Euphorbia Pekinensis Rupr. sensitizes colorectal cancer to PD-1 blockade by remodeling the tumor microenvironment and enhancing peripheral immunity.

BACKGROUND: Immune checkpoint blockade, such as monoclonal antibodies targeting programmed cell death protein 1 (PD-1), has been a major breakthrough in the treatment of several cancers, but has limited effect in colorectal cancer (CRC), which is a highly prevalent cancer worldwide. Current chemotherapy-based strategies to boost PD-1 response have many limitations. And the role of peripheral immunity in boosting PD-1 response continues to attract attention. Therefore, candidate combinations of PD-1 blockade need to be drugs with multi-targets and multi-modulatory functions. However, it is still unknown whether traditional Chinese medicines with such property can enhance the applicability and efficacy of PD-1 blockade in colorectal cancer. METHODS: Euphorbia Pekinensis extract (EP) was prepared and the constituents were analyzed by HPLC. CRC cells were used for in vitro experiments, including cell viability assay, colony formation assay, flow cytometry for 7-AAD staining, western blotting for caspase 3 and caspase 7, HMGB1 and ATP detection. An orthotopic CT26 mouse model was subsequently used to investigate the combination of EP and PD-1 blockade therapy. Tumor volume and tumor weight were assessed, tumor tissues were subjected to histopathological HE staining and TUNEL staining, and tumor-infiltrating immune cells were evaluated by immunofluorescence staining. RNA-sequencing, target prediction and pathway analysis were further employed to explore the mechanism. Molecular docking and cellular thermal shift assay (CETSA) were utilized to verify the direct target of the core component of EP. And, loss-of-function analysis was carried to confirm the upstream-downstream relationship. Flow cytometry was employed to analyze CD8+ T cells in the peripheral blood and spleen. RESULTS: The main constituents of EP are diterpenoids and flavonoids. EP dramatically suppresses CRC cell growth and exerts its cytotoxic effect by triggering immunogenic cell death in vitro. Moreover, EP synergizes with PD-1 blockade to inhibit tumorigenesis in tumor-bearing mice. Disruption of ISX nuclear localization by helioscopinolide E is a central mechanism of EP-induced apoptosis in CRC cell. Meanwhile, EP activates immune response by upregulating Phox2b to reshape the immune microenvironment. In addition, EP regulates peripheral immunity by regulating the T cell activation and proliferation, and the ratio of CD8+ T cells in peripheral blood is drastically increased, thereby enhancing the therapeutic efficacy of anti-PD1 immunotherapy. CONCLUSION: EP triggers intra-tumor immunogenic cell death and modulates the immunoregulatory signaling to elicit the tumor immunogenicity. Moreover, EP participates in transcriptional activation of immune response-related pathways. Consequently, multiple stimulating functions of EP on macro- and micro-immune potentiates the anti-tumor effect of PD-1 blockade in CRC.

SARS-CoV-2 NSP12 utilizes various host splicing factors for replication and splicing regulation.

The RNA-dependent RNA polymerase (RdRp) is a crucial element in the replication and transcription of RNA viruses. Although the RdRps of lethal human coronaviruses severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV) have been extensively studied, the molecular mechanism of the catalytic subunit NSP12, which is involved in pathogenesis, remains unclear. In this study, the biochemical and cell biological results demonstrate the interactions between SARS-CoV-2 NSP12 and seven host proteins, including three splicing factors (SLU7, PPIL3, and AKAP8). The entry efficacy of SARS-CoV-2 considerably decreased when SLU7 or PPIL3 was knocked out, indicating that abnormal splicing of the host genome was responsible for this occurrence. Furthermore, the polymerase activity and stability of SARS-CoV-2 RdRp were affected by the three splicing factors to varying degrees. In addition, NSP12 and its homologues from SARS-CoV and MERS-CoV suppressed the alternative splicing of cellular genes, which were influenced by the three splicing factors. Overall, our research illustrates that SARS-CoV-2 NSP12 can engage with various splicing factors, thereby impacting virus entry, replication, and gene splicing. This not only improves our understanding of how viruses cause diseases but also lays the foundation for the development of antiviral therapies.

A three-dimensional integration strategy for Q-markers identification: Taken Euphorbia Pekinensis Radix as an example.

Euphorbia Pekinensis Radix (EPR) is an important antitumor medicinal resource. However, quality control of EPR has not been well established due to the lack of quality markers (Q-markers) research. In this study, a three-dimensional integration strategy was developed to systematically characterize Q-markers and this method was successfully applied to identify Q-markers of EPR. Firstly, three core quality attributes-effectiveness, testability and specificity-were considered as three dimensions, and the weights of each dimension were calculated by analytical hierarch process. Then, the values of each dimension were evaluated by multi-indicators. For EPR with antitumor activity, cytotoxic assay and network pharmacology, UPLC analysis and literature search, compound belonging search were employed to calculate the values of effectiveness, testability and specificity, respectively. Finally, the weights and values were multiplied as the scores of each component on that dimension, and the total scores of the three dimensions were further integrated based on the radar plot and expressed as regression area, by which Q-markers were quantified and visualized. Five components were identified as Q-markers of EPR due to their high-ranked antitumor capacity, ease of measurement and excellent specificity, which laid an important foundation for the quality control improvement of EPR. Furthermore, the integrated strategy summarized here is helpful for the quantitative identification of Q-markers and promote the quality standard of traditional Chinese medicine.

[Quantitative identification of Q-markers of Euphorbiae Humifusae Herba based on AHP-CRITIC comprehensive weighting method].

This study investigated the quality markers(Q-markers) of Euphorbiae Humifusae Herba based on the analytic hierarchy process(AHP)-criteria importance through intercriteria correlation(CRITIC) comprehensive weighting method. The Q-markers evaluation system was constructed based on the AHP-CRITIC comprehensive weighting method with quantitative identification of Q-markers of Euphorbiae Humifusae Herba as the target layer. The index weights of the factor layer and the control layer were integrated based on the weights of three indicators(effectiveness, testability, and specificity) in the factor layer calculated by the AHP method and weights of eight indicators(anti-inflammatory inhibitory rate, coagulation shortening rate, anti-cancer inhibition rate, component degree value, component test batch, component average content, content variation coefficient, and number of medicinal materials retrieved according to components) in the control layer calculated by the CRITIC method. The comprehensive score of the chemical components of Euphorbiae Humifusae Herba was weighted and ranked to identify the Q-markers of Euphorbiae Humifusae Herba. In terms of comprehensive scores, top 10 potential Q-markers of Euphorbiae Humifusae Herba were ranked as cynaroside > quercetin > gallic acid > apigenin > luteolin > apigenin-7-O-glucoside > quercetin-7-O-glucoside > ellagic acid > astragalin > ethyl gallate. This study provides a reference for the quality control of Euphorbiae Humifusae Herba and a methodological reference for the quantitative identification of Q-markers of Chinese medicine.

Pimarane, abietane, and labdane diterpenoids from Euphorbia pekinensis Rupr. and their anti-tumor activities.

Chemical investigation of the roots of Euphorbia pekinensis Rupr. led to the isolation of five undescribed labdane diterpenoids "(4S, 5S, 9R, 10S, 13R)-18-O-galloyl-labda-8(17), 14(15)-dien-13-ol; (4S, 5S, 9R, 10S, 13R)-13-hydroxy-labda-8(17), 14(15)-dien-18-one; (4S, 5S, 9R, 10S, 13R)-18-O-acetyl-labda-8(17), 14(15)-dien-13-ol; (4S, 5S, 9R, 10S)-labda-8(17), 13(16), 14(15)-trien-18-ol; (5R, 6R, 9R, 10S, 13R)-labda-8(17), 14(15)-dien-6,13-diol", two undescribed pimarane diterpenoids "(2R, 5S, 9R, 10S, 12R, 13R)-2,12-dihydroxy-isopimara-7,15-dien-3-one; (5S, 9R, 10S, 12R, 13R)-2, 12-dihydroxy-isopimara-1, 7, 15-trien-3-one)", together with nine known diterpenoids, including three pimarane-type "(3ß,11α,13α)-3,11-dihydroxypimara-7,15-diene-2,12-dione; (11R, 12S)-2,11,12-trihydroxy-ent-isopimara-1,7,15-trien-3-one; isopimara-7,15-dien-3ß-ol)", five abietane-type "helioscopinolide A-C; helioscopinolide E; helioscopinolide I″, and one lathyrane-type "jolkinol B". The structures of these compounds were elucidated by analysis of HRESIMS, 1D NMR, 2D NMR, and X-ray diffraction. These sixteen compounds were evaluated for cytotoxic activity in vitro against three human cancer cell lines, U-937, LOVO, and K-562. Jolkinol B exhibited IC50 of 3.60 µM and 8.44 µM against U-937 and LOVO cell lines, (4S, 5S, 9R, 10S, 13R)-18-O-galloyl-labda-8(17), 14(15)-dien-13-ol displayed IC50 of 5.92 µM against U-937 cell lines, isopimara-7,15-dien-3ß-ol showed IC50 of 0.87 µM against K-562 cell lines.

The interactions of ZDHHC5/GOLGA7 with SARS-CoV-2 spike (S) protein and their effects on S protein's subcellular localization, palmitoylation and pseudovirus entry.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein determines virus entry and the palmitoylation of S protein affects virus infection. An acyltransferase complex ZDHHC5/GOGAL7 that interacts with S protein was detected by affinity purification mass spectrometry (AP-MS). However, the palmitoylated cysteine residues of S protein, the effects of ZDHHC5 or GOLGA7 knockout on S protein's subcellular localization, palmitoylation, pseudovirus entry and the enzyme for depalmitoylation of S protein are not clear. METHODS: The palmitoylated cysteine residues of S protein were identified by acyl-biotin exchange (ABE) assays. The interactions between S protein and host proteins were analyzed by co-immunoprecipitation (co-IP) assays. Subcellular localizations of S protein and host proteins were analyzed by fluorescence microscopy. ZDHHC5 or GOGAL7 gene was edited by CRISPR-Cas9. The entry efficiencies of SARS-CoV-2 pseudovirus into A549 and Hela cells were analyzed by measuring the activity of Renilla luciferase. RESULTS: In this investigation, all ten cysteine residues in the endodomain of S protein were palmitoylated. The interaction of S protein with ZDHHC5 or GOLGA7 was confirmed. The interaction and colocalization of S protein with ZDHHC5 or GOLGA7 were independent of the ten cysteine residues in the endodomain of S protein. The interaction between S protein and ZDHHC5 was independent of the enzymatic activity and the PDZ-binding domain of ZDHHC5. Three cell lines HEK293T, A549 and Hela lacking ZDHHC5 or GOLGA7 were constructed. Furthermore, S proteins still interacted with one host protein in HEK293T cells lacking the other. ZDHHC5 or GOLGA7 knockout had no significant effect on S protein's subcellular localization or palmitoylation, but significantly decreased the entry efficiencies of SARS-CoV-2 pseudovirus into A549 and Hela cells, while varying degrees of entry efficiencies may be linked to the cell types. Additionally, the S protein interacted with the depalmitoylase APT2. CONCLUSIONS: ZDHHC5 and GOLGA7 played important roles in SARS-CoV-2 pseudovirus entry, but the reason why the two host proteins affected pseudovirus entry remains to be further explored. This study extends the knowledge about the interactions between SARS-CoV-2 S protein and host proteins and probably provides a reference for the corresponding antiviral methods.

Fish herpesvirus protein (CaHV-138L) can target to mitochondrial protein FoF1 ATPase.

Herpesvirus infection usually relies on the interaction between viral protein and host protein to enhance replication of the enveloped virus. Fish Carassius auratus herpesvirus (CaHV) is highly pathogenic pathogen causing gill acute hemorrhages of crucian carp (Carassius auratus) and high moritality rates among those infected fish. The protein of CaHV (CaHV-138 L) containing two transmembrane (TM) domains and an immunoglobulin C-2 Type (IGc2) domain was predicted as a viral membrane protein. In this investigation, fluorescence observation showed that full-length CaHV-138 L mainly localized on the plasma membrane or around nuclear membrane of fish fathead minnow (FHM) cells in a punctate pattern. The TM domain deletion mutants of CaHV-138 L (ΔTM1, ΔTM2, and ΔTM1&ΔTM2) diffusely distributed in both the cytoplasm and the nucleus, mainly presented patchy fashion in the cytoplasm, and mainly presented both in the nucleus and in the cytoplasm, respectively. Obviously, the TM domain deletion mutants significantly affected CaHV-138 L subcellular localization. Meanwhile, colocalization assay showed that the full-length viral protein colocalized with mitochondria. Furthermore, the interaction between CaHV-138 L and host protein was identified by yeast two-hybrid (Y2H) and co-immunoprecipitation (co-IP) assays. The host mitochondrial protein FoF1 ATP synthase (FoF1-ATPase) that interacts with this viral protein was screened. The data indicated that CaHV-138 L can target to mitochondrial protein FoF1-ATPase, which might provide energy for virus replication through mediating mitochondrial ATP synthesis. This study has provided valuable information for better understanding of the links of herpesvirus proteins with aquaculture animal proteins.

Interaction between Two Iridovirus Core Proteins and Their Effects on Ranavirus (RGV) Replication in Cells from Different Species.

The two putative proteins RGV-63R and RGV-91R encoded by Rana grylio virus (RGV) are DNA polymerase and proliferating cell nuclear antigen (PCNA) respectively, and are core proteins of iridoviruses. Here, the interaction between RGV-63R and RGV-91R was detected by a yeast two-hybrid (Y2H) assay and further confirmed by co-immunoprecipitation (co-IP) assays. Subsequently, RGV-63R or RGV-91R were expressed alone or co-expressed in two kinds of aquatic animal cells including amphibian Chinese giant salamander thymus cells (GSTCs) and fish Epithelioma papulosum cyprinid cells (EPCs) to investigate their localizations and effects on RGV genome replication. The results showed that their localizations in the two kinds of cells are consistent. RGV-63R localized in the cytoplasm, while RGV-91R localized in the nucleus. However, when co-expressed, RGV-63R localized in both the cytoplasm and the nucleus, and colocalized with RGV-91R in the nucleus. 91R△NLS represents the RGV-91R deleting nuclear localization signal, which is localized in the cytoplasm and colocalized with RGV-63R in the cytoplasm. qPCR analysis revealed that sole expression and co-expression of the two proteins in the cells of two species significantly promoted RGV genome replication, while varying degrees of viral genome replication levels may be linked to the cell types. This study provides novel molecular evidence for ranavirus cross-species infection and replication.

Rana grylio virus 43R encodes an envelope protein involved in virus entry.

Rana grylio virus (RGV), a member of genus Ranavirus in the family Iridoviridae, is a viral pathogen infecting aquatic animal. RGV 43R has homologues only in Ranavirus and contains a transmembrane (TM) domain, but its role in RGV infection is unknown. In this study, 43R was determined to be associated with virion membrane. The transcripts encoding 43R and the protein itself appeared late in RGV-infected EPC cells and its expression was blocked by viral DNA replication inhibitor, indicating that 43R is a late expressed protein. Subcellular localization showed that 43R-EGFP fusion protein distributed in cytoplasm of EPC cells and that TM domain is essential for its distribution in cytoplasm. 43R-EGFP fusion protein colocalized with viral factories in RGV-infected cells. A recombinant RGV deleting 43R (Δ43R-RGV) was constructed by homologous recombination to investigate its role in virus infection. Compared with wild type RGV, the ability of Δ43R-RGV to induce the cytopathic effect and its virus titers were significantly reduced. Furthermore, it is revealed that 43R deletion significantly inhibited viral entry but did not influence viral DNA replication by measuring and comparing the DNA levels of RGV and Δ43R-RGV in the infected cells at the early stage of infection. RGV neutralization with anti-43R serum reduced the virus titer. Therefore, these data showed that RGV 43R is a late gene that encodes an envelope protein involved in RGV entry.

Complete genome sequence and architecture of crucian carp Carassius auratus herpesvirus (CaHV).

Crucian carp Carassius auratus herpesvirus (CaHV) was isolated from diseased crucian carp with acute gill hemorrhages and high mortality. The CaHV genome was sequenced and analyzed. The data showed that it consists of 275,348 bp and contains 150 predicted ORFs. The architecture of the CaHV genome differs from those of four cyprinid herpesviruses (CyHV1, CyHV2, SY-C1, CyHV3), with insertions, deletions and the absence of a terminal direct repeat. Phylogenetic analysis of the DNA polymerase sequences of 17 strains of Herpesvirales members, and the concatenated 12 core ORFs from 10 strains of alloherpesviruses showed that CaHV clustered together with members of the genus Cyprinivirus, family Alloherpesviridae.