
Next, interaction of Vps16 with Rab7, assessed by co-immunoprecipitation, was enhanced in CA1 after HPC (Figure 7F). The strength of this interaction was weakened in UVRAG knockdown rats (Figure 7G). Furthermore, the activation of Rab7 was largely reduced by UVRAG knockdown either in HPC or Sham-operated rats (Figure 7H).To test the causal relationship between the UVRAG downregulation and the impairment of AP maturation in CA1 after HPC, we then investigated the impact of UVRAG knockdown on the AP maturation. With UVRAG knockdown both LC3-II/LC3-I ratio and SQSTM1/p62 were remarkably increased either in Sham-operated or HPCrats (Figure 8A and B).
Figure 8
Knockdown of UVRAG decreases autophagic flux and causes neuronal damage in CA1 of hypoxic preconditioned rats. (A) The effects of UVRAG knockdown on LC3 in hypoxic preconditioned rats. Same amount of protein lysates from CA1 was subjected to immunoblot analysis using anti-LC3 antibody at 4 h after reperfusion. The histogram presents the quantitative analyses of the ratio of LC3-II/LC3-I. (B) The effects of UVRAG knockdown on SQSTM1/p62 in hypoxic preconditioned rats. The histogram presents the quantitative analyses of SQSTM1/p62 levels. Data are expressed as percentage of value of Sham-operated animals. The values are expressed as mean±S.D. *P<0.05 versus Sham+NC animals, and &P<0.05 versus HPC+NC group (n=6 in each group). (C) Representative microphotographs of cresyl violet staining, immunostaining of NeuN and FJ-B staining showed that knockdown of UVRAG increased neuronal damage in the hippocampus at 7 days after HPC. Sham+NC group (a–d), Sham-operated animals with injection of negative control siRNA vector; Sham+siRNA-425 group, Sham-operated animals with injection of UVRAG-rat-425 vector (e–h); tGCI+NC group (i–l); tGCI+ siRNA-425 group (m–p); HPC+NC group (q–t); HPC+ siRNA-425 group (u–x). Scale bar: a, e, i, m, q, u: 250 μm, b–d, f–h, j–l, n–p, r–t, v–x: 25 μm. (D–F) Quantitative analyses of survival neurons, NeuN-positive cells and FJ-B-positive cells in CA1. Each bar represents the mean±S.D. *p<0.05 versus Sham+NC animals, #P<0.05 versus tGCI+NC group and &P<0.05 versus HPC+NC group (n=8 in each group)
Finally, we examined the effect of UVRAG downregulation on neuroprotection induced by HPC (Figure 8C–F). UVRAG knockdown completely abolished the neuroprotection of HPC, revealed by the percentages of survival cells, NeuN-positive cells and FJ-B-positive cells in CA1. Notably, no obvious cell damage was found in Sham-operated rats either with UVRAG knockdown or the negative control siRNA.
Discussion
The present study supports the hypothesis that HPC activates Rab7 by mediating UVRAG-Vps16 interaction after tGCI in rats. As a result, the restoration of autophagic flux and promotion of AP maturation, and the ensuing decrease of AP accumulation in CA1 may have contributed to the neuroprotection induced by HPC against tGCI.HPC has been described as an endogenous strategy by which a sublethal hypoxic exposure can protect brain tissues from further severe ischemic insult.[2, 29, 30] Several studies have demonstrated that cardioprotection elicited by IPC was mediated via upregulation of autophagy.[31, 32] An in vitro study found that autophagy contributed to IPC-induced neuroprotection against ischemia in PC12 cells.[33] Also, Sheng et al.[34] reported that the activation of autophagy had an important part in the neuroprotection induced by IPC following focal ischemia in rats. Our previous study demonstrated that the downregulation of autophagy may induce ischemic tolerance after hypoxic postconditioning.[35] However, the exact effects of autophagy on cerebral ischemia were still controversial.[7, 36, 37, 38, 39, 40, 41] Undoubtedly, the autophagy pathway is activated after cerebral ischemia.[39, 40, 41, 42] In this study we have demonstrated that APs and LC3-II increased in CA1 after tGCI. In contrast, HPC decreased APs and LC3-II after tGCI. It is noteworthy that a remarkable increase in ALs was revealed after HPC.APs can fuse with lysosomes to form ALs where the contents are degraded by lysosomal hydrolases. Therefore, the accumulation of APs after tGCI may imply either an increase in AP formation, or a reduction in autophagic flux. The nucleation and elongation of phagophore are two major steps of autophagy initiation. The latter depends on the transition of LC3-I to LC3-II, which is mediated by Atg5-Atg12 conjugation. Therefore, the loss of Atg5 would effectively inhibit autophagy.[43] However, there were no significant differences in the level of Atg5 after tGCI with or without HPC. This result indicates that decreased AP accumulation induced by HPC may not be attributed to the suppression of autophagy initiation.We then examined whether impaired autophagic flux was involved in the accumulation of APs after tGCI. LC3-II is degraded after the fusion of APs with lysosomes. SQSTM1/p62, which is able to bind to ubiquitinated damaged proteins, is designated for autophagic degradation. Therefore, the increase in both the LC3-II/LC3-I ratio and SQSTM1/p62 implies the impairment of autophagic flux.[44] This study demonstrated increased ratio of LC3-II/LC3-I in CA1 after tGCI, accompanied by an increase of SQSTM1/p62, indicating the autophagic flux was blocked. In contrast, HPC eliminated these effects induced by tGCI. Moreover, both the LC3-II/LC3-I ratio and SQSTM1/p62 remarkably increased after HPC pretreatment with BFA. These results indicate that HPC restored autophagic flux after tGCI.In addition, the defective AP removal can be caused either by inadequate lysosomal proteolytic activities or defective fusion between APs and lysosomes. Accordingly, levels of the lysosome membrane proteins LAMP2 and the soluble lysosomal enzyme Cathepsin D were examined. The level of LAMP2 did not alter until 48 h of reperfusion after tGCI with or without HPC, suggesting that the size of the lysosomal compartment and total number of lysosomes remained unchanged. Interestingly, LAMP2 was mainly found in neurons at 48 h of reperfusion. However, LAMP2 decreased significantly in neurons at 168 h. Lysosomes are composed of soluble acidic hydrolases, integral membrane proteins and membrane-associated proteins.[45] Dysfunction in any of these components may cause lysosomal deficits, leading to accumulation of undegraded metabolites. Therefore, decrease of LAMP2 in neurons led to lysosomal dysfunction and ultimately neuronal damage at 168 h after tGCI. Moreover, our results revealed that LAMP2 localized mainly in microglia. Our previous study confirmed that microglia can be activated in CA1 after tGCI.[29] These findings indicate that the lysosomal activation and alteration in microglia may have played crucial roles in the pathogenesis of tGCI. After tGCI, microglial cells are activated and may attempt to digest damaged components, including debris or misfolded proteins through the autophagy-lysosome pathway. To the best of our knowledge, this is the first report on LAMP2 expression in microglia after cerebral ischemia.Unlike LAMP2, a persistent increase of Cathepsin D in HPC groups was observed. However, the pretreatment with PA did not block the neuroprotection induced by HPC. These results demonstrate that HPC-induced neuroprotection is neither mediated via the increase in the number of lysosome nor the improvement of lysosomal function after tGCI. Taken together, a defect in autophagosome-lysosome fusion may have caused AP accumulation after tGCI. Indeed, as compared with HPCrats, the number of total autophagic vacuoles in CA1 of tGCIrats substantially increased while the number of ALs did not, suggesting impaired autophagosome-lysosome fusion after tGCI. Furthermore, the inhibition of autophagosome-lysosome fusion via CQ remarkably aggravated neuronal death in CA1, with increases in both the ratio of LC3-II/LC3-I and SQSTM1/p62. These data indicates that AP maturation may have contributed to the HPC-induced neuroprotection against neuronal injury with tGCI.The regulation of AP maturation induced by HPC is still poorly understood. In an in vitro study, Rab7 was shown to be involved in the regulation of autophagosome-lysosome fusion and lysosomal genesis.[5] The upregulation of Rab7 has an important role in mediating autophagic flux increases induced by starvation in cardiomyocytes.[46] Besides, the downregulation of Rab7 impairs AP maturation and exacerbates cell death in cultured cardiomyocytes.[47] These studies support the involvement of Rab7 in the regulation of AP maturation. However, our results failed to demonstrate any effect of HPC on the expression of Rab7 during reperfusion. Noteworthily, the distribution of Rab7 between ischemic and HPC brains was different after reperfusion. Rab7 of CA1 in tGCIrats was expressed mainly in glia at 168 h of reperfusion. Alternatively, most Rab7 in HPC was expressed in neurons. This is the first report on the transition of Rab7 expression from neurons to astrocytes after tGCI. It remains unclear whether this difference of Rab7 distribution between HPC and tGCIrats contributes to neuroprotection and AP maturation induced by HPC.Rab7 activity depends on its functional states. In an in vitro study with cultured Purkinje neurons, Rab7 activity decreased after prolonged trophic factor withdrawal, with AP accumulation and cell death. However, the deactivation of Rab7 was prevented and autophagic flux was restored by adding insulin-like growth factor-I.[48] Our study showed that the RILP-Rab7 interaction in HPCrats was higher than that in tGCI, revealing that HPC enhanced Rab7 activity during tGCI-induced autophagy activation.The C-Vps complex is thought to be a GEF of Rab7 and promotes Rab7 transiting from GDP-bound to GTP-bound state, resulting in activation of Rab7.[49] Moreover, the Vps39p subunit of HOPS complex has GEF activity on Ypt7p, the yeast ortholog of Rab7.[24] In this study, the interaction of Vps16 with Rab7 in tGCIrats was enhanced after HPC. This interaction may have mediated the activation of Rab7 after HPC. Previous evidence indicated that the C-Vps complex was positively regulated by UVRAG[25] and UVRAG had a vital role in autophagosome-lysosome fusion.[25, 50] Therefore, it is speculated that UVRAG interaction with the C-Vps complex may enhance the ability of C-Vps in GDP-to-GTP conversion of Rab7, thereby promoting the fusion of APs with lysosomes. Our results support that UVRAGdeficiency disrupts the interaction between Vps16 and Rab7, decreases the activation of Rab7, prevents AP fusion with lysosomes and ultimately abrogates the neuroprotection provided by HPC.In summary, we found that HPC attenuated neuronal death by enhancing AP maturation in CA1 after tGCI. The activation of Rab7 played an essential role in AP maturation, which mediated neuroprotection of HPC against tGCI. Moreover, given that UVRAG deficiency disrupted the interaction between Vps16 and Rab7 and decreased the activation of Rab7, UVRAG might be deemed as a potential molecular target for protection against tGCI.
Materials and methods
All surgical procedures and animal experiments were performed according to the ARRIVE guidelines, and were approved and monitored by the Animals Care and Use Committee of Guangzhou Medical University. Adult male Wistar rats weighing 220–280 g (7–8 weeks old) were obtained from Southern Medical University. Rats for experiment were housed under standard temperature (22±1 °C) in a 12 h light/dark controlled environment with free access to food and water.In this study, 442 rats were used, and nine of which in tGCI and five in HPC groups died during the procedure of tGCI, and three in tGCI and two in HPC groups died after tGCI. Also, three rats died after intracerebroventricular injection of BFA; five died after the injection of CQ; six died after the injection of HVJ-E vectors, and two died during the procedure of hypoxia. Nine rats that convulsed during ischemia and four during 72 h post-ischemia were also excluded.
Transient global ischemia and HPC
To induce acute cerebral ischemia, tGCI was carried out according to the four-vessel occlusion method.[51] All procedures in our study were performed under aseptic conditions. In brief, anesthesia was induced with 3–4% isoflurane in a chamber and maintained during the operation with a mask using 2–3% isoflurane. Vertebral arteries were electrocauterized, and common carotid arteries were isolated. A teflon/silastic occluding device was placed loosely around each carotid artery without interrupting carotid blood flow. Forebrain ischemia was induced 24 h after surgery in the rats which were awake, by occluding both common carotid arteries for 10 min. After occlusion, rats that lost their righting reflex within 1 min and whose pupils dilated were selected for experiments. Rectal temperature was maintained at 37~38 °C throughout the procedure. Sham-operated rats received the same surgical procedure except the occlusion of arteries. Rats that convulsed during ischemia or post-ischemia were excluded from this study.Twenty-four hours before ischemia, rats were placed in a hypoxia chamber through which air containing 8% O2 and 92% N2 flowed continuously at the temperature of 23–25 °C, and preconditioned for 30 min.[2]
Pharmacological interventions
The implantation of the intracerebroventricular injection cannula into the right lateral ventricle was performed stereotaxically under isoflurane anesthesia. The cannula was stereotaxically placed through a burr hole opened on the right parietal skull at 1.5 mm lateral, 0.8 mm posterior and 4.0 mm dorsal with respect to the bregma, and affixed to the skull with stainless steel screws and cranioplastic cement. Rats were allowed to recover from surgery for one week before treatment. All animals displayed normal feeding and drinking behaviors postoperatively. For pharmacological interventions, BFA (4 μmol/l, 10 μl, intracerebroventricularly, Sigma Aldrich, St Louis, MO, USA.) was administered at 30 min before tGCI or HPC and PA (32 μg/kg, intraperitoneally, Sigma), CQ (60 mg/kg, intraperitoneally, Sigma) or the vehicle (25% dimethylsulfoxide in phosphate-buffered saline (PBS)) was administered at 2 h before HPC. To evaluate the toxicity of PA and CQ to the cells of hippocampus, six animals without hypoxia or ischemia were treated with PA and CQ intraperitoneally, respectively.
Assessment of cellular damage
As studied previously,[2] Nissl, FJ-B and NeuN staining were performed to determine the hippocampal cell damage at 7 days after reperfusion. The sections from Nissl and NeuN staining were examined under a light microscope (× 660). FJ-B stained images were observed with a fluorescence microscope (Leica Microsystems, Wetzlar, Hessen, Germany). Cell counts were conducted as described previously.[52] Cells in the CA1 pyramidal layer were quantitatively analyzed within three non-repeated rectangular areas of 0.037 mm2 in the typical dorsal hippocampus between stereotaxic planes AP 4.5 and 4.9 mm (interaural). Data were quantified bilaterally in sections from each brain and assessed double-blindedly. Also, four sections per animal were evaluated.
TEM
For electronic microscopic analysis, samples were pre-fixed in 0.1 M sodium cacodylate-buffered (PH 7.4) 2.5% glutaraldehyde solution for 2 h and post-fixed in 0.1 M sodium cacodylate-buffered 1% OsO4 solution for 1 h. After dehydrated in a step-wised ethanol gradient solution (50% ethanol 20 min, 70% ethanol 20 min, 80% ethanol 20 min, 90% ethanol 20 min, 95% ethanol 20 min and 100% ethanol 20 min), samples were incubated with propylene oxide for 10 min, then impregnated with a mixture of propylene oxide/Spurr (1:1) and embedded in Spurr resin. Ultrathin sections were examined using JEOL JEM-1200EX TEM. Five rats were used at each experimental group. Four tissue samples were taken for thin sections from each animal. Five sections were cut per sample. Ten electron micrographs taken from the cell layer areas between the nuclei and apical dendrites of the CA1 pyramidal neurons were taken per section at a primary magnification of × 25000. Morphometrical measurements of APs and ALs were carried out using the point-counting method.[44, 53] Data from each electron micrograph taken from the samples of the same experimental point were used to calculate the mean and its variation. The cytoplasmic volume fractions of the autophagic vacuole were expressed as percentage of the cytoplasmic volume.
Immunohistochemistry
The animals were killed at 0, 4, 24, 48 and 168 h after reperfusion with or without hypoxia (n=6 in each group). Single-label immunohistochemistry was conducted by the avidin-biotin-peroxidase complex method.[2] The primary antibodies used in these studies include LC3 (1:100; NOVUS, Littleton, CO, USA), Atg5 (1:100; Abcam, Cambridge, UK), LAMP2 (1:2000; Sigma), Cathepsin D (1:1000; Sigma), Rab7 (1:100; Abcam), UVRAG (1:100; Millipore, Bedford, MA, USA) and NeuN (1:4000; Millipore). Immunopositive cells in which the reaction product was present within a clear and regular-shaped cytoplasmic or nuclear border were quantified under a light microscope with × 660 magnification. The average intensity of Cathepsin D-positive fiber staining in which reaction product presented in the cell processes in the CA1 subregion was determined using Image-Pro Plus software for Windows, version 6.0 (Media Cybernetics, Inc. Warrendale, PA, USA). Four non-repeated × 200 magnification microscopic random fields (141.15 μm2 per field) in the pyramidal layer, stratum radiatum and stratum lacunosum-moleculare of each subject were assessed in four coronal tissue sections. Measures of staining intensity of fibers were averaged across tissue sections to provide a single mean value for each structure for each rat. These mean values were used for statistical analysis.Double-fluorescent immunohistochemistry was conducted to demonstrate cell types and the exact position where LAMP2 and Rab7 were expressed. NeuN, MAP-2, GFAP, OX-42 and Iba-1 were used to identify NeuN, neuronal cell bodies, astrocyte and microglia, respectively. Double-fluorescent immunohistochemistry was performed as described previously.[2] Antibodies used in these studies include LAMP2 (1:1000; Sigma), Rab7 (1:50; Abcam), NeuN (1:1000; Millipore), GFAP (1:2000; Millipore), OX-42 (1:100; Millipore), Iba-1 (1:100; Millipore), MAP-2 (1:300; Millipore), Cy3-conjugated goat anti-mouse IgG antibody (1:100; Invitrogen, Carlsbad, CA, USA) and FITC-conjugated goat anti-rabbit antibody (1:50; Invitrogen). Slides were analyzed with a confocal laser microscope (Leica Microsystems).
Western blotting
Rats were killed under chloral hydrate anesthesia at 0, 4, 24 and 48 h after reperfusion with or without hypoxia (n=5 in each group). The brain tissue was cut into 2 mm coronal slices using a brain matrix and the CA1 subregions of bilateral hippocampi were quickly dissected under a stereomicroscope. Proteins of the hippocampal CA1 subregion were extracted as described previously.[2] Western blot was performed as described previously[2] using the antibodies including LC3 (1:10000; Novus), Atg5 (1:1000; Abcam), SQSTM1/p62 (1:2000; Abcam), LAMP2 (1:6000; Sigma), Cathepsin D (1:1000; Sigma), Rab7 (1:1000; Abcam), Vps16 (1:1000; Proteintech Group, Inc. Chicago, IL, USA), UVRAG (1:1000; Millipore) and GAPDH (1:10000; Proteintech). Densitometric analysis for the quantification of the bands was performed using image analysis software (Quantity One, Bio-Rad Laboratories, Inc., Hercules, CA, USA). Relative optical densities of protein bands were calibrated with β-actin or GAPDH and normalized to those in Sham-operated rats.
Immunoprecipitation
Tissues from the CA1 subregions of bilateral hippocampi were lysed in a buffer composed of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Triton X-100 and 1 mM EDTA supplemented with a protease inhibitor tablet (Roche, Mannheim, Germany). A total of 0.4 mg of protein was incubated with 8 μg of RILP antibody (ProSci, San Diego, CA, USA) or Vps16 antibody (Proteintech) overnight at 4 °C followed by incubation with 120 μl of protein G-Sepharose (Millipore) for 2 h at 4 °C. Immunocomplexes were collected by centrifugation and examined with western blot after separation by SDS-PAGE.
Reverse transcription quantitative RT-PCR
Total RNA was extracted from RGC-5 transfected with siRNAs using Trizol reagent (Fermentas, Pittsburgh, PA, USA). RT-qPCR was performed according to the standard protocol. The primers of UVRAG were 5′-ACTCCAGACTTGAGGCAAAC-3′ (forward) and 5′-ACAGATACTCACCATCTGACC-3′ (reverse). The primers of β-actin were 5′-AGGGAAATCGTGCGTGACAT-3′ (forward) and β-actin-R 5′-GAACCGCTCATTGCCGATAG-3′ (reverse). Quantitative PCRs were conducted by LightCycler Fast-Start DNA Master SYBR Green 1 kit and on a LightCycler 1.5 PCR machine (Roche). Data were analyzed using the comparative Cmethod (2-ΔΔ) and normalized to β-actin. All reactions were performed in triplicate. Results were expressed as fold changes compared to negative control.
UVRAG RNAi knockdown
Three different sequences of UVRAG siRNAs (UVRAG-rat-558: 5′-GCUGUUGAUAGAGUGGAAATT-3′, 5′-UUUCCACUCUAUCAACAGCTT-3′UVRAG-rat-371: 5′-CCUACUUCACUCUUCAUUUTT-3′, 5′-AAAUGAAGAGUGAAGUAGGTT-3′UVRAG-rat-425: 5′-GGAGUGAAGUGAUUAAGAATT-3′, 5′-UUCUUAAUCACUUCACUCCTT-3′) and negative control siRNA (5′-UUCUCCGAACGUGUCACGUTT-3′, 5′-ACGUGACACGUUCGGAGAATT-3′) were purchased from ViewSolid Biotech (Beijing, China). To select the highest knockdown effects of UVRAG siRNAs, three UVRAG siRNA sequences at 25 nM, 50 nM and 100 nM were transferred into rat RGC-5. Then, UVRAG mRNA expression levels were detected by RT-qPCR in transferred cell lysates, and the UVRAG siRNAs with the lower express levels of UVRAG mRNA were selected for transferring into rat brain. Hemagglutinating virus of Japan HVJ-E vector kit (CosmoBio, Tokyo, Japan) was used to deliver of siRNA plasma into target tissues. Following with manufacturer’ protocols, HVJ-E was incorporated with UVRAG siRNAs to yield an HVJ-E UVRAG siRNA complex (vector). The vectors were introduced into target tissues by membrane-fusion activity of fusion protein. Four μg of UVRAG siRNAs or negative control siRNA were combined with HVJ-E, respectively. The complexes in PBS were administered bilaterally into the hippocampal CA1 region of rats by stereotaxic injection 60 min before HPC. A 5-μl volume containing 2 μg of siRNA was injected into the CA1 region of dorsal hippocampus (3.5 mm posterior to bregma, 2.3 mm lateral to bregma, 2.6 mm below the dura) using a 10-μl Hamilton syringe with 34-gauge needle at a flow rate of 0.3 μl/min. For immunoblot analysis, rats were killed at 4 h after reperfusion in HPC or 24 h after injection of vectors in Sham-operated group.
Data analyses
Data were expressed as mean±S.D. Statistical significance was determined by one-way ANOVA or the two-tailed Student’s t-test. The Mann–Whitney U-test was used for morphometrical measurements of TEM. A two-tailed P-value<0.05 was considered statistically significant.
Authors: Narasimman Gurusamy; Istvan Lekli; Nikolai V Gorbunov; Mihaela Gherghiceanu; Lawrence M Popescu; Dipak K Das Journal: J Cell Mol Med Date: 2008-09-13 Impact factor: 5.310
Authors: Craig M Smith; Yaming Chen; Mara L Sullivan; Patrick M Kochanek; Robert S B Clark Journal: Neurobiol Dis Date: 2010-09-27 Impact factor: 5.996
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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
Authors: Mario A Lauterbach; Victor Saavedra; Matthew S J Mangan; Anke Penno; Christoph Thiele; Eicke Latz; Lars Kuerschner Journal: Autophagy Date: 2020-08-24 Impact factor: 16.016
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