Developing a multi-epitope vaccine candidate to combat porcine epidemic diarrhea virus and porcine deltacoronavirus co-infection by employing an immunoinformatics approach.

Coinfection of porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) is common in pig farms, but there is currently no effective vaccine to prevent this co-infection. In this study, we used immunoinformatics tools to design a multi-epitope vaccine against PEDV and PDCoV co-infection. The epitopes were screened through a filtering pipeline comprised of antigenic, immunogenic, toxic, and allergenic properties. A new multi-epitope vaccine named rPPMEV, comprising cytotoxic T lymphocyte-, helper T lymphocyte-, and B cell epitopes, was constructed. To enhance immunogenicity, the TLR2 agonist Pam2Cys and the TLR4 agonist RS09 were added to rPPMEV. Molecular docking and dynamics simulation were performed to reveal the stable interactions between rPPMEV and TLR2 as well as TLR4. Additionally, the immune stimulation prediction indicated that rPPMEV could stimulate T and B lymphocytes to induce a robust immune response. Finally, to ensure the expression of the vaccine protein, the sequence of rPPMEV was optimized and further performed in silico cloning. These studies suggest that rPPMEV has the potential to be a vaccine candidate against PEDV and PDCoV co-infection as well as a new strategy for interrupting the spread of both viruses.

Natural Evolution of Porcine Epidemic Diarrhea Viruses Isolated from Maternally Immunized Piglets.

The porcine epidemic diarrhea virus (PEDV) can cause severe piglet diarrhea or death in some herds. Genetic recombination and mutation facilitate the continuous evolution of the virus (PEDV), posing a great challenge for the prevention and control of porcine epidemic diarrhea (PED). Disease materials of piglets with PEDV vaccination failure in some areas of Shanxi, Henan and Hebei provinces of China were collected and examined to understand the prevalence and evolutionary characteristics of PEDV in these areas. Forty-seven suspicious disease materials from different litters on different farms were tested by multiplex PCR and screened by hematoxylin-eosin staining and immunohistochemistry. PEDV showed a positivity rate of 42.6%, infecting the small and large intestine and mesenteric lymph node tissues. The isolated strains infected Vero, PK-15 and Marc-145 multihost cells and exhibited low viral titers in all three cell types, as indicated by their growth kinetic curves. Possible putative recombination events in the isolates were identified by RDP4.0 software. Sequencing and phylogenetic analysis showed that compared with the classical vaccine strain, PEDV SX6 contains new insertion and mutations in the S region and belongs to genotype GIIa. Meanwhile, ORF3 has the complete amino acid sequence with aa80 mutated wild strains, compared to vaccine strains CV777, AJ1102, AJ1102-R and LW/L. These results will contribute to the development of new PEDV vaccines based on prevalent wild strains for the prevention and control of PED in China.

Designing a multi-epitope vaccine to control porcine epidemic diarrhea virus infection using immunoinformatics approaches.

Porcine epidemic diarrhea virus (PEDV), a continuously evolving pathogen, causes severe diarrhea in piglets with high mortality rates. However, current vaccines cannot provide complete protection against PEDV, so vaccine development is still necessary and urgent. Here, with the help of immunoinformatics approaches, we attempted to design a multi-epitope vaccine named rPMEV to prevent and control PEDV infection. The epitopes of rPMEV were constructed by 9 cytotoxic T lymphocyte epitopes (CTLs), 11 helper T lymphocyte epitopes (HTLs), 6 linear B cell epitopes (LBEs), and 4 conformational B cell epitopes (CBEs) based on the S proteins from the four representative PEDV G2 strains. To enhance immunogenicity, porcine ß-defensin-2 (PBD-2) was adjoined to the N-terminal of the vaccine as an adjuvant. All of the epitopes and PBD-2 were joined by corresponding linkers and recombined into the multivalent vaccine, which is stable, antigenic, and non-allergenic. Furthermore, we adopted molecular docking and molecular dynamics simulation methods to analyze the interaction of rPMEV with the Toll-like receptor 4 (TLR4): a stable interaction between them created by 13 hydrogen bonds. In addition, the results of the immune simulation showed that rPMEV could stimulate both cellular and humoral immune responses. Finally, to raise the expression efficiency, the sequence of the vaccine protein was cloned into the pET28a (+) vector after the codon optimization. These studies indicate that the designed multi-epitope vaccine has a potential protective effect, providing a theoretical basis for further confirmation of its protective effect against PEDV infection in vitro and in vivo studies.

Cycloartane-type triterpenoids and steroids from Trichilia connaroides and their multidrug resistance reversal activities.

Four undescribed cycloartane-type triterpenoids (1-4) and seven undescribed steroids (6-12), along with five known analogues (5 and 13-16), were isolated from the leaves of Trichilia connaroides. Their structures were identified based on the NMR data and HRESIMS, and the absolute configurations were determined through single-crystal X-ray diffraction analysis, Mosher's method, and ECD calculations. The multidrug resistance (MDR) reversal activities of all the isolates were assessed, and compounds 10 and 11 showed significant activities to reverse the MDR of MCF-7/DOX cells with IC50 values of 2.90 and 3.76 µM, respectively. These bioactive compounds may bring fresh insights into the research and development of MDR reversal agents.

Design, Synthesis, Biological Evaluation, and Preliminary Mechanistic Study of a Novel Mitochondrial-Targeted Xanthone.

α-Mangostin, a natural xanthone, was found to have anticancer effects, but these effects are not sufficient to be effective. To increase anticancer potential and selectivity, a triphenylphosphonium cation moiety (TPP) was introduced to α-mangostin to specifically target cancer cell mitochondria. Compared to the parent compound, the cytotoxicity of the synthesized compound 1b increased by one order of magnitude. Mechanistic analysis revealed that the anti-tumor effects were involved in the mitochondrial apoptotic pathway by prompting apoptosis and arresting the cell cycle at the G0/G1 phase, increasing the production of reactive oxygen species (ROS), and reducing mitochondrial membrane potential (Δψm). More notably, the antitumor activity of compound 1b was further confirmed by zebrafish models, which remarkably inhibited cancer cell proliferation and migration, as well as zebrafish angiogenesis. Taken together, our results for the first time indicated that TPP-linked 1b could lead to the development of new mitochondrion-targeting antitumor agents.

Small GTPase-A Key Role in Host Cell for Coronavirus Infection and a Potential Target for Coronavirus Vaccine Adjuvant Discovery.

Small GTPases are signaling molecules in regulating key cellular processes (e.g., cell differentiation, proliferation, and motility) as well as subcellular events (e.g., vesicle trafficking), making them key participants, especially in a great array of coronavirus infection processes. In this review, we discuss the role of small GTPases in the coronavirus life cycle, especially pre-entry, endocytosis, intracellular traffic, replication, and egress from the host cell. Furthermore, we also suggest the molecules that have potent adjuvant activity by targeting small GTPases. These studies provide deep insights and references to understand the pathogenesis of coronavirus as well as to propose the potential of small GTPases as targets for adjuvant development.

Tubocapsenolide A targets C-terminal cysteine residues of HSP90 to exert the anti-tumor effect.

Heat shock protein 90 (HSP90) is a chaperone protein that has been shown to regulate cancer progression. As a result, HSP90 has emerged as an attractive target for cancer therapy. Tubocapsenolide A (TA) is an anti-tumor component isolated from Tubocapsicum anomalum. Although the anti-tumor activity of TA was considered to be related to HSP90, the binding site and deep anti-tumor mechanisms still need to be elucidated. In this study, we found that TA is a covalent inhibitor of HSP90, which inhibits HSP90 ATPase activity without blocking ATP binding. Further studies indicated that TA targets the C-terminal Cys521 site, which led to HSP90 partial oligomerization and hindered its anti-aggregation and refolding activity. The damage of the chaperone activity disrupted the interaction between HSP90 and its cochaperone CDC37 as well as its client proteins, thereby inducing cell cycle arrest and apoptosis. Moreover, TA was found to have therapeutic effects on the xenograft tumor model by inducing the degradation of HSP90 client proteins. Together, our results identified HSP90 as the direct target of TA for mediating the anti-tumor activity. TA could serve as a lead compound for developing novel HSP90 C-terminal covalent inhibitors with binding site different from the ATP-binding domain.

New dimeric phthalides from the rhizomes of Ligusticum sinense Oliv.

Two new phthalide dimers (1 and 2) were obtained from the rhizomes of Ligusticum sinense Oliv., along with three known dimeric phthalides (3-5). Their structures were determined with the aid of the spectroscopic data, and their absolute configurations were elucidated based on the comparison of calculated and experimental electronic circular dichroism (ECD) spectra. All the compounds were evaluated in vitro for their inhibitory activities against NO production in LPS-treated RAW264.7 macrophages. Among them, compounds 1 and 3 showed potent NO prohibitive activity with IC50 values at 4.86 ± 0.29 µM and 4.87 ± 0.32 µM, respectively.

Osteosarcoma cell proliferation suppression via SHP-2-mediated inactivation of the JAK/STAT3 pathway by tubocapsenolide A.

Introduction: Previously, we have reported a withanolide-type steroid, named tubocapsenolide A (TA), which shows potent anti-proliferative activity in several cancer cell lines. However, its inhibitory effect on the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway and therapeutic potential on osteosarcoma have not been reported. Objectives: In the present study, we aimed to investigate the effect and molecular mechanism of TA in osteosarcoma. Methods: The biological functions of TA in U2OS cells were investigated using colony formation, 5-ethynyl-20-deoxyuridine (EDU) staining, and cell cycle/apoptosis assays. The interaction between TA and Src homology 2 phosphatase 2 (SHP-2) was detected by enzyme activity and validated by target-identification methods such as drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), and biolayer interferometry (BLI). The in vivo anti-tumor efficacy of TA was analyzed in the xenograft tumor model. Western blotting analysis was performed to detect the protein expression levels. Results: TA exhibited antitumor activity against osteosarcoma both in vitro and in vivo by regulating the JAK/STAT3 signaling pathway. Mechanically, TA interacted with SHP-2 directly and activated its phosphatase activity. Importantly, protein tyrosine phosphatase (PTP) inhibitor, SHP-2 inhibitor, and SHP-2 siRNA could reverse the inhibitory effect of TA on the JAK/STAT3 signaling pathway and restored the TA-induced cell death. Conclusion: TA activated the phosphatase activity of SHP-2, which resulted in the inhibition of the JAK/STAT3 pathway and contributed to the antitumor efficacy of TA. Collectively, these findings suggested that TA could serve as a novel therapeutic agent for the treatment of osteosarcoma.

Acetophenone derivatives from the roots of Melicope ptelefolia.

Five new prenylated acetophenones, melicoptelins A-E (1-5), along with one known congener (6) were isolated from the roots of Melicope ptelefolia. Among them, compounds 2a/2b, 3a/3b, and 4a/4b were obtained as inseparable interconverting mixtures of keto and enol tautomers. Their structures were elucidated on the basis of extensive spectroscopic methods, including 1D, 2D NMR and HRESIMS. Compouds 2a/2b, 4a/4b and 5 exhibit protein tyrosine phosphatase 1B (PTP1B) inhibitory activity with IC50 values of 34.4, 55.2 and 66.6 µM, respectively.

Cytotoxic withanolides from the aerial parts of Tubocapsicum anomalum.

Ten new withanolides (1-10) and three artificial withanolides (11-13) were isolated from the aerial parts of Tubocapsicum anomalum, together with five known analogues (14-18). Their structures were determined on the basis of extensive spectroscopic and chemical methods. They include seven acnistin-type (1-4, 11, 14 and 15), three withajardin-type (5-7), and eight normal-type (8-10, 12, 13 and 16-18) withanolides. Of normal-type withanolides, a chemical conversion from the 16α,17α-epoxywithanolide (16) to Δ13,14-16α-hydroxywithanolide (18) was achieved by Wagner-Meerwein rearrangement. All isolates were evaluated for their cytotoxicity against four human tumor cell lines (HCT-116, HepG2, MCF-7 and A375). Among them, compounds 1-3, 6-8, 14, 16-18 showed cytotoxic activity with IC50 values of 0.24-8.71 µM.