Binding of Human ACE2 and RBD of Omicron Enhanced by Unique Interaction Patterns Among SARS-CoV-2 Variants of Concern

The 2019 coronavirus disease (COVID-19) pandemic has had devastating impacts on our global health. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing COVID-19, has continued to mutate and spread worldwide despite global vaccination efforts. In particular, the Omicron variant, first identified in South Africa in late November 2021, has now overtaken the Delta variant and become the dominant strain worldwide. Compared to the original strain identified in Wuhan, Omicron features 50 genetic mutations, with 15 mutations in the receptor-binding domain (RBD) of the spike protein, which binds to the human angiotensin-converting enzyme 2 (ACE2) receptor for viral entry. However, it is not completely understood how these mutations alter the interaction and binding strength between the Omicron RBD and ACE2. In this study, we used a combined steered molecular dynamics (SMD) simulation and experimental microscale thermophoresis (MST) approach to quantify the interaction between Omicron RBD and ACE2. We report that the Omicron brings an enhanced RBD-ACE2 interface through N501Y, Q493K/R, and T478K mutations; the changes further lead to unique interaction patterns, reminiscing the features of previously dominated variants, Alpha (N501Y) and Delta (L452R and T478K). Our MST data confirmed that the Omicron mutations in RBD are associated with a five-fold higher binding affinity to ACE2 compared to the RBD of the original strain. In conclusion, our result could help explain the Omicron variants prevalence in human populations, as higher interaction forces or affinity for ACE2 likely promote greater viral binding and internalization, leading to increased infectivity. TOC GRAPHIC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/477633v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1997fcborg.highwire.dtl.DTLVardef@951e96org.highwire.dtl.DTLVardef@b3956org.highwire.dtl.DTLVardef@e15ef9_HPS_FORMAT_FIGEXP M_FIG C_FIG

Differential Interactions Between Human ACE2 and Spike RBD of SARS-CoV-2 Variants of Concern

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. It is known that the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 interacts with the human angiotensin-converting enzyme 2 (ACE2) receptor, initiating the entry of SARS-CoV-2. Since its emergence, a number of SARS-CoV-2 variants have been reported, and the variants that show high infectivity are classified as the variants of concern according to the US CDC. In this study, we performed both all-atom steered molecular dynamics (SMD) simulations and microscale thermophoresis (MST) experiments to characterize the binding interactions between ACE2 and RBD of all current variants of concern (Alpha, Beta, Gamma, and Delta) and two variants of interest (Epsilon and Kappa). We report that the RBD of the Alpha (N501Y) variant requires the highest amount of force initially to be detached from ACE2 due to the N501Y mutation in addition to the role of N90-glycan, followed by Beta/Gamma (K417N/T, E484K, and N501Y) or Delta (L452R and T478K) variant. Among all variants investigated in this work, the RBD of the Epsilon (L452R) variant is relatively easily detached from ACE2. Our results combined SMD simulations and MST experiments indicate what makes each variant more contagious in terms of RBD and ACE2 interactions. This study could help develop new drugs to inhibit SARS-CoV-2 entry effectively. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/453598v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@fb2424org.highwire.dtl.DTLVardef@1d7c9org.highwire.dtl.DTLVardef@fdfb49org.highwire.dtl.DTLVardef@7c8db9_HPS_FORMAT_FIGEXP M_FIG TOC Graphic C_FIG

Dynamic Interactions of Fully Glycosylated SARS-CoV-2 Spike Protein with Various Antibodies

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a public health crisis, and the vaccines that can induce highly potent neutralizing antibodies are essential for ending the pandemic. The spike (S) protein on the viral envelope mediates human angiotensin-converting enzyme 2 (ACE2) binding and thus is the target of a variety of neutralizing antibodies. In this work, we built various S trimer-antibody complex structures on the basis of the fully glycosylated S protein models described in our previous work, and performed all-atom molecular dynamics simulations to get insight into the structural dynamics and interactions between S protein and antibodies. Investigation of the residues critical for S-antibody binding allows us to predict the potential influence of mutations in SARS-CoV-2 variants. Comparison of the glycan conformations between S-only and S-antibody systems reveals the roles of glycans in S-antibody binding. In addition, we explored the antibody binding modes, and the influences of antibody on the motion of S protein receptor binding domains. Overall, our analyses provide a better understanding of S-antibody interactions, and the simulation-based S-antibody interaction maps could be used to predict the influences of S mutation on S-antibody interactions, which will be useful for the development of vaccine and antibody-based therapy.

Prediction Method of Elastic Modulus of Trabecular Bone Based on SE-DenseVoxNet / 中国医疗器械杂志

Osteoporosis is one of the common metabolic diseases, which can easily lead to osteoporotic fractures. Accurate prediction of bone biomechanical properties is of great significance for the early prevention and diagnosis of osteoporosis. Bone mineral density measurement is currently used clinically as the gold standard for assessing bone strength and diagnosing osteoporosis, but studies have shown that bone mineral density can only explain 60% to 70% of bone strength changes, and trabecular bone microstructure is an important factor affecting bone strength. In order to establish the connection between trabecular bone microstructure and bone strength, this paper proposes a prediction method of trabecular bone modulus based on SE-DenseVoxNet. This method takes three-dimensional binary images of trabecular bone as input and predicts its elastic modulus in the z-axis direction. Experiments show that the error and bias between the predicted value of the method and the true value of the sample are small and have good consistency.

Structure, Dynamics, Receptor Binding, and Antibody Binding of Fully-glycosylated Full-length SARS-CoV-2 Spike Protein in a Viral Membrane

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mediates host cell entry by binding to angiotensin-converting enzyme 2 (ACE2), and is considered the major target for drug and vaccine development. We previously built fully-glycosylated full-length SARS-CoV-2 S protein models in a viral membrane including both open and closed conformations of receptor binding domain (RBD) and different templates for the stalk region. In this work, multiple s-long all-atom molecular dynamics simulations were performed to provide deeper insight into the structure and dynamics of S protein, and glycan functions. Our simulations reveal that the highly flexible stalk is composed of two independent joints and most probable S protein orientations are competent for ACE2 binding. We identify multiple glycans stabilizing the open and/or closed states of RBD, and demonstrate that the exposure of antibody epitopes can be captured by detailed antibody-glycan clash analysis instead of a commonly-used accessible surface area analysis that tends to overestimate the impact of glycan shielding and neglect possible detailed interactions between glycan and antibody. Overall, our observations offer structural and dynamic insight into SARS-CoV-2 S protein and potentialize for guiding the design of effective antiviral therapeutics.

Modeling and Simulation of a Fully-glycosylated Full-length SARS-CoV-2 Spike Protein in a Viral Membrane

This technical study describes all-atom modeling and simulation of a fully-glycosylated full-length SARS-CoV-2 spike (S) protein in a viral membrane. First, starting from PDB:6VSB and 6VXX, full-length S protein structures were modeled using template-based modeling, de-novo protein structure prediction, and loop modeling techniques in GALAXY modeling suite. Then, using the recently-determined most occupied glycoforms, 22 N-glycans and 1 O-glycan of each monomer were modeled using Glycan Reader & Modeler in CHARMM-GUI. These fully-glycosylated full-length S protein model structures were assessed and further refined against the low-resolution data in their respective experimental maps using ISOLDE. We then used CHARMM-GUI Membrane Builder to place the S proteins in a viral membrane and performed all-atom molecular dynamics simulations. All structures are available in CHARMM-GUI COVID-19 Archive (http://www.charmm-gui.org/docs/archive/covid19), so researchers can use these models to carry out innovative and novel modeling and simulation research for the prevention and treatment of COVID-19.

Wntless represses brown adipose tissue differentiation and energy expenditure / 中华内分泌代谢杂志

Objective To explore the effect of Wntless ( Wls)-mediated Wnt signaling on the development and energy metabolism of brown adipose tissue (BAT). Methods BAT-specific Wls knockout (WlsMyf5Δ/Δ) mice were generated by Cre-loxP system. The differentiations of BAT in WlsMyf5Δ/Δ knockout mice and Wlsfl/fl control mice were analyzed by histological morphology, immunohistochemistry, real-time PCR, and Western blot. After stromal vascular fraction (SVF) cells in BAT were induced to differentiate, oil red O staining, real-time PCR, and cell respiration experiments were performed for analyzing in-vitro cell differentiation and oxygen consumption. The energy metabolism of mice was monitored by rectal temperature, oxygen consumption rate in BAT, and energy expenditure. The adiposity of mice was evaluated by NMR while the glucose metabolism was analyzed by the glucose and insulin tolerance tests. Results The WlsMyf5Δ/Δ knockout mice appeared smaller body size, lower weight, higher percentage of lean fat, lower size of BAT, with higher body temperature on the back as compared to Wlsfl/fl control mice. The differentiation and thermogenesis of BAT in Wls-deficient mice were relatively augmented, along with an increase in Ucp1 mRNA and protein expressions. SVF cells from BAT in WlsMyf5Δ/Δ knockout mice revealed enhanced brown differentiation. Adiposity was decreased and glucose metabolic capacity was enhanced in the WlsMyf5Δ/Δknockout mice, without significant change in the whole body. Conclusion Wls-mediated Wnt signaling decreases the thermogenesis and glucose metabolism of BAT by suppressing its differentiation.

The roles of holothurian glycosaminoglycan combined with cisplatin on proliferation and chemotherapeutic response in A549 human lung adenocarcinoma cell / 中华肿瘤杂志

Objective@#To investigate the effects and mechanism of Holothurian Glycosaminoglycan (hGAG) alone in combination with cisplatin (DDP) on apoptosis of pulmonary adenocarcinoma cell A549.@*Methods@#A549 cells were separately treated with blank, hGAG, DDP and hGAG combined with DDP (hGAG + DDP). The cell morphology in 4 groups was observed using light microscope. CCK8 assay was used to determine the cell viability. Flow cytometry by Hoechst 33258 and AnnexinV-FITC/PI staining was applied to detect cell apoptosis. Western blot was then used to detect the protein expression of Bax, Bcl-2, survivin and caspase-3.@*Results@#After treatment for 24 h, the inhibitory rates of A549 cells in control, hGAG, DDP and hGAG + DDP groups were 0, (19.74±5.39)%, (42.01±2.57)% and (53.89±4.58)%, respectively. Moreover, after treatment for 48 h and 72 h, the inhibitory rates in each group were 0, (23.17±4.78)% and (29.17±4.21 )%, (54.00±7.64)% and (59.35±7.31)%, as well as (77.58±4.26)% and (79.94±4.58)%, respectively. The cell viability was significantly lower in drug treatment groups compared with those in control group at the same time point (P<0.05). Hochest 33258 staining showed that no obvious apoptotic cells were detected in the control group, while apoptotic cells were visible in hGAG, cisplatin and combination groups. Flow cytometry showed that cell apoptotic rates were (2.38±0.59)%, (12.59±4.22)%, (16.36±3.63)% and (44.60±5.45)% in the control, hGAG, DDP and hGAG + DDP groups, respectively. The cell apoptosis was significantly lower in drug treatment groups compared with those in control group at the same time point (P<0.05). Furthermore, western blot results showed that the expression of Bax and caspase-3 protein was increased (P<0.05), whereas Bcl-2 and survivin was decreased (P<0.05) in the hGAG+ DDP group compared with cisplatin alone (P<0.05).@*Conclusions@#HGAG can inhibit the proliferation and promote the apoptosis of human lung adenocarcinoma A549 cells. Meanwhile, it can strengthen the chemosensitivity of A549 cells to DDP via up-regulation of Bax, caspase-3 and down-regulation of Bcl-2 and survivin.

Effects of radioactive 125I-seeds on the growth inhibition and apoptosis induction of human lung adenocarcinoma cells A549 in nude mice / 中华放射医学与防护杂志

Objective To investigate the inhibitory effects of radioactive 125I seeds on the growth and apoptosis of human lung adenocarcinoma cells A 549 in nude mice.Methods Human lung adenocarcinoma A549 cells were cultured in vitro and subcutaneously transplanted in BALA/c nude mice.When the tumor size reached(300 ±50)mm3,40 tumor-bearing mice were divided into 4 groups by the random number table method as 0,0.6,0.8 mCi(1 Ci＝3.7×1010Bq)groups and blank control group,with 10 in each group.The 125I seeds of 0,0.6,and 0.8 mCi were implanted into the transplanted tumors in nude mouse,respectively.The blank control group received no treatment.The weight of nude mice was measured regularly every 4 days.The mice were sacrificed on the 32 days after 125I seeds implication.The transplanted tumors were weighed and the weight gain curve for nude mice was plotted.Hematoxylin-eosin(HE)staining was used to observe the histopathological changes of the tumor tissue.Cell apoptosis was detected by TUNEL assay,and the expressions of the P21,Caspase-9,Survivin and Livin proteins were detected by immunohistochemical assay.Results There was no nude mice dead in each group.On the day 28 and 32 after 125I seeds treatment,the body weights of nude mice of 0.6 and 0.8 mCi groups became lighter than those of the blank control group(q＝4.26,9.19,4.11,11.59,P＜0.05),the tumor weights of the 0.6 and 0.8 mCi groups were significantly decreased(q＝5.021,5.692,P＜0.05)with tumor inhibition rates of about 49％and 62％.In the 0.6 and 0.8 mCi groups,a large number of tumor cells degenerated to be necrotic cells.In addition,the apoptotic indexes were(50.00 ±2.58)％and(62.33 ± 4.51)％in the 0.6 and 0.8 mCi groups,respectively,and higher than that of blank control group(27.00 ±4.69)％.The expressions of P21 and Caspase-9 proteins in the 0.6 and 0.8 mCi groups were significantly higher than that in the blank control group(χ2＝11.380,24.310,11.380,20.376,P＜0.05).The expressions of Survivin and Livin proteins in the 0.6 and 0.8 mCi groups was significantly lower than that in the blank control group(χ2＝9.643,23.254,15.429,26.667,P＜0.05).Conclusions Radioactive 125I seeds can inhibit the proliferation of tumor cells and promote the apoptosis of A 549 cells probably by up-regulating the expressions of P21 and Caspase-9 but down-regulating the expressions of Survivin and Livin.

Study on glycosaminoglycan from Apostichopus Japonius on cellular immuno-regulation function of peripheral blood cells from pulmonary tuberculosis patients in vitro / 中国医师杂志

Objective To investigate the effects of glycosaminoglycans (HGAG) on the immune function of peripheral blood cells from patients with pulmonary tuberculosis.Methods Peripheral blood monouclear cells (PBMC) were isolated from peripheral blood of 40 healthy people (healthy group) and 30 tuberculosis patients (tuberculosis group) and cocultured with HGAG in vitro for 24 hours.Flow cytometry was used to detect the expression of CD45RA and CD45RO,as well as the expression of CD1a and CD83.Results The results showed that the expression of CD45RA and CD45RO in the tuberculosis group was the most significant (P ＜ 0.05) at the concentration of 50 μg/m coculturing with HGAG.The expression of CD45RA and CD45RO were most obvious in the healthy group at the concentration of 10 μg/ml and 50 μg/ml respectively (P ＜0.001).The difference of CD45RA between the two groups was no significant (P ＞0.05),while the difference of CD45RO was statistically significant (P ＜ 0.01) before co-culturing.The expression of CD45RA and CD45RO at 10 μg/ml and 50 μg/ml after co-culturing with HGAG were statistically significant (P ＜ 0.05).There was no statistical difference in CD1a and CD83 in healthy group before and after co-culturing (P ＞ 0.05),while there was statistically difference (P ＜ 0.05) before and after culturing in tuberculosis group.Before co-culturing,there was no significant difference in the expression of CD1a between the healthy group and the tuberculosis group (P ＞ 0.05),but CD83 expression was statistically different (P ＜ 0.001).After co-culturing,there were no significant differences in CD1a and CD83 expression between healthy and healthy groups (P ＞ 0.05).Conclusions HGAG can down-regulate the expression of CD45RA and up-regulate the expression of CD45RO in a certain concentration range,and promote the maturation of dendritic cells (DC) in tuberculosis patients and regulate the cellular immunity of patients with pulmonary tuberculosis in vitro.