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Background: Biliary tract cancer is a highly lethal malignancy with poor clinical outcome. Accumulating evidence indicates targeted therapeutics may provide new hope for improving treatment response in BTC, hence better understanding the genomic profile is particularly important. Since tumor tissue may not be available for some patients, a complementary method is urgently needed. Circulating tumor DNA (ctDNA) provides a noninvasive means for detecting genomic alterations, and has been regarded as a promising tool to guide clinical therapies.Methods: Next-generation sequencing of 150 cancer-related genes was used to detect gene alterations in blood-derived ctDNA from 154 Chinese patients with BTC. Genomic alterations were analyzed and compared with an internal tissue genomic database and TCGA database.Results: 94.8% patients had at least one change detected in their ctDNA. The median maximum somatic allele frequency was 6.47% (ranging 0.1–34.8%). TP53 and KRAS were the most often mutated genes. The frequencies of single nucleotide variation in commonly mutated genes in ctDNA were similar to those detected in tissue samples, TP53 (35.1 vs. 40.4%) and KRAS (20.1 vs. 22.6%). Pathway analysis revealed that mutated genes were mapped to several key pathways including PI3K-Akt, p53, ErbB and Ras signaling pathway. In addition, patients harboring LRP1B, TP53, and ErbB family mutations presented significantly higher tumor mutation burden.Conclusions: These findings demonstrated that ctDNA testing by NGS was feasible in revealing genomic changes and could be a viable alternative to tissue biopsy in patients with metastatic BTC.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging and highly pathogenic coronavirus that causes coronavirus disease (COVID-19), and might even lead to death. Circular RNAs (circRNAs), a new type of RNAs, are implicated in viral pathogenesis and host immune responses. However, their dynamic expression patterns and functions during SARS-CoV-2 infection remain to be unclear. We herein performed genome-wide dynamic analysis of circRNAs in human lung epithelial cells infected with SARS-CoV-2 at four time points. A total of 6118 circRNAs were identified at different genomic locations, including 5641 known and 477 novel circRNAs. Notably, a total of 42 circRNAs were significantly dysregulated, wherein 17 were up-regulated and 25 were down-regulated following infection at multiple phases. The gene ontology and KEGG enrichment analyses revealed that the parental genes of circRNAs were mainly involved in immune and inflammatory responses. Further, the RNA binding protein (RBP) prediction analysis indicated that the dysregulated circRNAs could regulate mRNA stability, immunity, cell death by binding specific proteins. Additionally, the circRNA-miRNA-gene network analysis showed that circRNAs indirectly regulated gene expression by absorbing their targeted miRNAs. Collectively, these results shed light on the roles of circRNAs in virus-host interactions, facilitating future studies on SARS-CoV-2 infection and pathogenesis.

Objective Heat shock factor 1 (HSF1), a transcriptional regulator of heat shock proteins (HSPs), is an attractive therapeutic target for cancer. However, only a few HSF1 inhibitors have been identified so far.Methods The mRNA and protein levels of HSF1, HSPs, cleaved PARP, and phosphorylated HSF1 were examined by real-time PCR and Western blot. Forced expression, RNA interference, and immunofluorescence assay were used for mechanistic studies. Cell viability and apoptosis were measured by WST-8 assay and flow cytometry, respectively. Xenograft studies were performed in nude mice to evaluate the effect of dorsomorphin and an HSP90 inhibitor on tumor growth.Results Dorsomorphin suppressed multiple stimuli-induced and constitutive HSPs expression in cancer cells. Mechanistic studies revealed that dorsomorphin reduced heat-induced HSP expression independent of adenosine monophosphate activated protein kinase. Dorsomorphin reduced heat-stimulated HSF1 Ser320 phosphorylation and nuclear translocation, as well as resting nuclear HSF1 levels in cancer cells. Dorsomorphin induced cancer cell apoptosis by inhibiting HSF1 expression. A structure-activity study revealed that the 4-pyridyl at the 3-site of the pyrazolo [1, 5-a]pyrimidine ring is critical for the anti-HSF1 activities of dorsomorphin. Dorsomorphin sensitized cancer cells to HSP90 and proteasome inhibitors and inhibited HSP70 expression induced by these inhibitors in vitro. In tumor-bearing nude mice, dorsomorphin enhanced HSP90 inhibitor-induced cancer cell apoptosis, tumor growth inhibition, and HSP70 expression.Conclusions Dorsomorphin is an HSF1 inhibitor. It induces cancer cell apoptosis, sensitizes cancer cells to both HSP90 and proteasome inhibitors, and suppresses HSP upregulation by these drugs, which may prevent the development of drug resistance. Hence, dorsomorphin and its derivates may serve as potential precursors for developing drugs against cancer.

Obesity and related inflammation are critical for the pathogenesis of insulin resistance, but the underlying mechanisms are not fully understood. Formyl peptide receptor 2 (FPR2) plays important roles in host immune responses and inflammation-related diseases. We found that Fpr2 expression was elevated in the white adipose tissue of high-fat diet (HFD)–induced obese mice and db/db mice. The systemic deletion of Fpr2 alleviated HFD-induced obesity, insulin resistance, hyperglycemia, hyperlipidemia, and hepatic steatosis. Furthermore, Fpr2 deletion in HFD-fed mice elevated body temperature, reduced fat mass, and inhibited inflammation by reducing macrophage infiltration and M1 polarization in metabolic tissues. Bone marrow transplantations between wild-type and Fpr2−/− mice and myeloid-specific Fpr2 deletion demonstrated that Fpr2-expressing myeloid cells exacerbated HFD-induced obesity, insulin resistance, glucose/lipid metabolic disturbances, and inflammation. Mechanistic studies revealed that Fpr2 deletion in HFD-fed mice enhanced energy expenditure probably through increasing thermogenesis in skeletal muscle; serum amyloid A3 and other factors secreted by adipocytes induced macrophage chemotaxis via Fpr2; and Fpr2 deletion suppressed macrophage chemotaxis and lipopolysaccharide-, palmitate-, and interferon-γ–induced macrophage M1 polarization through blocking their signals. Altogether, our studies demonstrate that myeloid Fpr2 plays critical roles in obesity and related metabolic disorders via regulating muscle energy expenditure, macrophage chemotaxis, and M1 polarization.

Identification of new recombinant HCV strains and positive selection sites are crucially important for the formulation of virus intervention measures. However, little is known about the recombinant variant information and positive selection sites of circulating HCV strains in mainland China. In this study, we systematically identified recombinant variants and positive selection sites of HCV in mainland China during the 2010-2019. Phylogenetic analysis results indicated that HCV-6 was one of the dominant genotypes in mainland China during 2010-2019, whereas genotypes 7 and 8 were not detected. Recombinant analysis based on 102 full-length genome sequences of Chinese epidemic strains of HCV identified four intra-genotypic recombinants (strains WYHCV286, GB28, GZ2983, and HCV156) and one inter-genotypic recombinant (strain HH075). Specifically, two breakpoints in the 5' UTR of two recombinants, the strains HH075 and WYHCV286, are rather unusual and has not been described before. Further, selection pressure analyses revealed five positive selective sites, which were located in the core, E2, and NS5B protein. Notably, positive selective sites in NS5B and core protein may be partially responsible for the drug resistance and immune evasion. To the best of our knowledge, this study firstly reported five specific intertypic and intratypic recombinants of Chinese epidemic strains of HCV, which highlight their significance for anti-HCV treatment and vaccine development.

Surveillance of recombinant enterovirus 71 (EV71) and subgenotype replacement is vital for preventing and controlling hand, foot, and mouth disease (HFMD) outbreaks. Despite this, data on recombinant variants and phylogeny of circulating EV71 strains in mainland China are limited. In this study, recombinant variants of EV71 were identified in mainland China from 2009 to 2018. Phylogenetic analysis indicated that except for individual strains (CQ2014-86/CQ/CHN/2014 and EV71/Xiamen/2009 (B5)), almost all of the EV71 strains in mainland China belonged to the subgenotype C4a. Analysing complete genome sequences of 196 EV71 isolates, 3 intertypic recombination strains (VR1432, 30-2/2015/BJ, and Guangdong-2009) and 5 intratypic recombination strains (EV71/P1034/2013, VR1432, Henan-ZMD/CHN/2012, Hubei-WH/CHN/2012, and EV71/P868/2013/China) were identified among naturally circulating EV71. The breakpoints of these recombinant strains were located within the P1, P2, and P3 encoding regions. Notably, a double recombinant (VR1432) resulting from recombination between EV71 subgenotype C4a and C4b strain SHZH98 and a CA8 strain Donovan was identified. This study reports these specific intertypic and intratypic recombination events for the first time highlighting the importance of genetic recombination in the emergence of new enterovirus variants. Supplementary Information The online version of this article (10.1007/s11262-021-01830-3) contains supplementary material, which is available to authorized users.

Background: Bladder cancer is a common genitourinary malignancy worldwide. Immune checkpoint inhibitors(ICIs) including atezolizumab, pembrolizumab, nivolumab have shown durable responses and have been approved by FDA. However ICIs demonstrate antitumor effects only in a fraction of patients, and research exploring the association between gene mutation and clinical benefit is limited. Mutations of LRP1B(The low-density lipoprotein receptor-related protein 1B) are frequently seen in solid tumors and have been reported to be associated with higher tumor mutation burden(TMB) and prolonged survival in NSCLC, melanoma and liver cancer. The association between LRP1B mutation and survival in bladder cancer is unknown. Methods: The association between LRP1B mutation with TMB was analyzed in bladder cancer patients from the public immunotherapy-treated cohort published by Miao et. al as well as a clinical cohort from 3D Medicines database involving 118 Chinese patients. Wilcoxon test was used for the comparison of TMB. Overall survival(OS) and progression-free survival (PFS) analyses were conducted in the public cohort using Kaplan-Meier curves and log-rank tests. Statistical significance was set at p = 0.05. Results: 15%(5/33) bladder cancer patients in the public immunotherapy-treated cohort and 18.6% (22/118) patients in the clinical cohort harbored LRP1B mutation. LRP1B mutation is associated with higher TMB in both public cohort(p=0.028) and clinical cohort(p=0.003). Survival analysis in the public cohort demonstrated that LRP1B mutation resulted in significantly longer OS ( 16.8 vs 8.08 months; HR, 0.16; p=0.043) and a increasing trend on PFS without significant difference (15.9 vs 4.1 months; HR, 0.62; P = 0.39) in bladder cancer patients treated with ICIs. Conclusions: This study shows that LRP1B mutation is relatively common and correlated with higher TMB in bladder cancer. LRP1B mutation may serve as a predictive biomarker of ICI benefit in bladder cancer. Citation Format: Mengmei Yang, Wenzhuan Xie, Mengli Huang, Peng Wu. Association of LRP1B mutation with tumor mutation burden and survival in bladder cancer patients treated with immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 356.

Background: DNA damage repair (DDR) pathway have attracted much attention because they modulate cancer risk, progression and therapeutic response. DDR deficiency sensitize the tumor to poly ADP-ribose polymerase inhibitors (PARPi). Also alterations in DDR genes are associated with higher tumor mutation burden (TMB) and better response to immune checkpoint inhibitors (ICI). Here we conduct a large-scale genomic analysis to clarify the landscape of DDR gene mutations in hepatocellular carcinoma and the correlation between individual gene mutation and tumor mutation burden. Methods: Tumor samples from 1427 patients with advanced hepatocellular carcinoma were assayed through next-generation sequencing with 381 cancer-related genes including 30 predefined DDR genes: MRE11A, RAD50, BARD1, BLM, BRCA1, BRCA2, BRIP1, PALB2, MLH1, MSH2, MSH6, PMS2, POLD1, ATM, ATR, CHEK1, CHEK2, FANCA, FANCC, FANCD2, FANCG, FANCF, FANCL, RAD51, CUL3, ERCC1, POLE, MUTYH, and PRKDC. TMB (mutations per megabase [mut/Mb]) was calculated based on nonsilent somatic mutations in coding regions. Wilcoxon test was used for the comparisons and statistical significance was set at p = 0.05. Results: Somatic DDR gene alterations were found in 18.8% of the HCC patients. The most common mutational types were copy number variation (30.54%), frameshift (21.89%), and nonsense (20%). The most frequently altered DDR genes are ATM (2.6%) and BRCA2 (2.2%), followed by FANCA (0.9%), PALB2 (0.8%), and MLH1 (0.7%). As to different functional pathways of DDR, the gene alteration frequency from high to low is HRR (6.1%), DS (3.2%), FA (2.5%), MMR (2.2%), BER (0.6%), NHEJ (0.6%), and NER (0.3%). The TMB of patients carrying somatic DDR gene alterations was significantly higher than those without somatic DDR gene alterations (7.26 vs 6.45 mut/MB, p Conclusion: This is the largest study to clarify the landscape of DDR gene mutations in hepatocellular carcinoma. This study confirms that mutations in DDR genes are relatively common and are correlated with higher tumor mutation burden. This also indicates not all DDR genes are equal since mutations of certain individual genes were associated with higher TMB whereas others were not; further studies are needed to confirm this. Citation Format: Mengmei Yang, Wenzhuan Xie, Mengli Huang, Xinhua Zhu. Analysis of DNA damage repair gene alterations and their association with tumor mutation burden in 1427 hepatocellular carcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2040.

e21166 Background: Transforming growth factor beta receptor 2 (TGFBR2) is a transmembrane receptor that plays an important role in both cellular functions and immune reaction, including cell proliferation, cell differentiation and extracellular matrix production. It forms a heterodimeric complex with TGF-beta receptor type-1 and binds TGF-beta. The association between TGFBR2 gene mutations and efficiency of immune checkpoint inhibitors (ICIs) has not been investigated in non-small cell lung cancer (NSCLC). Methods: Two published cohorts of NSCLC patients treat with ICIs were used to explore the immunotherapeutic predictive role of TGFBR2 gene mutations, including a discovery cohort (Gandara et al., Nature Genetics, 2018) and a validation cohort(Samstein et al. Nature Genetics, 2019). The Cancer Genome Altas (TCGA) Pan -Lung Cancer dataset was used for prognostic analysis. Results: TGFBR2 mutations were identified in 1.6% (7/429) and 2.3% (8/344) patients with NSCLC in the discovery and validation cohort, respectively. In the discovery cohort, patients with TGFBR2 mutations had shorter progression-free survival (PFS, median, 1.3 months vs 2.7 months; HR = 2.83; 95% CI, 1.33–6; p = 0.0046) and dramatically shorter overall survival (OS, median, 2.4 months vs 11.1 months; HR = 3.46; 95% CI, 1.63–7.35; p = 0.00058) than those with wild-type TGFBR2. Consistent with this, TGFBR2 mutations were significantly correlated with shorter OS (3 months vs 10 months; HR = 3.46; 95% CI, 1.63–7.35; p = 0.00058) in the validation cohort. In addition, no significant difference was found in OS between TGFBR2-mutant and TGFBR2-wild-type NSCLC patients with standard treatment in TCGA cohort (p = 0.821). Conclusions: TGFBR2 gene mutations are associated with shorter survival in NSCLC patients treated with ICIs, suggesting that TGFBR2 mutations be used as a potential biomarker of predicting the efficiency of immunotherapy and guiding NSCLC systemic treatment.

e16528 Background: Immune checkpoint inhibitors (ICIs) targeting PD-1/L1 have been approved as first-line treatment for cisplatin-ineligible patients and as second-line therapy for patients with metastatic urothelial carcinoma of the bladder. Biomarkers can help select patients who are more likely to response to ICIs. RNF43 is an E3 ubiquitin ligase that acts as a negative regulator of Wnt/β-catenin signaling pathway. In colorectal cancer (CRC) patients treated with immune checkpoint inhibitors (ICIs), RNF43 mutations predicted longer overall survival (OS). The impact of RNF43 mutations on the efficiency of ICIs in bladder cancer(BLC) remains to be explored. Methods: We downloaded the mutation and clinical data of 211 BLC patients treated with ICIs from the immunotherapeutic cohort published by Samstein et al. (2019). OS analyses were conducted using Kaplan-Meier curves and log-rank tests. Wilcoxon test was used for the comparison of TMB. We also downloaded a TCGA cohort for prognostic analysis. The correlations between RNF43 and immune infiltrates were analyzed in the TIMER2.0 database. Statistical significance was set at p = 0.05. Results: RNF43 mutations were identified in 4.3%(9/211) and 3%(13/438) BLC patients in the immunotherapeutic and TCGA cohort, respectively. In the immunotherapeutic cohort, patients with RNF43 mutations had significantly longer OS (25 months vs 8 months; p = 0.015) and higher tumor mutation burden(TMB, 42.3 vs 7.9; p = 3.15E-06) than RNF43-wild-type patients. Different from this, no significant difference was found in OS between RNF43-mutant and RNF43-wild-type BLC patients with standard treatment in the TCGA cohort (p = 0.696). These results indicated that RNF43 was not a prognostic factor but a predictive biomarker of survival in BLC treated with ICIs. No difference was observed in subsets of immune cells between RNF43-mutant and the RNF43-wide-type BLC patients, including neutrophils, macrophages, CD8+ T cells, Tregs, B cells and NK cells. Conclusions: RNF43 mutations may be a predictor of survival benefit from ICIs in bladder cancer and correlated with higher TMB. Further studies in other ICI-treated cohorts are needed to confirm these results.

e21587 Background: Immunotherapies targeting immune checkpoint receptors have become the cornerstone of systemic treatment options for malignant melanoma. The response to these immunotherapies may correlate with driver mutations. MAP2K1/2 genes are mutated in approximately 10% of melanomas, however, the impact of MAP2K1/2 gene alterations on the efficiency of immunotherapy has not been clarified. Methods: Six metastatic melanoma clinical cohorts treated with ICIs were included to investigate the association between clinical efficacy of immunotherapy and MAP2K1/2 mutations. Survival analyses were conducted in cohorts receiving two kinds of ICB agents, namely anti-CTLA-4 or anti-PD-1. RNA expression profiling from these cohorts and from the TCGA melanoma cohort were used to explore the potential mechanism related to immune activation. Results: In an independent anti-CTLA-4-treated cohort (n = 110), we found that MAP2K1/2 mutations are predictive of high objective response rate (17.6% vs 1.3%, p = 0.0185) and long progression-free survival [median OS, 49.2 months vs 8.3 months; hazard ratio (HR) = 0.37; 95% CI, 0.15–0.91; p = 0.0307] and overall survival (median PFS, 19.4 months vs 2.8 months; HR = 0.2; 95% CI, 0.05–0.83; p = 0.0262). This predictive value was further validated in a pooled anti-CTLA-4-treated cohort (n = 235) in terms of overall survival (median OS, 49.3 months vs 22.0 months; HR = 0.44; 95% CI, 0.22–0.91; p = 0.0255). However, no correlation between MAP2K1/2 mutations and overall survival was observed in the anti-PD-1-treated cohort (n = 285). Subgroup Cox regression analysis indicated that MAP2K-mutated patients receive less benefit from the anti-PD-1 monotherapy than from the anti-CTLA-4 treatment (median OS, 27.0 months vs 49.3 months; HR = 3.26; 95% CI, 1.18–9.02; p = 0.0225), which was contrary to the result obtained for the total population. Furthermore, transcriptome profiling analysis revealed that MAP2K-mutated tumors are enriched in CD8+ T cells, B cells, and neutrophil cells and also express high levels of CD33 and IL10, which might be the underlying mechanism for melanoma patients with MAP2K1/2-mutated benefit more from anti-CTLA-4 treatment. Conclusions: We identified mutations in MAP2K1/2 genes as the independent predictive factors for anti-CTLA-4 therapy in melanoma patients and found that anti-CTLA-4 treatment in patient harbouring MAP2K1/2 mutations might be more effective than the anti-PD-1 therapy.

Impairment of the immune system is a developing concern in evaluating the toxicity of cadmium (Cd). In the present study, we investigated if Cd could impair cutaneous wound healing through interfering with inflammation after injury. We found that exposure of mice to CdCl2 through drinking water at doses of 10, 30, and 50 mg/l for 8 weeks significantly impaired cutaneous wound healing. Chronic 30 mg/l CdCl2 treatment elevated murine blood Cd level comparable to that of low dose Cd-exposed humans, had no effect on blood total and differential leukocyte counts, but reduced neutrophil infiltration, chemokines (CXCL1 and CXCL2), and proinflammatory cytokines (TNFα, IL-1β, and IL-6) expression in wounded tissue at early stage after injury. Wounded tissue homogenates from CdCl2-treated mice had lower chemotactic activity for neutrophils than those from untreated mice. Mechanistic studies showed that chronic Cd treatment suppressed ERK1/2 and NF-κB p65 phosphorylation in wounded tissue at early stage after injury. Compared with neutrophils isolated from untreated mice, neutrophils from CdCl2 treated mice and normal neutrophils treated with CdCl2 invitro both had lower chemotactic response, calcium mobilization and ERK1/2 phosphorylation upon chemoattractant stimulation. Collectively, our study indicate that chronic low-dose Cd exposure impaired cutaneous wound healing by reducing neutrophil infiltration through inhibiting chemokine expression and neutrophil chemotactic response, and suppressing proinflammatory cytokine expression. Cd may suppress chemokine and proinflammatory expression through inactivating ERK1/2 and NF-κB, and inhibit neutrophil chemotaxis by attenuating calcium mobilization and ERK1/2 phosphorylation in response to chemoattractants.

Superparamagnetic polymer composite microspheres Fe3O4/P(GMA-AA-MMA) prepared by soap free emulsion polymerization were used to support Schiff base palladium complex. Fourier transform infrared (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM), vibrating sample magnetometer (VSM), X-ray photoelectronic spectrography (XPS) and thermal gravity (TG) studies have been used to characterize the supported magnetic Schiff base catalyst. The magnetic catalyst could provide excellent reactivity in the Suzuki coupling reactions of aryl halides with phenylboronic acids under mild condition. Meanwhile, the supported magnetic catalyst acted as a true heterogeneous catalyst in the Suzuki coupling reactions. Furthermore, the novel catalyst could be conveniently recovered by an external magnet and reused at least seven times without significant loss of its catalytic activity or Pd leaching.

e13663 Background: Cyclin-dependent kinase 12 (CDK12) is a cyclin-dependent kinase that regulates transcription and RNA splicing, thereby modulating multiple cellular processes. It has been suggested that CDK12 loss-of-function mutations lead to a higher neoantigen burden and favorable responses to PD-1 inhibitors in advanced prostate cancer. Given this potentially actionable molecular subtype, we sought to determine the prevalence of CDK12 alterations in Chinese cancer patients and the association with TMB and overall survival(OS). Methods: The prevalence of CDK12 alterations were queried in 3D Medicines database with 15,745 Chinese cancer patients involved. Whole-exome sequencing data of 464 patients with prostate adenocarcinoma(PRAD) from the Cancer Genome Altas (TCGA) were downloaded to explore the association between CDK12 gene alteration and OS. And the association with TMB were analyzed in a cohort of 731 patients with various cancer types published by Memorial Sloan Kettering (MSKCC) (Samstein et al., Nature Genetics, 2019). Results: Any CDK12 and known or likely deleterious CDK12 mutations were identified in 598(3.8%) and 98(0.62%) patients, respectively. Across all cancer types, prostate adenocarcinoma(PRAD) was found to have the highest frequency of deleterious mutations(8.75%, 23/263), followed by breast cancer (4.97%, 25/503). Mutations were also detected in multiple cancer types including bladder cancer, ovarian cancer, lung cancer, colorectal cancer and so on with a frequency of less than 1%. CDK12 mutations were associated with shorter OS (HR = 15.25; 95% CI, 2.88-80.73; p < 0.001) in TCGA PRAD and cholangiocarcinoma datasets, which was not seen in other cancer types. Patients harboring CDK12 mutation had a significant higher TMB(p < 0.001) in the pan-cancer study of publicly-available cohort from MSKCC. Conclusions: CDK12 alterations existed across tumor types in Chinese patients with relatively high frequencies detected in PRAD and breast cancer and represent extremely rare events in multiple cancers. CDK12 mutation was a poor prognostic factor in PRAD and cholangiocarcinoma. In a pan cancer analysis patients with CDK12 mutation tended to have a significant higher TMB and may benefit from PD-1/L1 blockade immunotherapy.

FAM3B, also named PANDER, is a cytokine-like protein identified in 2002. Previous studies showed that FAM3B regulates glucose and lipid metabolism through interaction with liver and endocrine pancreas. FAM3B is also expressed by other tissues but its basic function is unclear. In this study, we found that FAM3B was expressed in mouse colon, intestine, liver and lung tissues and multiple types of cell lines, including murine pancreatic β-cell (Min6), microglia (N9) and muscle cell (C2C12); human colon cancer cells (HCT8, HCT116, HT29), hepatocyte (HL-7702), hepatocellular carcinoma cell (SMMC-7721) and lung carcinoma cell (A549). Inhibition of FAM3B expression by RNA interference induced apoptotic cell death of HCT8, HCT116, A549, N9, C2C12 and Min6 cells and decreased cell viability of HL-7702 and murine primary hepatocytes. Further studies with HCT8 cells showed that knockdown of FAM3B increased the protein levels of membrane-bound Fas and Bax, reduced the expression of Bcl-2, promoted the cleavage of caspases-8, -3, -9 and PARP, and the nuclear translocation of cleaved PARP. These results suggest that FAM3B silencing activates both extrinsic and intrinsic apoptotic pathways. Mechanistic studies showed that neutralizing antibody against Fas or silencing Fas-associated death domain had no effect on, while caspase inhibitors could significantly reverse FAM3B knockdown induced apoptosis, suggesting Fas and death receptor mediated extrinsic apoptotic pathway is not involved in FAM3B silencing induced apoptosis. Further studies showed that p53 was significantly upregulated after FAM3B knockdown. Silencing p53 could almost completely reverse FAM3B knockdown induced upregulation of Bax, downregulation of Bcl-2, cleavage of caspases-8, -9, -3, and apoptotic cell death, suggesting p53-dependent pathway plays critical roles in FAM3B silencing induced apoptosis. Studies with HCT116 cells confirmed that inhibition of FAM3B expression induced apoptosis through p53-dependent pathway. Furthermore, knockdown of FAM3B reduced the protein level of Mdm2 and promoted p53 phosphorylation. Taken together, our studies demonstrated that silencing FAM3B promoted p53 phosphorylation and induced p53 accumulation by decreasing Mdm2 expression, which resulted in apoptotic cell death.

MicroRNAs (miRNAs) are a new class of regulatory molecules implicated in type 2 diabetes, which is characterized by insulin resistance and hepatic glucose overproduction. We show that miRNA-451 (miR-451) is elevated in the liver tissues of dietary and genetic mouse models of diabetes. Through an adenovirus-mediated gain- and loss-of-function study, we found that miR-451 negatively regulates hepatic gluconeogenesis and blood glucose levels in normal mice and identified glycerol kinase (Gyk) as a direct target of miR-451. We demonstrate that miR-451 and Gyk regulate hepatic glucose production, the glycerol gluconeogenesis axis, and the AKT-FOXO1-PEPCK/G6Pase pathway in an opposite manner; Gyk could reverse the effect of miR-451 on hepatic gluconeogenesis and AKT-FOXO1-PEPCK/G6Pase pathway. Moreover, overexpression of miR-451 or knockdown of Gyk in diabetic mice significantly inhibited hepatic gluconeogenesis, alleviated hyperglycemia, and improved glucose tolerance. Further studies showed that miR-451 is upregulated by glucose and insulin in hepatocytes; the elevation of hepatic miR-451 in diabetic mice may contribute to inhibiting Gyk expression. This study provides the first evidence that miR-451 and Gyk regulate the AKT-FOXO1-PEPCK/G6Pase pathway and play critical roles in hepatic gluconeogenesis and glucose homeostasis and identifies miR-451 and Gyk as potential therapeutic targets against hyperglycemia in diabetes.

The C—C coupling reactions catalyzed by supported N-heterocyclic carbene palladium complex have attracted considerable attention because catalytic activity is excellent and supported catalyst can be separated easily and reused several times. The performance of the carriers has an important effect on the catalytic activity and reusability of the supported catalyst. In this paper, the advances in the C—C coupling reactions catalyzed by supported N-eterocyclic carbene palladium complex have been reviewed. Polymer, silica, carbon and magnetic nanoparticles were used to support the N-heterocyclic carbene pal- ladium catalysts. Finally, the direction of the supported N-heterocyclic carbene palladium complex was also put forward.