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Background: c-Met encoded by the proto-oncogene MET, also known as hepatocyte growth factor (HGF) receptor, plays a crucial role in cellular processes. MET exon 14 skipping alteration (METΔ14EX) is a newly discovered MET mutation. SMAD2 is an important downstream transcription factor in TGF-β pathway. Unfortunately, the mechanisms by which METΔ14EX leads to oncogenic transformation are scarcely understood. The relationship between METΔ14EX and SMAD2 has not been studied yet., Methods: We generate METΔ14EX models by CRISPR-Cas9. In vitro transwell, wound-healing, soft-agar assay, in vivo metastasis and subcutaneous recurrence assay were used to study the role of METΔ14EX in tumour progression. RNA-seq, Western blotting, co-immunoprecipitation (CO-IP) and immunofluorescent were performed to explore the interaction between c-Met and SMAD2., Results: Our results demonstrated that METΔ14EX, independent of HGF, can prolong the constitutive activation of c-Met downstream signalling pathways by impeding c-Met degradation and facilitating tumour metastasis and recurrence. Meanwhile, METΔ14EX strengthens the interaction between c-Met and SMAD2, promoting SMAD2 phosphorylation. Therapeutically, MET inhibitor crizotinib impedes METΔ14EX-mediated tumour metastasis by decreasing SMAD2 phosphorylation., Conclusions: These data elucidated the previously unrecognised role of METΔ14EX in cancer progression via activation of SMAD2 independent of TGF-β, which helps to develop more effective therapies for such patients. METΔ14EX alteration significantly triggers tumour progression via activation of SMAD2 signalling that are involved in activating tumour invasion, metastasis and recurrence. On the left, in the MET wild-type (METWT), the juxtamembrane (JM) domain is involved in the regulation of tyrosine kinase activity, receptor degradation, and caspase cleavage. On the right, the METΔ14EX mutation leads to the loss of the juxtamembrane domain, resulting in an abnormal MET protein lacking a CBL-binding site. This causes the accumulation of truncated MET receptors followed by constitutive activation of the MET signalling pathway. Thus, the METΔ14EX-mutated protein has strong binding and phosphorylation to SMAD2, which results in the phosphorylation of a large number of SMAD2/3 proteins that combine with SMAD4 to form a complex in the nucleus, activating downstream signalling pathways, such as EMT and ECM remodelling, resulting in tumour progression and recurrence. TF transcription factor., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

There are several technical challenges and public issues concerning genome editing applications before they become viable in commercial aquaculture. Recently, we developed a novel strategy to generate all-female (AF) common carp, which exhibited a growth advantage over the control carp, using genetic editing through single gene-targeting manipulation. Here, we found that the body weight of the AF common carp was higher by 22.58% than that of the control common carp. Because the genotype of the AF common carp was cyp17a1+/-;XX, the contents of sex steroids were normally synthesized, as they were comparable to that of the control female carp. To evaluate the food safety of the AF carp, Wistar rats were fed a diet containing control female carp (control, C) or all-female (AF) carp at an incorporation rate of 5, 10 and 20% (w/w) for 90 days. Compared with those fed control carp, the rats fed AF common carp exhibited no significant difference in body weight, food intake, feed conversion ratio, hematology, serum biochemistry, urine test, relative organ weight, gross necropsy, and histopathological examination. This is the first food safety assessment of the farmed fish strain cultured using CRISPR/Cas9, which will further advance the fishery development of genome-edited animals., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Tea trees have a high demand for nitrogen (N) fertilizer to improve the yield and quality of tea. In this research, transcriptome analysis revealed the effect of N starvation and resupply upon N uptake in tea plants. We identified 4098 differentially expressed genes (DEGs) that were significantly enriched in amino acid and N metabolism and were extensively mapped to the tea genome. The CsNRT gene family plays vital roles in the nitrogen uptake of tea plants. The full CDS sequences of CsNRT1.1, CsNRT1.2, CsNRT1.5, CsNRT1.7, CsNRT2.4, CsNRT2.5, CsNRT3.1 and CsNRT3.2 were cloned. One-year-old cutting seedlings of Zhongcha302 (ZC302) were selected for hydroponic culture and were used for gene expression analysis. The seedlings were resupplied with 0.2 and 2 mM N after N starvation. The results of the gene expression under different N treatments and in various tissues indicated that the expression of CsNRT2.4 was highly expressed in tea roots and was greatly induced by N. Overexpressed CsNRT2.4 in transgenic Arabidopsis thaliana increased the root lengths and fresh weights and improved the NO 3 - uptake rate in the Arabidopsis roots at a low NO 3 - level. Thus, we inferred that CsNRT2.4 was a key gene for N uptake in tea plant roots. This study provides new insights into the molecular mechanisms of tea plant responses to N resupply and reveals hub genes for improving nitrogen usage efficiency (NUE) in tea plants., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Although the blockade of the programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) pathway has become a promising treatment strategy for several types of cancers, the constitutive activation of c-Met in tumors may cause a low overall response rate to PD-1 inhibitors. Increasing evidence indicates that the dual inhibition of c-Met and PD-1 could improve the efficacy of anti-PD-1/PD-L1 monoclonal antibodies for tumor immunotherapy. In this study, we developed two bispecific single-chain diabodies targeting c-Met and PD-1 for the treatment of solid tumors based on protein homology modeling, and we identified that the binding affinity of diabody-mp to c-Met was 50-folds higher than that of diabody-pm. The results of in vitro studies revealed that both diabodies suppressed HGF-induced proliferation, migration, and invasion of tumor cells, inhibiting the activation of c-Met signaling by antagonizing HGF binding to c-Met. Moreover, they promoted T cell activation by blocking the PD-1 pathway, mediating tumor cellular cytotoxicity through T cell engagement. In vivo studies with mice models demonstrated that diabody-mp exhibited higher therapeutic efficacy than other structural antibodies, greatly enhancing the survival of c-Met-positive tumor-bearing mice compared to single or combined c-Met and PD-1 blockade therapy. Furthermore, diabody-mp, which had a higher c-Met binding affinity, showed better anti-tumoral activity than diabody-pm, which had a lower c-Met binding affinity. In conclusion, bispecific anti-PD-1/c-Met diabody-mp, with high c-Met-associated affinity, inhibited tumor growth by activating T cells, suggesting its therapeutic potential for c-Met-positive solid tumors., Competing Interests: No potential conflicts of interest were disclosed., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Seven isatin-thiosemicarbazone analogues bearing different substituents (R) attached at C-5 of the indoline ring, TSC-ISA-R (R = -H, -CH 3 , -OCH 3 , -OCF 3 , -F, -Cl and -NO 2 ), were synthesized and evaluated as inhibitors of mushroom tyrosinase (TYR). The inhibitory behaviour and performance of TSC-ISA-R were investigated spectroscopically in relation to the substituent modifications through examining their inhibition against the diphenolase activity of TYR using L-DOPA as a substrate. The IC 50 values of TSC-ISA-R were determined to be in the range of 81-209 μM. The kinetic analysis showed that TSC-ISA-R were reversible and mixed type inhibitors. Three potential non-covalent interactions rather than complexation including the binding of TSC-ISA-R with free TYR, TYR-L-DOPA complex, and with substrate L-DOPA were found to be involved in the inhibition. The substituent modifications affected these interactions by varying the characters of the resulting TSC-ISA-R in different degrees. The thiosemicarbazido moiety of each TSC-ISA-R contributed predominantly to the inhibition, and the isatin moiety seemed to play a regulatory role in the binding of TSC-ISA-R to the target molecules. The results of theoretical calculations using density functional theory method indicated a different effect of -R on the electron distribution in HOMO of TSC-ISA-R. The LUMO-HOMO energy gap of TSC-ISA-R almost accords with the trend of their experimental inhibition potency., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Purpose Programmed cell death 1 (PD-1), which is upregulated under the continuous induction of the tumor microenvironment, causes chimeric antigen receptor (CAR)-T cell hypofunction via interaction with programmed death ligand 1 (PD-L1). This study aimed to construct CAR-T cells that are resistant to PD-1 inhibition to improve the effect of CAR-T cells in solid tumors. Methods We constructed a type of dual-function CAR-T cell that targets tumor-associated antigen c-Met and blocks the binding of PD-1 with PD-L1. The expression of c-Met, PD-L1, and inhibitory receptors was measured using flow cytometry. The cytotoxicity, cytokine release, and differentiation level of CAR-T cells were determined using lactate dehydrogenase release assay, enzyme-linked immunosorbent assay, and flow cytometry, respectively. The levels of p-Akt, p-MAPK, caspase-3, and Bcl2 were detected by western blot. The in vivo anti-tumor effect was evaluated using tumor xenograft models. Results Dual-function CAR-T cells could mediate enhanced active signals upon encountering target antigens and had targeted cytotoxicity to target cells. However, the cytotoxicity of c-Met-CAR-PD-1 + T cells was impaired due to the interaction of PD-1 with PD-L1. By blocking the binding of PD-1 and PD-L1, the novel dual-function CAR-PD-1 + T cells could maintain cytotoxicity to PD-L1 + tumor cells. In tumor tissue, the dual-function CAR-T cells showed lower inhibitory receptor expression and lower differentiation characteristics, which resulted in potent anti-tumor effects and prolonged survival in PD-L1 + tumor xenograft models compared to single-target CAR-T cells. Conclusion These results confirm that the novel dual-function CAR-T cells exhibit stronger anti-tumor activity against solid tumors than traditional single-target CAR-T cells and present a new approach that enhance the activity of CAR-T cells in solid tumors.

Background Redirecting T cells to tumor cells using bispecific antibodies (BsAbs) is emerging as a potent cancer therapy. The main concept of this strategy is to cross-link tumor cells and T cells by simultaneously binding to cell surface tumor-associated antigen (TAA) and the CD3ƹ chain. However, immune checkpoint programmed cell death ligand-1 (PD-L1) on tumor cells or other myeloid cells upreglulated remarkablely after the treatment of CD3-binding BsAbs, leads to the generation of suppressed microenvironment for immune evasion and tumor progression. Although this resistance could be partially reversed by anti-PD-L1 treatment, targeting two pathways through one antibody-based molecule may provide a strategic advantage over the combination of BsAbs and immune checkpoint inhibitors. Methods We developed two novel BsAbs PD-1/c-Met DVD-Ig and IgG-scFv both targeting PD-1 to restore the immune effector function of T cells and engaging them to tumor cells via binding to cellular-mesenchymal to epithelial transition factor (c-Met). Binding activities, T cell activation and proliferation were analyzed by flow cytometry. Cell Cytotoxicity and cytokine release were measured using LDH release assay and ELISA, respectively. Anti-tumor response in vivo was evaluated by generate xenograft models in NOD-SCID mice. Results These bispecific antibodies exhibited effective antitumor activity against high- and low- c-Met-expressing gastric cancer cell lines in vitro and mediated strong tumor growth inhibition in human gastric cancer xenograft models. Conclusion The engagement of the PD-1/PD-L1 blockade to c-Met-overexpressing cancer cells is a promising strategy for the treatment of gastric cancer and potentially other malignancies.

Background: Bispecific antibodies, BsAbs, are molecules with the ability to bind to two different epitopes on the same or different antigens. c-MET, cellular-mesenchymal to epithelial transition factor, is deregulated in many types of human malignancies. Abnormal c-MET activation in cancer correlates with poor prognosis. PD-1, programmed death-1, is an additional inhibitory receptor expressed by T cells. Blocking the interactions between PD-1 and PD-L1 has emerged as a promising immunotherapy for treating cancer., Objectives: The goal of this study was to identify a novel bispecific antibody targeting both c-MET and PD-1 as an anti-cancer therapeutic candidate., Methods: The BsAb was produced using 293E expression system and purified by Protein A affinity chromatography. Then the binding specificity and affinity of the BsAb was examined by FACS and biolayer light interferometry. The ability of the BsAb to inhibit the proliferation of tuman cells was measured using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit; the potential signaling pathway involved was identified by Western Blot. Cytokine secreted by PHA-L stimulated PBMC was measured by ELISA. Effects of BsAb on PBMC-mediated lysis of MKN45 cells was measured by LDH cytotoxicity assay., Results: Based on the original sequences of PD-1 and c-MET mAb, a BsAb gene was designed, cloned into pCEP4 vector for expression in 293E cells. The BsAb was obtained after purification of the cell culture supernatant. It can bind to PD-1 and c-MET simultaneously, the calculated affinity was 11.5 nM for PD-1 and 9.09 nM for c-MET. The BsAb enhanced IFN-γ production over control IgG by 2-3 folds. It also inhibit the c-MET pathway activation and the proliferation of tumor cells significantly, comparable to JnJ-38877605. The BsAb showed dose-dependent cytotoxic activity against MKN45 cells., Conclusion: Our results indicated that a novel BsAb recognizing PD-1 and c-MET was successfully generated. It could redirect T cells to kill tumor cells, while retaining its inherent ability to restore T cells and inhibit tumor cells. With this potential, this BsAb could be developed as a therapeutic candidate for the treatment of various solid tumors., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)