The Vespertilionidae is the largest family in the order Chiroptera and has a worldwide distribution in the temperate and tropical regions. In order to further clarify the karyotype relationships at the lower taxonomic level in Vespertilionidae, genome-wide comparative maps have been constructed between Myotis myotis (MMY, 2n = 44) and six vesper bats from China: Myotis altarium (MAL, 2n = 44), Hypsugo pulveratus (HPU, 2n = 44), Nyctalus velutinus (NVE, 2n = 36), Tylonycteris robustula (TRO, 2n = 32), Tylonycteris sp. (TSP, 2n = 30)and Miniopterus fuliginosus (MFU, 2n = 46) by cross-species chromosome painting with a set of painting probes derived from flow-sorted chromosomes of Myotis myotis. Each Myotis myotis autosomal probe detected a single homologous chromosomal segment in the genomes of these six vesper bats except for MMY chromosome 3/4 paint which hybridized onto two chromosomes in the genome of M. fuliginosus. Our results show that Robertsonian translocation is the main mode of karyotype evolution in Vespertilionidae and that the addition of heterochromatic material also plays an important role in the karyotypic evolution of the genera Tylonycteris and Nyctalus. Two conserved syntenic associations (MMY9 + 23 and 18 + 19) could be the synapomorphic features for the genus Tylonycteris. The integration of our maps with the published maps has enabled us to deduce chromosomal homologies between human and these six vesper bats and provided new insight into the karyotype evolution of the family Vespertilionidae.

Rhinolophus (Rhinolophidae) is the second most speciose genus in Chiroptera and has extensively diversified diploid chromosome numbers (from 2n = 28 to 62). In spite of many attempts to explore the karyotypic evolution of this genus, most studies have been based on conventional Giemsa staining rather than G-banding. Here we have made a whole set of chromosome-specific painting probes from flow-sorted chromosomes of Aselliscus stoliczkanus (Hipposideridae). These probes have been utilized to establish the first genome-wide homology maps among six Rhinolophus species with four different diploid chromosome numbers (2n = 36, 44, 58, and 62) and three species from other families: Rousettus leschenaulti (2n = 36, Pteropodidae), Hipposideros larvatus (2n = 32, Hipposideridae), and Myotis altarium (2n = 44, Vespertilionidae) by fluorescence in situ hybridization. To facilitate integration with published maps, human paints were also hybridized to A. stoliczkanus chromosomes. Our painting results substantiate the wide occurrence of whole-chromosome arm conservation in Rhinolophus bats and suggest that Robertsonian translocations of different combinations account for their karyotype differences. Parsimony analysis using chromosomal characters has provided some new insights into the Rhinolophus ancestral karyotype and phylogenetic relationships among these Rhinolophus species so far studied. In addition to Robertsonian translocations, our results suggest that whole-arm (reciprocal) translocations involving multiple non-homologous chromosomes as well could have been involved in the karyotypic evolution within Rhinolophus, in particular those bats with low and medium diploid numbers.

Bats are a unique but enigmatic group of mammals and have a world-wide distribution. The phylogenetic relationships of extant bats are far from being resolved. Here, we investigated the karyotypic relationships of representative species from four families of the order Chiroptera by comparative chromosome painting and banding. A complete set of painting probes derived from flow-sorted chromosomes of Myotis myotis (family Vespertilionidae) were hybridized onto metaphases of Cynopterus sphinx (2n = 34, family Pteropodidae), Rhinolophus sinicus (2n=36, family Rhinolophidae) and Aselliscus stoliczkanus (2n=30, family Hipposideridae) and delimited 27, 30 and 25 conserved chromosomal segments in the three genomes, respectively. The results substantiate that Robertsonian translocation is the main mode of chromosome evolution in the order Chiroptera, with extensive conservation of whole chromosomal arms. The use of M. myotis (2n=44) probes has enabled the integration of C. sphinx, R. sinicus and A. stoliczkanus chromosomes into the previously established comparative maps between human and Eonycteris spelaea (2n=36), Rhinolophus mehelyi (2n=58), Hipposideros larvatus (2n=32), and M. myotis. Our results provide the first cytogenetic signature rearrangement that supports the grouping of Pteropodidae and Rhinolophoidea in a common clade (i.e. Pteropodiformes or Yinpterochiroptera) and thus improve our understanding on the karyotypic relationships and genome phylogeny of these bat species.

BackgroundGlypican-3 (GPC3), a cell surface glycoprotein that is pathologically highly expressed in hepatocellular carcinoma (HCC), is an attractive target for immunotherapies, including chimeric antigen receptor (CAR) T cells. The serum GPC3 is frequently elevated in HCC patients due to the shedding effect of cell surface GPC3. The shed GPC3 (sGPC3) is reported to block the function of cell-surface GPC3 as a negative regulator. Therefore, it would be worth investigating the potential influence of antigen shedding in anti-GPC3 CAR-T therapy for HCC.MethodsIn this study, we constructed two types of CAR-T cells targeting distinct epitopes of GPC3 to examine how sGPC3 influences the activation and cytotoxicity of CAR-T cells in vitro and in vivo by introducing sGPC3 positive patient serum or recombinant sGPC3 proteins into HCC cells or by using sGPC3-overexpressing HCC cell lines.ResultsBoth humanized YP7 CAR-T cells and 32A9 CAR-T cells showed GPC3-specific antitumor functions in vitro and in vivo. The existence of sGPC3 significantly inhibited the release of cytokines and the cytotoxicity of anti-GPC3 CAR-T cells in vitro. In animal models, mice carrying Hep3B xenograft tumors expressing sGPC3 exhibited a worse response to the treatment with CAR-T cells under both a low and high tumor burden. sGPC3 bound to CAR-T cells but failed to induce the effective activation of CAR-T cells. Therefore, sGPC3 acted as dominant negative regulators when competed with cell surface GPC3 to bind anti-GPC3 CAR-T cells, leading to an inhibitory effect on CAR-T cells in HCC.ConclusionsWe provide a proof-of-concept study demonstrating that GPC3 shedding might cause worse response to CAR-T cell treatment by competing with cell surface GPC3 for CAR-T cell binding, which revealed a new mechanism of tumor immune escape in HCC, providing a novel biomarker for patient enrolment in future clinical trials and/or treatments with GPC3-targeted CAR-T cells.

Background: Treatment of hepatocellular carcinoma (HCC) using antibody-based targeted therapies, such as antibody conjugates and chimeric antigen receptor T (CAR-T) cell therapy, shows potent antitumor efficacy. Glypican-3 (GPC3) is an emerging HCC therapeutic target; therefore, antibodies against GPC3 would be useful tools for developing immunotherapies for HCC. Methods: We isolated a novel human monoclonal antibody, 32A9, by phage display technology. We determined specificity, affinity, epitope and anti-tumor activity of 32A9, and developed 32A9-based immunotherapy technologies for evaluating the potency of HCC treatment in vitro or in vivo. Results: 32A9 recognized human GPC3 with potent affinity and specificity. The epitope of 32A9 was located in the region of the GPC3 protein core close to the modification sites of the HS chain and outside of the Wnt-binding site of GPC3. The 32A9 antibody significantly inhibited HCC xenograft tumor growth in vivo. We then pursued two 32A9-based immunotherapeutic strategies by constructing an immunotoxin and CART cells. The 32A9 immunotoxin exhibited specific cytotoxicity to GPC3-positive cancer cells, while 32A9 CART cells efficiently eliminated GPC3-positive HCC cells in vitro and caused HCC xenograft tumor regressions in vivo. Conclusions: Our study provides a rationale for 32A9 as a promising GPC3-specific antibody candidate for HCC immunotherapy.

Neutralizing antibody is one of the most effective interventions for acute pathogenic infection. Currently, over three million people have been identified for SARS-CoV-2 infection but SARS-CoV-2-specific vaccines and neutralizing antibodies are still lacking. SARS-CoV-2 infects host cells by interacting with angiotensin converting enzyme-2 (ACE2) via the S1 receptor-binding domain (RBD) of its surface spike glycoprotein. Therefore, blocking the interaction of SARS-CoV-2-RBD and ACE2 by antibody would cause a directly neutralizing effect against virus. In the current study, we selected the ACE2 interface of SARS-CoV-2-RBD as the targeting epitope for neutralizing antibody screening. We performed site-directed screening by phage display and finally obtained one IgG antibody (4A3) and several domain antibodies. Among them, 4A3 and three domain antibodies (4A12, 4D5, and 4A10) were identified to act as neutralizing antibodies due to their capabilities to block the interaction between SARS-CoV-2-RBD and ACE2-positive cells. The domain antibody 4A12 was predicted to have the best accessibility to all three ACE2-interfaces on the spike homotrimer.Pseudovirus and authentic SARS-CoV-2 neutralization assays showed that all four antibodies could potently protect host cells from virus infection. Overall, we isolated multiple formats of SARS-CoV-2-neutralizing antibodies via site-directed antibody screening, which could be promising candidate drugs for the prevention and treatment of COVID-19.

The study aims to analyze the phenolic compounds in Malus spp. and evaluate their antioxidant and pro‐apoptotic effects in BGC‐803 gastric cancer cells. The results showed that cyanidin‐3‐galactoside was the main polyphenol in Malus “Royalty” (MR), while catechin, epicatechin, and proanthocyanidin B1 and B2 contents were higher in Malus “Cinnabar” (MC) and Malus micromalus (MM) than in MR fruits. The total polyphenol content, total flavonoid content, and antioxidative properties of Malus spp. fruits followed an order of MR > MC > MM. Fruit extracts could inhibit BGC‐803 cells growth and induce apoptosis, with IC50 values of 0.47, 0.36, and 0.31 mg/ml for MR, MC, and MM, respectively. Furthermore, fruit extracts induced cell apoptosis through increasing Bcl‐2 and Bcl‐xl (pro‐apoptosis) expression and inhibiting Bax and Bak (anti‐apoptosis) expression, thereby accelerating cell apoptosis. Taken together, these results demonstrate that red fruits (i.e., MR and MC) were more effective against cancer cells than green fruits (i.e., MM). Practical applications Fruits of Malus spp. are ≤5 cm in size, considerably smaller than apples. They are rich in various natural bioactive compounds that are often consumed as a dietary supplement or used as natural raw materials for function food. In the current study, it is comprehensively characterized profile and bioactivity of phenolic metabolites in Malus spp. fruits of different colors, and found that red fruits are more effective in reducing the free radicals and inducing cancer cells apoptosis than green fruits. These findings are valuable for food technologists and food manufacturers, especially those who produce crabapple supplement. The study investigated the molecular mechanism of how Malus spp. fruits exert anti‐cancer functions. This lays a theoretical foundation for future research on developing anti‐cancer function food and provides helpful guidance for fruit market management and fruit processing industry.

Chromosomal aberrations in non-small-cell lung carcinomas (NSCLCs) are common events. In our study, the lung cancer cell lines (NCI-H446 and SPC-A-1) displayed numerous numerical and structural alterations in their chromosomes by G-banded karyotypic analysis, and abnormalities of chromosome 12 by fluorescence in situ hybridization. Sequentially, we used 14 microsatellite markers within 12q to analyze loss of heterozygosity (LOH) in lung cancer cell lines and NSCLCs. Possible LOH on 12q were statistically inferred to occur in five lung cell lines. Importantly, 17 out of 25 NSCLCs (68%) showed LOH at chromosome 12q. Frequencies of LOH for individual markers ranged from 18% to 44%. Several deletions which were marked with D12S1301, D12S2196, D12S398, D12S90, D12S1056, D12S1713, D12S375, D12S1040, D12S326, and D12S106 were newly detected. Allelic loss on 12q15-q21 detected with D12S1040 occurred at the later stages of NSCLC progression (p < 0.05, Fisher's exact test). LOH on 12q marked with D12S2196, D12S398, D12S326, and D12S106 were frequently found in NSCLCs from the patients without smoking history (p < 0.05, Fisher's exact test). These findings indicated that allelic loss on 12q is commonly involved in NSCLCs, and new tumor suppressor genes may occur within 12q.