A panel of multivalent nanobodies broadly neutralizing Omicron subvariants and recombinant.

The emerging Omicron subvariants have a remarkable ability to spread and escape nearly all current monoclonal antibody (mAb) treatments. Although the virulence of SARS-CoV-2 has now diminished, it remains a significant threat to public health due to its high transmissibility and susceptibility to mutation. Therefore, it is urgent to develop broad-acting and potent therapeutics targeting current and emerging Omicron variants. Here, we identified a panel of Omicron BA.1 spike receptor-binding domain (RBD)-targeted nanobodies (Nbs) from a naive alpaca VHH library. This panel of Nbs exhibited high binding affinity to the spike RBD of wild-type, Alpha B.1.1.7, Beta B.1.351, Delta plus, Omicron BA.1, and BA.2. Through multivalent Nb construction, we obtained a subpanel of ultrapotent neutralizing Nbs against Omicron BA.1, BA.2, BF.7 and even emerging XBB.1.5, and XBB.1.16 pseudoviruses. Protein structure prediction and docking analysis showed that Nb trimer 2F2E5 targets two independent RBD epitopes, thus minimizing viral escape. Taken together, we obtained a panel of broad and ultrapotent neutralizing Nbs against Omicron BA.1, Omicron BA.2, BF.7, XBB.1.5, and XBB.1.16. These multivalent Nbs hold great promise for the treatment against SARS-CoV-2 infection and could possess a superwide neutralizing breadth against novel omicron mutants or recombinants.

CD147 promotes breast cancer migration and invasion by inducing epithelial-mesenchymal transition via the MAPK/ERK signaling pathway.

BACKGROUND: CD147, a transmembrane glycoprotein, has been implicated in various cancer-related processes but its role in breast cancer remains poorly understood. Herein, we investigated the expression of CD147 in different breast cancer cell lines and explored its functional roles, including migration, invasion, drug resistance and modulation of key proteins associated with cancer progression. METHODS: The expression of CD147 was assessed in MCF-10 A, BT549, MDA-MB-231 and MCF-7 breast cancer cell lines using qRT-PCR and Western blotting, following which lyposome transfections were performed, leading overexpression of CD147 in BT549 cells and knockdown of CD147 in MCF-7 cells. Scratch assays and Transwell invasion and were performed to evaluate the cells' migration and invasion abilities. Sensitivity to 5-FU was determined via CCK-8 assays, and the expression of Snail1, E-cadherin, Vimentin, MMP-9 and the MAPK/ERK pathway were analyzed by qRT-PCR and Western blotting. RESULTS: Compared with normal beast epithelial cells, CD147 was highly expressed in all breast cancer cell lines, with the highest overexpression observed in MCF-7 cells and the lowest overexpression observed in BT549 cells. Overexpression of CD147 in BT549 cells increased, migration, invasion, viability and resistance to 5-FU of BT549 cells, while CD147 knockdown in MCF-7 cells reduced these properties of MCF-7 cells. Furthermore, CD147 influenced the expression of Snail1, Vimentin, E-cadherin, and MMP-9, suggesting its involvement in epithelial-mesenchymal transition (EMT) regulation. The MAPK/ERK pathway was activated by CD147 in BT549 cells, as indicated by increased p-MEK/MEK ratio and p-ERK/ERK ratio. In contrast, CD147 silencing in MCF-7 cells resulted in reduced p-MEK/MEK ratio and p-ERK/ERK ratio. CONCLUSION: In summary, our findings suggest CD147 as a potential therapeutic target in breast cancer treatment, particularly in cases where drug resistance and metastasis are concerns, worthy of further explorations.

The crystal structure of the emerging human-infecting hepatitis E virus E2s protein.

Hepatitis E virus (HEV) is a non-enveloped, globular particle that is responsible for acute hepatitis. HEV is classified into the Hepeviridae family and can be divided into four species (A-D). All HEV variants that infect humans are reported to belong to species A (HEV-A), except species C (HEV-C), which was reported to infect humans in December 2018. We determined the crystal structure of the HEV-C E2s domain at 1.8 Å resolution. It contains a classical 12-stranded ß-sandwich motif and forms dimers by hydrogen bonding, though the amino acid residues that form hydrogen bonds are quite different from the residues of HEV-A. The HEV-C E2s domain shares the common groove region with other structurally related viruses, and some subtle differences in this region may be related to host adoption or antibody binding. Antibody binding experiments and structural analysis revealed that HEV-C E2s is able to bind to the previously reported broad-spectrum antibody 8G12 but not bind to the antibody 8C11. Meanwhile, the structure analysis shows that HEV-C E2s does not have the key sites for binding to host cells as displayed by HEV-A (Genotype 1) E2s. These structural and biological findings present important implications for understanding the molecular mechanisms of host recognition and entry of HEV-C, as well as provide clues to the development of therapeutic antibodies and vaccines against HEV-C infection.

Postfusion structure of human-infecting Bourbon virus envelope glycoprotein.

Thogotoviruses are important zoonotic viruses infecting a variety of domestic animals, as well as humans. Among these viruses, Bourbon virus (BRBV) is one of the several human-infecting members, which emerged in the US in recent years and caused human deaths. Here, we report the crystal structure of the BRBV envelope glycoprotein in the postfusion conformation. The structure adopts the typical fold of a class III viral fusion protein and displays an extensive positively charged electrostatic potential pattern, which resembles the glycoprotein of Dhori virus and is consistent with our previous predictions. In addition, compared to other previously defined class III viral fusion proteins, the structures of all thogotovirus glycoproteins and homologs are more similar to herpes virus glycoprotein Bs than to the rhabdovirus G proteins. Thus, class III viral fusion proteins are quite diverse in structure, and sub-classes may have developed during evolution.

Flavonoids from tartary buckwheat induce G2/M cell cycle arrest and apoptosis in human hepatoma HepG2 cells.

The cytotoxicity and antioxidant activity on human hepatoma cell line HepG2 of three flavonoids homogenous compounds from tartary buckwheat seeds and bran, namely quercetin, isoquercetin, and rutin, were investigated. The total antioxidant competency detection results indicated that the antioxidant capacity of quercetin was the strongest in a biological response system. A [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay showed that quercetin exhibited the strongest cytotoxic effects against the HepG2 cell line. Flow cytometric analysis indicated that quercetin significantly increased the production of reactive oxygen species, and led to the G2/M phase arrest accompanied by an increase of apoptotic cell death after 48 h of incubation. Quercetin-induced cell apoptosis was shown to involve p53 and p21 up-regulation, Cyclin D1, Cdk2, and Cdk7 down-regulation. These results suggested that the induction of G2/M arrest, apoptosis, and cell death by quercetin may associate with increased expression of p53 and p21, decrease of Cyclin D1, Cdk2, and Cdk7 levels, and generation of reactive oxygen species in cells. This study will help to better understand and fully utilize medicinal resources of plant flavonoids.

[Protective effect of baicalin against rotenone induced injury on PC12 cells].

OBJECTIVE: To explore the protective effect of baicalin against rotenone-induced injury on PC12 cells, and the po-tential mechanism of action action was also explored. METHOD: PC12 cells were injured by rotenone and were treated with different concentrations (0.1, 1, 10 µmol x L(-1)) of baicalin at the same time. Cell viability was analyzed by MTT, and morphology was observed by phase-contrast microscopy. The cell apoptosis was detected by flow cytometry by Annexin V-FITC/PI staining. The intracellular ROS level was determined by fluorescence microscope with DCF-DA staining. The expression of Bcl-2, Bax and Caspase-3 was analyzed by Western blot. RESULT: The viability of PC12 cells exposure to rotenone for 24 hour was gradually decreased with dose escalating and 1.5 µmol x L was adopted to do the following experiment. Baicalin increased cell viability, improved cell morphology and decreased intracellular ROS level. Moreover, FACS indicated baicalin attenuated the apoptosis induced by rotenone significantly. Western blot showed that Bcl-2, Bax and Caspase-3 expression in rotenone-induced PC12 cells was reversed by baicalin. CONCLUSION: This study has demonstrated that baicalin protects PC12 cells against rotenone-induced apoptosis, at least in part, by scavenging excessive ROS and inhibiting the mitochondrion-dependent apoptotic pathway.

Overview of SARS-CoV-2 genome-encoded proteins.

Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) has spread rapidly throughout the world. SARS-CoV-2 is an enveloped, plus-stranded RNA virus with a single-stranded RNA genome of approximately 30,000 nucleotides. The SARS-CoV-2 genome encodes 29 proteins, including 16 nonstructural, 4 structural and 9 accessory proteins. To date, over 1,228 experimental structures of SARS-CoV-2 proteins have been deposited in the Protein Data Bank (PDB), including 16 protein structures, two functional domain structures of nucleocapsid (N) protein, and scores of complexes. Overall, they exhibit high similarity to SARS-CoV proteins. Here, we summarize the progress of structural and functional research on SARS-CoV-2 proteins. These studies provide structural and functional insights into proteins of SARS-CoV-2, and further elucidate the daedal relationship between different components at the atomic level in the viral life cycle, including attachment to the host cell, viral genome replication and transcription, genome packaging and assembly, and virus release. It is important to understand the structural and functional properties of SARS-CoV-2 proteins as it will facilitate the development of anti-CoV drugs and vaccines to prevent and control the current SARS-CoV-2 pandemic.

Omicron SARS-CoV-2 mutations stabilize spike up-RBD conformation and lead to a non-RBM-binding monoclonal antibody escape.

Omicron SARS-CoV-2 is rapidly spreading worldwide. To delineate the impact of emerging mutations on spike's properties, we performed systematic structural analyses on apo Omicron spike and its complexes with human ACE2 or S309 neutralizing antibody (NAb) by cryo-EM. The Omicron spike preferentially adopts the one-RBD-up conformation both before and after ACE2 binding, which is in sharp contrast to the orchestrated conformational changes to create more up-RBDs upon ACE2 binding as observed in the prototype and other four variants of concern (VOCs). Furthermore, we found that S371L, S373P and S375F substitutions enhance the stability of the one-RBD-up conformation to prevent exposing more up-RBDs triggered by ACE2 binding. The increased stability of the one-RBD-up conformation restricts the accessibility of S304 NAb, which targets a cryptic epitope in the closed conformation, thus facilitating the immune evasion by Omicron. These results expand our understanding of Omicron spike's conformation, receptor binding and antibody evasion mechanism.

Molecular insights into receptor binding of recent emerging SARS-CoV-2 variants.

Multiple SARS-CoV-2 variants of concern (VOCs) have been emerging and some have been linked to an increase in case numbers globally. However, there is yet a lack of understanding of the molecular basis for the interactions between the human ACE2 (hACE2) receptor and these VOCs. Here we examined several VOCs including Alpha, Beta, and Gamma, and demonstrate that five variants receptor-binding domain (RBD) increased binding affinity for hACE2, and four variants pseudoviruses increased entry into susceptible cells. Crystal structures of hACE2-RBD complexes help identify the key residues facilitating changes in hACE2 binding affinity. Additionally, soluble hACE2 protein efficiently prevent most of the variants pseudoviruses. Our findings provide important molecular information and may help the development of novel therapeutic and prophylactic agents targeting these emerging mutants.

Preparation of Astragalus membranaceus lectin and evaluation of its biological function.

Astragalus membranaceus lectin (AML) was abstracted as a supposedly novel agglutinin of 67 kDa from the seeds of Astragalus membranaceus. The seeds of Astragalus membranaceus were treated with acetate, ammonium sulfate precipitation, and purified by HiTrap SP XL ion column and Superdex G25 gel filtration chromatography to obtain the AML. AML contained 16.4% sugar, ~70% polar amino acids and ~30% hydrophobic amino acids. The AML exhibited agglutination activity toward human and animal erythrocytes, particularly human blood type O and rabbit erythrocytes. It also exhibited acid/alkali resistance and thermal denaturation above 64°C. Compared with human normal liver HL-7702 cells, different concentrations of AML (6.25, 12.50, 25.00 and 50.00 µg/ml) exhibited superior inhibitory effects on the growth of SGC-7901, HepG2 and H22 carcinoma cell lines, and displayed marked antibacterial effects on bacteria; the half maximal inhibitory concentration for B. dysenteriae, S. aureus and E. coli were 85.4, 80.2 and 65.3 µg/ml, respectively.

Anti-tumoral effects of a trypsin inhibitor derived from buckwheat in vitro and in vivo.

Native buckwheat, a common component of food products and medicine, has been observed to inhibit cancer cell proliferation in vitro. The aim of the present study was to evaluate the in vitro and in vivo anti-tumoral effects of recombinant buckwheat trypsin inhibitor (rBTI) on hepatic cancer cells and the mechanism of apoptosis involved. Apoptosis in the H22 cell line induced by rBTI was identified using MTT assays, DNA electrophoresis, flow cytometry, morphological observation of the nuclei, measurement of cytochrome C and assessment of caspase activation. It was identified that rBTI decreases cell viability by inducing apoptosis, as evidenced by the formation of apoptotic bodies and DNA fragmentation. rBTI-induced apoptosis occurred in association with mitochondrial dysfunction, leading to the release of cytochrome C from the mitochondria to the cytosol, as well as the activation of caspase-3, -8 and -9. In conclusion, the results of the present study suggested that rBTI specifically inhibited the growth of the H22 hepatic carcinoma cell line in vitro and in vivo in a concentration-dependent and time-dependent manner, while there were minimal effects on the 7702 normal liver cell line. In addition, rBTI­induced apoptosis in H22 cells was, at least in part, mediated by a mitochondrial pathway via caspase-9.