Nanobodies from camelid mice and llamas neutralize SARS-CoV-2 variants.

Since the start of the COVID-19 pandemic, SARS-CoV-2 has caused millions of deaths worldwide. Although a number of vaccines have been deployed, the continual evolution of the receptor-binding domain (RBD) of the virus has challenged their efficacy. In particular, the emerging variants B.1.1.7, B.1.351 and P.1 (first detected in the UK, South Africa and Brazil, respectively) have compromised the efficacy of sera from patients who have recovered from COVID-19 and immunotherapies that have received emergency use authorization1-3. One potential alternative to avert viral escape is the use of camelid VHHs (variable heavy chain domains of heavy chain antibody (also known as nanobodies)), which can recognize epitopes that are often inaccessible to conventional antibodies4. Here, we isolate anti-RBD nanobodies from llamas and from mice that we engineered to produce VHHs cloned from alpacas, dromedaries and Bactrian camels. We identified two groups of highly neutralizing nanobodies. Group 1 circumvents antigenic drift by recognizing an RBD region that is highly conserved in coronaviruses but rarely targeted by human antibodies. Group 2 is almost exclusively focused to the RBD-ACE2 interface and does not neutralize SARS-CoV-2 variants that carry E484K or N501Y substitutions. However, nanobodies in group 2 retain full neutralization activity against these variants when expressed as homotrimers, and-to our knowledge-rival the most potent antibodies against SARS-CoV-2 that have been produced to date. These findings suggest that multivalent nanobodies overcome SARS-CoV-2 mutations through two separate mechanisms: enhanced avidity for the ACE2-binding domain and recognition of conserved epitopes that are largely inaccessible to human antibodies. Therefore, although new SARS-CoV-2 mutants will continue to emerge, nanobodies represent promising tools to prevent COVID-19 mortality when vaccines are compromised.

mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants.

Here we report on the antibody and memory B cell responses of a cohort of 20 volunteers who received the Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) vaccine against SARS-CoV-21-4. Eight weeks after the second injection of vaccine, volunteers showed high levels of IgM and IgG anti-SARS-CoV-2 spike protein (S) and receptor-binding-domain (RBD) binding titre. Moreover, the plasma neutralizing activity and relative numbers of RBD-specific memory B cells of vaccinated volunteers were equivalent to those of individuals who had recovered from natural infection5,6. However, activity against SARS-CoV-2 variants that encode E484K-, N501Y- or K417N/E484K/N501-mutant S was reduced by a small-but significant-margin. The monoclonal antibodies elicited by the vaccines potently neutralize SARS-CoV-2, and target a number of different RBD epitopes in common with monoclonal antibodies isolated from infected donors5-8. However, neutralization by 14 of the 17 most-potent monoclonal antibodies that we tested was reduced or abolished by the K417N, E484K or N501Y mutation. Notably, these mutations were selected when we cultured recombinant vesicular stomatitis virus expressing SARS-CoV-2 S in the presence of the monoclonal antibodies elicited by the vaccines. Together, these results suggest that the monoclonal antibodies in clinical use should be tested against newly arising variants, and that mRNA vaccines may need to be updated periodically to avoid a potential loss of clinical efficacy.

Myc Regulates Chromatin Decompaction and Nuclear Architecture during B Cell Activation.

50 years ago, Vincent Allfrey and colleagues discovered that lymphocyte activation triggers massive acetylation of chromatin. However, the molecular mechanisms driving epigenetic accessibility are still unknown. We here show that stimulated lymphocytes decondense chromatin by three differentially regulated steps. First, chromatin is repositioned away from the nuclear periphery in response to global acetylation. Second, histone nanodomain clusters decompact into mononucleosome fibers through a mechanism that requires Myc and continual energy input. Single-molecule imaging shows that this step lowers transcription factor residence time and non-specific collisions during sampling for DNA targets. Third, chromatin interactions shift from long range to predominantly short range, and CTCF-mediated loops and contact domains double in numbers. This architectural change facilitates cognate promoter-enhancer contacts and also requires Myc and continual ATP production. Our results thus define the nature and transcriptional impact of chromatin decondensation and reveal an unexpected role for Myc in the establishment of nuclear topology in mammalian cells.

SAMHD1-mediated dNTP degradation is required for efficient DNA repair during antibody class switch recombination.

Sterile alpha motif and histidine-aspartic acid domain-containing protein 1 (SAMHD1), a dNTP triphosphohydrolase, regulates the levels of cellular dNTPs through their hydrolysis. SAMHD1 protects cells from invading viruses that depend on dNTPs to replicate and is frequently mutated in cancers and Aicardi-Goutières syndrome, a hereditary autoimmune encephalopathy. We discovered that SAMHD1 localizes at the immunoglobulin (Ig) switch region, and serves as a novel DNA repair regulator of Ig class switch recombination (CSR). Depletion of SAMHD1 impaired not only CSR but also IgH/c-Myc translocation. Consistently, we could inhibit these two processes by elevating the cellular nucleotide pool. A high frequency of nucleotide insertion at the break-point junctions is a notable feature in SAMHD1 deficiency during activation-induced cytidine deaminase-mediated genomic instability. Interestingly, CSR induced by staggered but not blunt, double-stranded DNA breaks was impaired by SAMHD1 depletion, which was accompanied by enhanced nucleotide insertions at recombination junctions. We propose that SAMHD1-mediated dNTP balance regulates dNTP-sensitive DNA end-processing enzyme and promotes CSR and aberrant genomic rearrangements by suppressing the insertional DNA repair pathway.

Microinvasion in hepatocellular carcinoma: predictive factor and application for definition of clinical target volume for radiotherapy.

BACKGROUND: To investigate the correlation between microinvasion and various features of hepatocellular carcinoma (HCC), and to clarify the microinvasion distance from visible HCC lesions to subclinical lesions, so as to provide clinical basis for the expandable boundary of clinical target volume (CTV) from gross tumor volume (GTV) in the radiotherapy of HCC. METHODS: HCC patients underwent hepatectomy of liver cancer in our hospital between July 2019 and November 2021 were enrolled. Data on various features and tumor microinvasion distance were collected. The distribution characteristics of microinvasion distance were analyzed to investigate its potential correlation with various features. Tumor size compared between radiographic and pathologic samples was analyzed to clarify the application of pathologic microinvasion to identify subclinical lesions of radiographic imaging. RESULTS: The average microinvasion distance was 0.6 mm, with 95% patients exhibiting microinvasion distance less than 3.0 mm, and the maximum microinvasion distance was 4.0 mm. A significant correlation was found between microinvasion and liver cirrhosis (P = 0.036), serum albumin level (P = 0.049). Multivariate logistic regression analysis revealed that HCC patients with cirrhosis had a significantly lower risk of microinvasion (OR = 0.09, 95%CI = 0.02 ~ 0.50, P = 0.006). Tumor size was overestimated by 1.6 mm (95%CI=-12.8 ~ 16.0 mm) on radiographic size compared to pathologic size, with a mean %Δsize of 2.96% (95%CI=-0.57%~6.50%). The %Δsize ranged from - 29.03% to 34.78%. CONCLUSIONS: CTV expanding by 5.4 mm from radiographic GTV could include all pathologic microinvasive lesions in the radiotherapy of HCC. Liver cirrhosis was correlated with microinvasion and were independent predictive factor of microinvasion in HCC.

Identity-Based Matchmaking Encryption with Equality Test.

The identity-based encryption with equality test (IBEET) has become a hot research topic in cloud computing as it provides an equality test for ciphertexts generated under different identities while preserving the confidentiality. Subsequently, for the sake of the confidentiality and authenticity of the data, the identity-based signcryption with equality test (IBSC-ET) has been put forward. Nevertheless, the existing schemes do not consider the anonymity of the sender and the receiver, which leads to the potential leakage of sensitive personal information. How to ensure confidentiality, authenticity, and anonymity in the IBEET setting remains a significant challenge. In this paper, we put forward the concept of the identity-based matchmaking encryption with equality test (IBME-ET) to address this issue. We formalized the system model, the definition, and the security models of the IBME-ET and, then, put forward a concrete scheme. Furthermore, our scheme was confirmed to be secure and practical by proving its security and evaluating its performance.

Protective Functions of ZO-2/Tjp2 Expressed in Hepatocytes and Cholangiocytes Against Liver Injury and Cholestasis.

BACKGROUND & AIMS: Liver tight junctions (TJs) establish tissue barriers that isolate bile from the blood circulation. TJP2/ZO-2-inactivating mutations cause progressive cholestatic liver disease in humans. Because the underlying mechanisms remain elusive, we characterized mice with liver-specific inactivation of Tjp2. METHODS: Tjp2 was deleted in hepatocytes, cholangiocytes, or both. Effects on the liver were assessed by biochemical analyses of plasma, liver, and bile and by electron microscopy, histology, and immunostaining. TJ barrier permeability was evaluated using fluorescein isothiocyanate-dextran (4 kDa). Cholic acid (CA) diet was used to assess susceptibility to liver injury. RESULTS: Liver-specific deletion of Tjp2 resulted in lower Cldn1 protein levels, minor changes to the TJ, dilated canaliculi, lower microvilli density, and aberrant radixin and bile salt export pump (BSEP) distribution, without an overt increase in TJ permeability. Hepatic Tjp2-defcient mice presented with mild progressive cholestasis with lower expression levels of bile acid transporter Abcb11/Bsep and detoxification enzyme Cyp2b10. A CA diet tolerated by control mice caused severe cholestasis and liver necrosis in Tjp2-deficient animals. 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene ameliorated CA-induced injury by enhancing Cyp2b10 expression, and ursodeoxycholic acid provided partial improvement. Inactivating Tjp2 separately in hepatocytes or cholangiocytes showed only mild CA-induced liver injury. CONCLUSION: Tjp2 is required for normal cortical distribution of radixin, canalicular volume regulation, and microvilli density. Its inactivation deregulated expression of Cldn1 and key bile acid transporters and detoxification enzymes. The mice provide a novel animal model for cholestatic liver disease caused by TJP2-inactivating mutations in humans.

Emodin inhibits invasion and migration of hepatocellular carcinoma cells via regulating autophagy-mediated degradation of snail and ß-catenin.

BACKGROUND: Previous studies reported that emodin extracted from Rheum palmatum L. exerts antiproliferation and antimetastatic effects in a variety of human cancer types. However, the role of emodin in hepatocellular carcinoma (HCC) remain unknown. METHODS: EdU and colony formation assays were performed to evaluate the effects of emodin on proliferation. The mobility capacities of HCC treated with emodin were evaluated using wound healing assay. Transwell invasion and migration assays were performed to evaluate anti-migratory and anti-invasive effects of emodin on HCC. Annexin V-FITC/PI was performed to analyze the apoptosis. PI stain was performed to analyze cell cycle. RNA sequencing technology was used to identify the differentially expressed genes (DEGs) induced by emodin in HCC. The impact of emodin on autophagic flux in HepG2 cells was examined by mCherry-GFP-LC3 analysis. Western blot was used to assess the protein expressions of epithelial-mesenchymal transition (EMT), autophagy, PI3K/AKT/mTOR and Wnt/ß-catenin signaling pathway. RESULTS: We found that emodin inhibited the growth of HepG2 cells in a dose- and time-dependent manner. In addition, emodin inhibited cell proliferation, induced S and G2/M phases arrest, and promoted apoptosis in HepG2 cells. The migration and invasion of HepG2 cells were also suppressed by emodin. Enrichment analysis revealed that DEGs involved in cell adhesion, cancer metastasis and cell cycle arrest. Moreover, western bolt results show that emodin-induced autophagy promotes Snail and ß-catenin degradation. We also found that blocking autophagic flux after emodin treatment caused EMT reversal. Furthermore, the PI3K agonist Y-P 740 significantly reversed the phosphorylation levels of GSK3ß and mTOR. These results indicated that emodin induced autophagy and inhibited the EMT in part through suppression of the PI3K/AKT/mTOR and Wnt/ß-catenin pathways. CONCLUSION: Our study indicated that emodin inhibited cell metastasis in HCC via the crosstalk between autophagy and EMT.

Comparison of anatomical thermal ablation and routine thermal ablation for hepatocellular carcinoma≤50 mm: A propensity score matching.

AIM: The present study was to evaluated the clinical value of anatomical thermal ablation to treat hepatocellular carcinoma compared with routine thermal ablation. METHODS: Hepatocellular carcinoma patients with tumor diameter ≤50 mm treated by thermal ablation at our center were retrospectively enrolled from October 2015 to December 2018. Enrolled patients were grouped into the anatomical ablation group and routine ablation group, respectively. To minimize the effects of potential confounders from selection bias, a propensity score matching was carried out. Technical efficacy, recurrence and survivals rates were compared. RESULTS: Altogether 101 patients (119 lesions) were grouped into the anatomical ablation group and 101 patients (131 lesions) into the routine ablation group. The ablation zone volume of the anatomical ablation group was 36.8 (2.5-176.9) ml, significantly larger than that of the routine ablation group (28.5 [28.5 (2.8-184.3) ml] (p = 0.005)). Adjusted with propensity score matching, The 1-, 2-, and 3-year local recurrence rates were 0.0%, 0.0%, and 0.0% for the anatomical ablation group and 6.9%, 10.1%, and 10.1% for the routine ablation group, respectively (p = 0.013). The cumulative 1-, 2-, and 3-year progression-free survival rates were 93.4%, 82.7%, and 79.0% for the anatomical ablation group, 74.2%, 56.9%, and 51.6% for the routine ablation group (p = 0.001). CONCLUSIONS: Anatomical ablation could be a favorable ablation strategy to improve therapeutic effect of thermal ablation for HCC with visible feeding vessels and reserved liver function.

Neutrophil-to-Apolipoprotein A1 Ratio Predicted Overall Survival in Hepatocellular Carcinoma Receiving Transarterial Chemoembolization.

PURPOSE: The purpose of this study was to investigate the predictive capability of neutrophil-to-apolipoprotein A1 ratio (NAR) for predicting overall survival (OS) among patients with hepatocellular carcinoma (HCC) receiving transarterial chemoembolization (TACE). PATIENTS AND METHODS: We investigated the clinical features of 554 patients with HCC receiving TACE and assessed NAR's predictive value for OS with 222 patients (the discovery cohort) and 332 patients (the validation cohort). The association of NAR with circulation lectin-type oxidized low-density lipoprotein receptor-1-positive (LOX-1+ ) polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) was illustrated. RESULTS: Multivariate Cox regression revealed that lymphocyte count; Tumor, Node, Metastasis (TNM) stage; and NAR were independent prognostic factors in the discovery cohort. The validation cohort confirmed the independent prognostic value of TNM stage and NAR. Patients with low NAR (<2.7) displayed significantly increased OS in the discovery cohort (59.8 months vs. 21 months), the validation group (38.0 months vs. 23.6 months), and the total cohort (44.1 months vs. 22.0 months). A Cox proportional hazards model was used to combine Cancer of the Liver Italian Program (CLIP) score with discretized NAR. C-index illustrated that NAR-integrated CLIP score was the best model compared with NAR and CLIP score. Furthermore, NAR-CLIP presented superior predictive capacity for 10-, 20-, 30-, 40-, 50-, and 60-month survival compared with CLIP score by survival receiver-operator characteristic analysis in the discovery cohort, validation cohort, and total cohort. NAR was significantly associated with LOX-1+ PMN-MDSCs by linear regression. CONCLUSION: This study identified NAR as an independent predictor for OS among patients with HCC receiving TACE. NAR reflected circulation LOX-1+ PMN-MDSC level. IMPLICATIONS FOR PRACTICE: The present study identified neutrophil-to-apolipoprotein A1 ratio (NAR) as an independent predictor for overall survival among patients with hepatocellular carcinoma receiving transarterial chemoembolization. NAR reflected circulation level of lectin-type oxidized low-density lipoprotein receptor-1-positive polymorphonuclear myeloid-derived suppressor cells.

Expression and prognostic roles of PRDXs gene family in hepatocellular carcinoma.

BACKGROUND: As the fourth leading cause of cancer-related death in the world, the therapeutic effect and 5-year overall survival of hepatocellular carcinoma (HCC) are not optimistic. Previous researches indicated that the disorder of PRDXs was related to the occurrence and development of cancers. METHODS: In this study, PRDXs were found in various tumor cell lines by CCLE database analysis. The analysis results of UALCAN, HCCDB and Human Protein Atlas databases showed the expression of PRDXs mRNA and protein in HCC tissues was dysregulated. Besides, UALCAN was used to assess the correlations between PRDXs mRNA as well as methylation levels and clinical characterization. RESULTS: High expression of PRDX1 or low expression of PRDX2/3 suggested poor prognosis for HCC patients which was demonstrated by Kaplan-Meier Plotter. The genetic alterations and biological interaction network of PRDXs in HCC samples were obtained from c-Bioportal. In addition, LinkedOmics was employed to analyze PRDXs related differentially expressed genes, and on this basis, enrichment of KEGG pathway and miRNAs targets of PRDXs were conducted. The results indicated that these genes were involved in several canonical pathways and certain amino acid metabolism, some of which may effect on the progression of HCC. CONCLUSIONS: In conclusion, the disordered expression of some PRDX family members was associated with the prognosis of HCC patients, suggesting that these PRDX family members may become new molecular targets for the treatment and prognosis prediction of HCC.

Occurrence and distribution of per- and polyfluoroalkyl substances (PFASs) in human livers with liver cancer.

Per- and polyfluoroalkyl substances (PFASs) are anthropogenic compounds that are widely accumulated in human tissues, and the liver is considered a primary target organ for PFASs exposure. The occurrence and distribution of 21 PFASs in liver tissues with tumors (n = 55) and without tumors (n = 55) are investigated in this study. Eleven perfluorinated carboxylic acids (PFCAs) and five perfluorinated sulfonic acids (PFSAs) were detected at high frequencies (45.5%-100 %), while the detection frequencies of five perfluoroalkyl phosphate (PFPAs) were relatively lower (≤29.1 %). PFSAs and PFCAs accounted for up to 82.5%-92.7 % of the total PFASs. Although it was not found to be statistically significant, the concentrations of the total PFASs were slightly higher in the tumor liver samples (mean 64.3, range 5.70-303 ng/g) than those in the non-tumor liver samples (mean 62.7, range 4.08-240 ng/g).The perfluorooctanoic acid (PFOA), perfluorotridecanoic acid (PFTrDA), and perfluorobutanesulphonate (PFBS) showed significant differences (p < 0.05) between the tumor and non-tumor liver samples, and the different distribution levels of these three PFASs may have been a consequence of oxidative stress. The total concentrations of PFASs in the three age groups were in the decreasing order of middle-aged people (45-60) > old people (>60) > young people (<45). The PFASs in females were generally lower than in males, which may have been related to women's special excretion methods (such as childbirth and breastfeeding). The results should be valuable for further mechanistic studies regarding the toxic effects of PFASs in human livers.

Development of a transcriptomic database for 14 species of scleractinian corals.

BACKGROUND: Scleractinian corals are important reef builders, but around the world they are under the threat of global climate change as well as local stressors. Molecular resources are critical for understanding a species' stress responses and resilience to the changing environment, but such resources are unavailable for most scleractinian corals, especially those distributed in the South China Sea. We therefore aimed to provide transcriptome resources for 14 common species, including a few structure forming species, in the South China Sea. DESCRIPTION: We sequenced the transcriptome of 14 species of scleractinian corals using high-throughput RNA-seq and conducted de novo assembly. For each species, we produced 7.4 to 12.0 gigabases of reads, and assembled them into 271 to 762 thousand contigs with a N50 value of 629 to 1427 bp. These contigs included 66 to 114 thousand unigenes with a predicted open reading frame, and 74.3 to 80.5% of the unigenes were functionally annotated. In the azooxanthelate species Tubastraea coccinea, 41.5% of the unigenes had at least a best-hit sequence from corals. In the other thirteen species, 20.2 to 48.9% of the annotated unigenes had best-hit sequences from corals, and 28.3 to 51.6% from symbiotic algae belonging to the family Symbiodinaceae. With these resources, we developed a transcriptome database (CoralTBase) which features online BLAST and keyword search for unigenes/functional terms through a user friendly Internet interface. SHORT CONCLUSION: We developed comprehensive transcriptome resources for 14 species of scleractinian corals and constructed a publicly accessible database ( www.comp.hkbu.edu.hk/~db/CoralTBase ). CoralTBase will facilitate not only functional studies using these corals to understand the molecular basis of stress responses and adaptation, but also comparative transcriptomic studies with other species of corals and more distantly related cnidarians.

Inhibition of cyclin E1 sensitizes hepatocellular carcinoma cells to regorafenib by mcl-1 suppression.

BACKGROUND: To clarify the effects of cylcin E1 expression on HCC tumor progression, we studied the expression of cyclin E1 and inhibitory efficacy of regorafenib and sorafenib in HCC cells, and investigated a potential therapy that combines regorafenib treatment with cyclin E1 inhibition. METHODS: Western blotting for caspase-3 and Hoechst 33225 staining was used to measure the expression level of apoptosis-related proteins under drug treatment. RESULTS: Our results showed that enhanced expression of cyclin E1 after transfection compromised apoptosis in HCC cells induced by regorafenib or sorafenib. Conversely, down-regulation of cyclin E1 gene expression or inhibition of cyclin E1 by the cyclin-dependent kinase (CDK) inhibitors dinaciclib (DIN) or flavopiridol sensitized HCC cells to regorafenib and sorafenib by inducing apoptosis. The expression of Mcl-1, which is modulated by STAT3, plays a key role in regulating the therapeutic effects of CDK inhibitors. Xenograft experiments conducted to test the efficacy of regorafenib combined with DIN showed dramatic tumor inhibitory effects due to induction of apoptosis. Our results suggested that the level of cyclin E1 expression in HCCs may be used as a pharmacodynamic biomarker to assess the antitumor effects of regorafenib or sorafenib. CONCLUSIONS: Combining regorafenib and CDK inhibitors may enhance the clinical efficiency of the treatment of HCCs.

HOXA-AS2 Promotes Proliferation and Induces Epithelial-Mesenchymal Transition via the miR-520c-3p/GPC3 Axis in Hepatocellular Carcinoma.

BACKGROUND/AIMS: Previous studies have demonstrated that long non-coding RNAs (lncRNAs) may play critical roles in cancer biology, including Hepatocellular carcinoma (HCC). The HOXA cluster antisense RNA2 (HOXA-AS2) lncRNA plays an important role in carcinogenesis, however, the underlying role of HOXA-AS2 in HCC remains unknown. The present study examined the effects of HOXA-AS2 on the progression of HCC, and explored the underlying molecular mechanisms. METHODS: Quantitative real-time PCR was used to detect HOXA-AS2 expression in HCC tissues and cell lines. Furthermore, the effects of HOXA-AS2 silencing and overexpression on cell proliferation, cell cycle, apoptosis, migration, and invasion were assessed in HCC in vitro and in vivo. Furthermore, bioinformatics online programs predicted and luciferase reporter assay were used to validate the association of HOXA-AS2 and miR-520c-3p in HCC cells. RESULTS: We observed that HOXA-AS2 was up-regulated in HCC tissues and cell lines. In vitro experiments revealed that HOXA-AS2 knockdown significantly inhibited HCC cells proliferation by causing G1 arrest and promoting apoptosis, whereas HOXA-AS2 overexpression promoted cell growth. Further functional assays indicated that HOXA-AS2 significantly promoted HCC cell migration and invasion by promoting EMT. Bioinformatics online programs predicted that HOXA-AS2 sponge miR-520c-3p at 3'-UTR with complementary binding sites, which was validated using luciferase reporter assay. HOXA-AS2 could negatively regulate the expression of miR-520c-3p in HCC cells. MiR-520c-3p was down-regulated and inversely correlated with HOXA-AS2 expression in HCC tissues. miR-520c-3p suppressed cell proliferation, invasion and migration in HCC cells, and enforced expression of miR-520c-3p attenuated the oncogenic effects of HOXA-AS2 in HCC cells. By bioinformatic analysis and dual-luciferase reporter assay, we found that miR-223-3p directly targeted the 3'-untranslated region (UTR) of Glypican-3 (GPC3), one of the key players in HCC. GPC3 was up-regulated in HCC tissues, and was negatively correlated with miR-520c-3p expression and positively correlated with HOXA-AS2 expression. CONCLUSION: In summary, our results suggested that the HOXA-AS2/miR-520c-3p/GPC3 axis may play an important role in the regulation of PTC progression, which could serve as a biomarker and therapeutic target for HCC.

Measurement of Serum and Hepatic Eicosanoids by Liquid Chromatography Tandem-Mass Spectrometry (LC-MS/MS) in a Mouse Model of Hepatocellular Carcinoma (HCC) with Delivery of c-Met and Activated ß-Catenin by Hepatocyte Hydrodynamic Injection.

BACKGROUND Most forms of cancer, including hepatocellular carcinoma (HCC), are associated with varying degrees of chronic inflammation. The association between the expression of eicosanoids, which are bioactive lipid mediators of inflammation, and HCC remains unknown. The aim of this study was to measure serum and hepatic eicosanoids in a mouse model of HCC with the delivery of c-Met and activated b-catenin by hepatocyte hydrodynamic injection. MATERIAL AND METHODS The HCC mouse model, and normal control mice, were used in this study with co-delivery of human c-Met combined with activated ß-catenin into hepatocytes through hydrodynamic injection. Liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis was used to measure serum and hepatic eicosanoid levels. RESULTS The combined activation of c-Met and ß-catenin was induced in the HCC mouse model. LC-MS/MS showed that a total of 13 eicosanoids in serum and 12 eicosanoids in liver tissue were significantly increased in the HCC mice, when compared with control mice. CONCLUSIONS In a mouse model of HCC, co-activation of the c-Met and ß-catenin signaling pathway resulted in increased levels of serum and hepatic eicosanoids.

Lipopolysaccharide promotes angiogenesis in mice model of HCC by stimulating hepatic stellate cell activation via TLR4 pathway.

Angiogenesis plays a key role in the progression of hepatocellular carcinoma (HCC). This study aimed to investigate whether lipopolysaccharide (LPS) could promote HCC angiogenesis and the role of hepatic stellate cell (HSC) in this process. In vivo orthotopic HCC model and the effect of LPS on HSC in vitro were studied. Our results demonstrated that LPS-induced HSC activation during the promotion of HCC growth and angiogenesis in mice. The LPS-TLR4 (Toll-like receptor 4) pathway in HSC is responsible for HCC angiogenesis. LPS-induced secretion of pro-angiogenic factors from HSC could promote endothelial cell migration and tubulogenesis. This study suggests that LPS acts with HSC in tumor stroma and promotes the secretion of pro-angiogenic factors that increase angiogenesis in HCC.

APE1 is dispensable for S-region cleavage but required for its repair in class switch recombination.

Activation-induced cytidine deaminase (AID) is essential for antibody diversification, namely somatic hypermutation (SHM) and class switch recombination (CSR). The deficiency of apurinic/apyrimidinic endonuclease 1 (Ape1) in CH12F3-2A B cells reduces CSR to ∼20% of wild-type cells, whereas the effect of APE1 loss on SHM has not been examined. Here we show that, although APE1's endonuclease activity is important for CSR, it is dispensable for SHM as well as IgH/c-myc translocation. Importantly, APE1 deficiency did not show any defect in AID-induced S-region break formation, but blocked both the recruitment of repair protein Ku80 to the S region and the synapse formation between Sµ and Sα. Knockdown of end-processing factors such as meiotic recombination 11 homolog (MRE11) and carboxy-terminal binding protein (CtBP)-interacting protein (CtIP) further reduced the remaining CSR in Ape1-null CH12F3-2A cells. Together, our results show that APE1 is dispensable for SHM and AID-induced DNA breaks and may function as a DNA end-processing enzyme to facilitate the joining of broken ends during CSR.

C-terminal region of activation-induced cytidine deaminase (AID) is required for efficient class switch recombination and gene conversion.

Activation-induced cytidine deaminase (AID) introduces single-strand breaks (SSBs) to initiate class switch recombination (CSR), gene conversion (GC), and somatic hypermutation (SHM). CSR is mediated by double-strand breaks (DSBs) at donor and acceptor switch (S) regions, followed by pairing of DSB ends in two S regions and their joining. Because AID mutations at its C-terminal region drastically impair CSR but retain its DNA cleavage and SHM activity, the C-terminal region of AID likely is required for the recombination step after the DNA cleavage. To test this hypothesis, we analyzed the recombination junctions generated by AID C-terminal mutants and found that 0- to 3-bp microhomology junctions are relatively less abundant, possibly reflecting the defects of the classical nonhomologous end joining (C-NHEJ). Consistently, the accumulation of C-NHEJ factors such as Ku80 and XRCC4 was decreased at the cleaved S region. In contrast, an SSB-binding protein, poly (ADP)-ribose polymerase1, was recruited more abundantly, suggesting a defect in conversion from SSB to DSB. In addition, recruitment of critical DNA synapse factors such as 53BP1, DNA PKcs, and UNG at the S region was reduced during CSR. Furthermore, the chromosome conformation capture assay revealed that DNA synapse formation is impaired drastically in the AID C-terminal mutants. Interestingly, these mutants showed relative reduction in GC compared with SHM in chicken DT40 cells. Collectively, our data indicate that the C-terminal region of AID is required for efficient generation of DSB in CSR and GC and thus for the subsequent pairing of cleaved DNA ends during recombination in CSR.

Activation-induced cytidine deaminase is dispensable for virus-mediated liver and skin tumor development in mouse models.

Activation-induced cytidine deaminase (AID) not only promotes immune diversity by initiating somatic hypermutation and class switch recombination in immunoglobulin genes but also provokes genomic instability by introducing translocations and mutations into non-immunoglobulin genes. To test whether AID is essential for virus-induced tumor development, we used two transgenic tumor models: mice expressing hepatitis C virus (HCV) core proteins (HCV-Tg), driven by the hepatitis B virus promoter, and mice expressing human papillomavirus type 8 proteins (HPV8-Tg), driven by the Keratin 14 promoter. Both strains were analyzed in the absence and presence of AID by crossing each with AID (-/-) mice. There was no difference in the liver tumor frequency between the HCV-Tg/AID (+/+) and HCV-Tg/AID (-/-) mice at 20 months of age although the AID (+/+) mice showed more severe histological findings and increased cytokine expression. Furthermore, a low level of AID transcript was detected in the HCV-Tg/AID (+/+) liver tissue that was not derived from hepatocytes themselves but from intra-hepatic immune cells. Although AID may not be the direct cause of HCV-induced oncogenesis, AID expressed in B cells, not in hepatocytes, may prolong steatosis and cause increased lymphocyte infiltration into HCV core protein-induced liver lesions. Similarly, there was no difference in the time course of skin tumor development between the HPV8-Tg/AID (-/-) and HPV8-Tg/AID (+/+) groups. In conclusion, AID does not appear to be required for tumor development in the two virus-induced tumor mouse models tested although AID expressed in infiltrating B cells may promote inflammatory reactions in HCV core protein-induced liver pathogenesis.