Shuwen Liu

Bio: Shuwen Liu is an academic researcher from Southern Medical University. The author has contributed to research in topics: Craton & Medicine. The author has an hindex of 51, co-authored 367 publications receiving 16630 citations. Previous affiliations of Shuwen Liu include New York Blood Center & South China University of Technology.

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Abstract: In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

Structural and functional properties of SARS-CoV-2 spike protein: potential antivirus drug development for COVID-19.

Abstract: Coronavirus disease 2019 is a newly emerging infectious disease currently spreading across the world. It is caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The spike (S) protein of SARS-CoV-2, which plays a key role in the receptor recognition and cell membrane fusion process, is composed of two subunits, S1 and S2. The S1 subunit contains a receptor-binding domain that recognizes and binds to the host receptor angiotensin-converting enzyme 2, while the S2 subunit mediates viral cell membrane fusion by forming a six-helical bundle via the two-heptad repeat domain. In this review, we highlight recent research advance in the structure, function and development of antivirus drugs targeting the S protein.

The spike protein of SARS-CoV — a target for vaccine and therapeutic development

Abstract: Severe acute respiratory syndrome (SARS) is a newly emerging infectious disease caused by a novel coronavirus, SARS-coronavirus (SARS-CoV). The SARS-CoV spike (S) protein is composed of two subunits; the S1 subunit contains a receptor-binding domain that engages with the host cell receptor angiotensin-converting enzyme 2 and the S2 subunit mediates fusion between the viral and host cell membranes. The S protein plays key parts in the induction of neutralizing-antibody and T-cell responses, as well as protective immunity, during infection with SARS-CoV. In this Review, we highlight recent advances in the development of vaccines and therapeutics based on the S protein.

Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion.

Abstract: The recent outbreak of coronavirus disease (COVID-19) caused by SARS-CoV-2 infection in Wuhan, China has posed a serious threat to global public health. To develop specific anti-coronavirus therapeutics and prophylactics, the molecular mechanism that underlies viral infection must first be defined. Therefore, we herein established a SARS-CoV-2 spike (S) protein-mediated cell–cell fusion assay and found that SARS-CoV-2 showed a superior plasma membrane fusion capacity compared to that of SARS-CoV. We solved the X-ray crystal structure of six-helical bundle (6-HB) core of the HR1 and HR2 domains in the SARS-CoV-2 S protein S2 subunit, revealing that several mutated amino acid residues in the HR1 domain may be associated with enhanced interactions with the HR2 domain. We previously developed a pan-coronavirus fusion inhibitor, EK1, which targeted the HR1 domain and could inhibit infection by divergent human coronaviruses tested, including SARS-CoV and MERS-CoV. Here we generated a series of lipopeptides derived from EK1 and found that EK1C4 was the most potent fusion inhibitor against SARS-CoV-2 S protein-mediated membrane fusion and pseudovirus infection with IC50s of 1.3 and 15.8 nM, about 241- and 149-fold more potent than the original EK1 peptide, respectively. EK1C4 was also highly effective against membrane fusion and infection of other human coronavirus pseudoviruses tested, including SARS-CoV and MERS-CoV, as well as SARSr-CoVs, and potently inhibited the replication of 5 live human coronaviruses examined, including SARS-CoV-2. Intranasal application of EK1C4 before or after challenge with HCoV-OC43 protected mice from infection, suggesting that EK1C4 could be used for prevention and treatment of infection by the currently circulating SARS-CoV-2 and other emerging SARSr-CoVs.

Fusion mechanism of 2019-nCoV and fusion inhibitors targeting HR1 domain in spike protein.

Abstract: Very recently, a novel coronavirus, 2019-nCoV, emerged in Wuhan, China and then quickly spread worldwide, resulting in >17,388 confirmed cases and 361 deaths as of 3 February 2020, thus calling for the development of safe and effective therapeutics and prophylatics. Similar to severe acute respiratory syndrome (SARS)-CoV, 2019nCoV belongs to lineage B betacoronavirus, and it has the ability to utilize human angiotensin-converting enzyme 2 (ACE2) as a receptor to infect human cells. SARS-CoV spike (S) protein S2 subunit plays a key role in mediating virus fusion with and entry into the host cell, in which the heptad repeat 1 (HR1) and heptad repeat 2 (HR2) can interact to form six-helical bundle (6-HB), thereby bringing viral and cellular membranes in close proximity for fusion. Using S-HR1 as a target, we have previously designed and developed several potent fusion inhibitors against SARS-CoV (e.g., SARS-HR2P) and Middle East respiratory syndrome (MERS)-CoV (e.g., MERS-HR2P). However, it is unclear whether 2019-nCoV also possesses a similar fusion and entry mechanism as that of SARS-CoV and MERS-CoV, and if so, whether a 2019-nCoV S-HR1 can also serve as an important target for the development of 2019-nCoV fusion/entry inhibitors. Through amino acid (aa) sequence alignment with SARS-CoV and 2019-nCoV S protein, we located the functional domain in 2019-nCoV S protein, including N-terminal domain (aa14–305), receptor-binding domain (aa319–541), and receptor-binding motif (aa437–508) in S1 subunit (aa14–685) and fusion peptide (aa788–806), HR1 (aa912–984), HR2 (aa1163–1213), transmembrane domain (aa1214–1237) and cytoplasm domain (aa1238–1273) in S2 subunit (aa686–1273) (Fig. 1a). In the post-fusion hairpin conformation of the SARS-CoV or MERS-CoV S protein, the HR2 domain forms both rigid helix and flexible loop to interact with HR1 domain (Fig. 1b). There are many strong interactions between HR1 and HR2 domains inside the helical region, which is thus designated “fusion core region” (HR1core and HR2core regions, respectively). According to the sequence alignment, the 2019-nCoV and SARS-CoV S2 subunits are highly conserved, with 92.6% and 100% overall identity in HR1 and HR2 domains, respectively. However, inside the HR1core region, 8 of the 21 residues show mutation (~38% difference). This is significantly different from the HR1core region of previously identified SARS-like viruses, such as WIV1, Rs3367, and RsSHC014, which are 100% identical to that of SARS-CoV (Fig. 1b). These novel point mutations in 2019-nCoV S2 subunit may change the interaction pattern between HR1 and HR2 domains in the post-fusion core, thus affecting the 6-HB formation. Based on our previous experience, we have now designed HR1and HR2-derived peptides, designated 2019nCoV-HR1P (aa924–965) and 2019-nCoV-HR2P (aa1168–1203), respectively (Fig. 1c), and explored their biological characteristics. Since the 2019-nCoV and SARS-CoV S-HR2 sequences are 100% identical, 2019-nCoV-HR2P may act as a fusion inhibitor in much the same way as our reported SARS-CoV fusion inhibitor, SARS-HR2P. Under native electrophoresis as described before, 2019-nCoV-HR2P, which carries negative charges, moved down to a lower gel position, while 2019-nCoV-HR1P, which carries positive charges, moved up and off the gel (Fig. 1d) in a manner similar to SARS-CoV-HR1P in native-polyacrylamide gel electrophoresis (PAGE) assay. Notably, in the 2019-nCoV-HR1P/2019nCoV-HR2P mixture, new bands emerged at the upper part in the native-PAGE gel in a 2019-nCoV-HR1P dose-dependent manner, indicating that 2019-nCoV-HR2P could interact with 2019-nCoV-HR1P to form a complex, possibly 6-HB. We then assessed the secondary structures of 2019-nCoV-HR1P, 2019-nCoV-HR2P, and 2019-nCoV-HR1P/2019-nCoV-HR2P complex, using circular dichroism as previously described. While 2019-nCoV-HR1P alone and 2019-nCoV-HR2P alone exhibited low helicity (<30%), the 2019-nCoV-HR1P/2019-nCoV-HR2P complex exhibited the characteristic helicity of 6-HB, with minimum values at 208 and 222 nm and helicity of 84.4% (Fig. 1e). Moreover, the 2019-nCoV-HR1P/2019-nCoV-HR2P complex showed good thermal stability with Tm of 66.2 C (Fig. 1f). These results confirm, for the first time, that 2019-nCoV HR1 and HR2 regions are able to interact with each other to form 6HB and suggest that 2019-nCoV-HR2P may inhibit 2019-nCoV fusion with and entry into the target cell, as we showed before with SARS-CoV, MERS-CoV, and other human CoVs. To confirm this hypothesis, we herein developed a 2019-nCoV Smediated cell–cell fusion assay as previously described. Using this assay, we demonstrated that 2019-nCoV-HR2P exhibited potent fusion-inhibitory activity with a half maximal inhibitory concentration (IC50) of 0.18 μM (Fig. 1g), indicating that the 2019-nCoV HR1 region could serve as an ideal target site. On the other hand, 2019-nCoV-HR1P exhibited no significant inhibitory effect at concentrations up to 40 μM, consistent with other coronavirus HR1-derived peptides, such as SARS-HR1P and MERS-HR1P. We previously reported that the HR1 region in various coronaviruses is a conserved target site, and based on that evidence, we designed a pan-coronavirus fusion inhibitor, denoted as EK1. Compared with 2019-nCoV-HR2P, EK1 shows

Aggregation-Induced Emission: Together We Shine, United We Soar!

Abstract: Ju Mei,†,‡,∥ Nelson L. C. Leung,†,‡,∥ Ryan T. K. Kwok,†,‡ Jacky W. Y. Lam,†,‡ and Ben Zhong Tang*,†,‡,§ †HKUST-Shenzhen Research Institute, Hi-Tech Park, Nanshan, Shenzhen 518057, China ‡Department of Chemistry, HKUST Jockey Club Institute for Advanced Study, Institute of Molecular Functional Materials, Division of Biomedical Engineering, State Key Laboratory of Molecular Neuroscience, Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China Guangdong Innovative Research Team, SCUT-HKUST Joint Research Laboratory, State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou 510640, China

Preexposure Chemoprophylaxis for HIV Prevention in Men Who Have Sex with Men

Abstract: The study subjects were followed for 3324 person-years (median, 1.2 years; maximum, 2.8 years). Of these subjects, 10 were found to have been infected with HIV at en rollment, and 100 became infected during follow-up (36 in the FTC–TDF group and 64 in the placebo group), indicating a 44% reduction in the incidence of HIV (95% confidence interval, 15 to 63; P = 0.005). In the FTC–TDF group, the study drug was detected in 22 of 43 of seronegative subjects (51%) and in 3 of 34 HIV-infected subjects (9%) (P<0.001). Nausea was reported more frequently during the first 4 weeks in the FTC–TDF group than in the placebo group (P<0.001). The two groups had similar rates of serious adverse events (P = 0.57). Conclusions Oral FTC–TDF provided protection against the acquisition of HIV infection among the subjects. Detectable blood levels strongly correlated with the prophylactic effect. (Funded by the National Institutes of Health and the Bill and Melinda Gates Foun dation; ClinicalTrials.gov number, NCT00458393.)