Intrinsic activation of ß-catenin signaling by CRISPR/Cas9-mediated exon skipping contributes to immune evasion in hepatocellular carcinoma.

Comprehensive analysis of clinical samples has recently identified molecular and immunological classification of hepatocellular carcinoma (HCC), and the CTNNB1 (ß-catenin)-mutated subtype exhibits distinctive characteristics of immunosuppressive tumor microenvironment. For clarifying the molecular mechanisms, we first established human and mouse HCC cells with exon 3 skipping of ß-catenin, which promoted nuclear translocation and activated the Wnt/ß-catenin signaling pathway, by using newly developed multiplex CRISPR/Cas9-based genome engineering system. Gene set enrichment analysis indicated downregulation of immune-associated gene sets in the HCC cells with activated ß-catenin signaling. Comparative analysis of gene expression profiles between HCC cells harboring wild-type and exon 3 skipping ß-catenin elucidated that the expression levels of four cytokines were commonly decreased in human and mouse ß-catenin-mutated HCC cells. Public exome and transcriptome data of 373 human HCC samples showed significant downregulation of two candidate cytokine genes, CCL20 and CXCL2, in HCC tumors with ß-catenin hotspot mutations. T cell killing assays and immunohistochemical analysis of grafted tumor tissues demonstrated that the mouse Ctnnb1Δex3 HCC cells evaded immunosurveillance. Taken together, this study discovered that cytokine controlled by ß-catenin signaling activation could contribute to immune evasion, and provided novel insights into cancer immunotherapy for the ß-catenin-mutated HCC subtype.

Identification and characterization of Stathmin 1 as a host factor involved in HIV-1 latency.

Latency remains a barrier to achieving a sterilizing cure to HIV infection. It is thus important to find new host factor(s) to better understand maintenance of HIV latency and be exploited to develop new and more efficient latency reversing agents (LRAs). Here we employed RNA interference screening with a latently HIV-1-infected cell-line to identify Stathmin 1 (STMN1) as a host factor required for maintaining HIV-1 latency. Depletion of STMN1 significantly enhanced HIV-1 expression in a STMN1 depletion-dependent manner and forced expression of exogenous STMN1 suppressed it. We further showed that STMN1 depletion increases HIV-1 proviral transcriptional elongation. Moreover, chromatin immunoprecipitation (ChIP)-qPCR assays revealed STMN1 accumulation on/near the HIV-1 5' LTR region compared to other regions on the HIV-1 provirus, suggesting the possible contribution of STMN1 to HIV-1 transcription. These results suggest that STMN1 is required for the maintenance of HIV-1 latency and implicates STMN1 as a novel therapeutic target to eradicate HIV-1.

Effect of low oxygen concentration on activation of inflammation by Helicobacter pylori.

The gastrointestinal tract of the human body is characterized by a highly unique oxygenation profile, where the oxygen concentration decreases toward the lower tract, not found in other organs. The epithelial cells lining the mucosa where Helicobacter pylori resides exist in a relatively low oxygen environment with a partial pressure of oxygen (pO2) below 58 mm Hg. However, the contribution of hypoxia to H. pylori-induced host immune responses remains elusive. In this study, we investigated the inflammasome activation induced by H. pylori under hypoxic, compared with normoxic, conditions. Our results indicated that the activation of caspase-1 and the subsequent secretion of IL-1ß were significantly enhanced in infected macrophages under 1% oxygen, compared with those under a normal 20% oxygen concentration. The proliferation of H. pylori under aerobic conditions was 3-fold higher than under microaerophilic conditions, and the bacterial growth was more dependent on CO2 than on oxygen. Also, we observed that hypoxia-induced cytokine production as well as HIF-1α accumulation were both decreased when murine macrophages were treated with an HIF-1α inhibitor, KC7F2. Furthermore, hypoxia enhanced the phagocytosis of H. pylori in an HIF-1α-dependent manner. IL-1ß production was also affected by the HIF-1α inhibitor in a mouse infection model, suggesting the important role of HIF-1α in the host defense system during infection with H. pylori. Our findings provide new insights into the intersection of low oxygen, H. pylori, and inflammation and disclosed how H. pylori under low oxygen tension can aggravate IL-1ß secretion.

Loss of ARID1A induces a stemness gene ALDH1A1 expression with histone acetylation in the malignant subtype of cholangiocarcinoma.

Genomic analyses have recently discovered the malignant subtype of human intrahepatic cholangiocarcinoma (ICC) characterized by frequent mutations of chromatin remodeling gene ARID1A; however, the biological and molecular functions still remain obscure. We here examined the clinical and biological significances of ARID1A deficiency in human ICC. Immunohistochemical analysis demonstrated that the loss of ARID1A was an independent prognostic factor for overall survival of ICC patients (P = 0.023). We established ARID1A-knockout (KO) cells by using the CRISPR/Cas9 system from two human cholangiocarcinoma cell lines. ARID1A-KO cells exhibited significantly enhanced migration, invasion, and sphere formation activity. Microarray analysis revealed that ALDH1A1, a stemness gene, was the most significantly elevated genes in ARID1A-KO cells. In addition, ALDH enzymatic activity as a hallmark of cancer stem cells was markedly high in the KO cells. ARID1A and histone deacetylase 1 were directly recruited to the ALDH1A1 promoter region in cholangiocarcinoma cells with undetectable ALDH1A1 expression by chromatin immunoprecipitation assay. The histone H3K27 acetylation level at the ALDH1A1 promoter region was increased in cells when ARID1A was disrupted (P < 0.01). Clinically, inverse correlation between ARID1A and ALDH1A1 expression was also identified in primary ICC (P = 0.018), and ARID1A-negative and ALDH1A1-positve ICCs showed worse prognosis than only ARID1A-negative cases (P = 0.002). In conclusion, ARID1A may function as a tumor suppressor in ICC through transcriptional downregulation of ALDH1A1 expression with decreasing histone H3K27 acetylation. Our studies provide the basis for the development of new epigenetic approaches to ARID1A-negative ICC. Immunohistochemical loss of ARID1A is an independent prognostic factor in intrahepatic cholangiocarcinoma patients. ARID1A recruits HDAC1 to the promoter region of ALDH1A1, a stemness gene, and epigenetically suppresses ALDH1A1 expression with decreasing histone H3K27 acetylation in cholangiocarcinoma cells.

Upregulation of cancer-associated gene expression in activated fibroblasts in a mouse model of non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH), characterized by chronic inflammation and fibrosis, is predicted to be the leading cause of cirrhosis and hepatocellular carcinoma (HCC) in the next decade. Although recent evidence suggests the importance of fibrosis as the strongest determinant of HCC development, the molecular mechanisms underlying NASH-induced carcinogenesis still remain unclear. Here we performed RNA sequencing analysis to compare gene expression profiles of activated fibroblasts prepared from two distinct liver fibrosis models: carbon tetrachloride-induced fibrosis as a model without obesity and HCC and genetically obese melanocortin 4 receptor-deficient (MC4R-KO) mice fed Western diet, which develop steatosis, NASH, and eventually HCC. Our data showed that activated fibroblasts exhibited distinct gene expression patterns in each etiology, and that the 'pathways in cancer' were selectively upregulated in the activated fibroblasts from MC4R-KO mice. The most upregulated gene in these pathways was fibroblast growth factor 9 (FGF9), which was induced by metabolic stress such as palmitate. FGF9 exerted anti-apoptotic and pro-migratory effects in fibroblasts and hepatoma cells in vitro and accelerated tumor growth in a subcutaneous xenograft model. This study reveals upregulation of cancer-associated gene expression in activated fibroblasts in NASH, which would contribute to the progression from NASH to HCC.

PATZ1 is required for efficient HIV-1 infection.

Successful HIV-1 infection and subsequent replication deeply depend on how the virus usurps the host cell machinery. Identification and functional characterization of these host factors may represent a critical strategy for developing novel anti-HIV-1 therapy. Here, expression cloning with a cDNA expression library identified as an inhibitor of HIV-1 infection, a carboxy-terminally truncated form of human POZ/BTB and AT-hook- containing Zinc finger protein 1 (PATZ1), a transcriptional regulatory factor implicated in development and cancer. Knockdown or knockout of endogenous PATZ1 revealed a supportive role of PATZ1 in HIV-1 infection, but not in transduction with murine leukemia virus-based retroviral vector. More specifically, knockdown or knockout of PATZ1 impaired the viral cDNA synthesis but not the entry process and expression of two PATZ1 isoforms in PATZ1-KO cells restored susceptibility to HIV-1 infection. These results indicate that PATZ1 plays an important role in HIV-1 infection.

Asymmetric dimethylation at histone H3 arginine 2 by PRMT6 in gastric cancer progression.

Histone modification plays important molecular roles in development and progression of cancers. Dysregulation of histone H3 arginine (R) methylation is still unknown in primary cancer, including gastric cancer (GC). Although PRMT6 contributes to asymmetric dimethylation at H3R2 (H3R2me2as) in cancer cells, its molecular functions are poorly understood in GC. In this study, we assessed H3R2me2as and PRMT6 expression levels in 133 primary GC tissues by immunohistochemistry. Increased H3R2me2as was found in 68 GC (51.1%) cases and independently related to poor prognosis. PRMT6 was overexpressed in 70 GC (52.6%) and strongly correlated with the global H3R2me2as levels (P < 0.001). By analyzing biological functions of PRMT6 in GC cell lines by lentivirus-based systems, PRMT6 overexpression enhanced global H3R2me2as and invasiveness in vitro, while PRMT6 knockout (PRMT6-KO) suppressed these effects and tumorigenicity in vivo. ChIP and microarray assays demonstrated that PRMT6-KO GC cells decreased the enrichments of H3R2me2as at the promoter regions of PCDH7, SCD and IGFBP5, resulting in upregulation of their gene expression. PRMT6 was recruited to the promoter regions of PCDH7 and SCD in the PRMT6-overexpressed cells. Knockdown of tumor suppressor PCDH7 in the PRMT6-KO GC cells elevated cell migration and invasion. PRMT6 expression inversely correlated with PCDH7 expression in primary GC (P = 0.021). Collectively, our findings strongly indicate that H3R2me2as is a strong prognostic indicator of GC patients, and PRMT6-overexpressing GC cells may acquire invasiveness through direct transcriptional inhibition of PCDH7 by increasing H3R2me2as level. Thus, inhibition of the PRMT6-H3R2me2as pathway could be a promising new therapeutic strategy in GC.

Loss of KDM6A characterizes a poor prognostic subtype of human pancreatic cancer and potentiates HDAC inhibitor lethality.

Although genomic analysis have recently discovered the malignant subtype of human pancreatic ductal adenocarcinoma (PDAC) characterized by frequent mutations of histone demethylase KDM6A, the biological and molecular roles still remain obscure. We herein elucidated the clinical and biological impacts of KDM6A deficiency on human PDAC and identified the therapeutic potential by pathological and molecular evaluation. Immunohistochemical analysis suggested that loss of KDM6A in cancerous tissues was an independent prognostic factor for both recurrence-free and overall survival in the 103 tumor specimens surgically resected from patients with PDAC. We established KDM6A knocked out cells by using the CRISPR/Cas9 system and KDM6A-expressed cells by doxycycline-inducible system from each two human PDAC cell lines, respectively. KDM6A knockout enhanced aggressive traits of human PDAC cell lines, whereas KDM6A overexpression suppressed them. Microarray analysis revealed reduced expression of 22 genes including five well-known tumor suppressors, such as CDKN1A, and ChIP-PCR analysis displayed depleted enrichment of histone H3 lysine 27 acetylation (H3K27ac) at the promoter regions of the five candidates. The epigenetic alterations were induced by the impaired recruitment of histone acetyltransferase p300, which cooperatively interacted with KDM6A. Consistent with these results, the KDM6A knockout cells demonstrated higher vulnerability to histone deacetylase (HDAC) inhibitors through the reactivation of CDKN1A in vitro and in vivo than the KDM6A wild-type. In conclusion, KDM6A exhibited essential roles in human PDAC as a tumor suppressor and KDM6A deficiency could be a promising biomarker for unfavorable outcome in PDAC patients and a potential surrogate marker for response to HDAC inhibitors.

Robust Enhancement of Lentivirus Production by Promoter Activation.

Lentiviral vectors are a valuable tool to deliver exogenous genes for stable expression in cells. While much progress has been made in processing lentiviral vector-containing culture medium, it remains to be explored how the production of lentiviral vector from producer cells can be increased. We initially found that co-expression of the SPRY domain-containing SOCS box protein 1 (SPSB1) promotes the production of human immunodeficiency virus type 1 (HIV-1) and lentiviral vector with increased expression of the Gag and envelope proteins and activation of the HIV-1 LTR and CMV promoter. The presence of AP-1, NF-κB and CREB/ATF recognition sites in these promoters prompted us to utilize human T-lymphotropic virus type 1 (HTLV-1) Tax for lentiviral vector production because Tax activates all these transcription factors. Co-expression of a small amount of Tax markedly increased both the expression of viral structural proteins in producer cells and release of lentiviral vector particles, resulting in a more than 10-fold enhancement of transduction efficiency. Of note, the Tax protein was not detected in the lentiviral vector particles concentrated by ultracentrifugation, supporting the safety of this preparation. Collectively, these results indicate that promoter activation in producer cells represents a promising approach to preparing high-titer lentiviral vectors.

Fatty Acid Binding Protein 4 (FABP4) Overexpression in Intratumoral Hepatic Stellate Cells within Hepatocellular Carcinoma with Metabolic Risk Factors.

Metabolic syndrome is a newly identified risk factor for hepatocellular carcinoma (HCC); however, tumor-specific biomarkers still remain unclear. We performed cross-species analysis to compare gene signatures of HCC from human patients and melanocortin 4 receptor-knockout mice, which develop HCC with obesity, insulin resistance, and dyslipidemia. Unsupervised hierarchical clustering and principle component analysis of 746 differentially expressed orthologous genes classified HCC of 152 human patients and melanocortin 4 receptor-knockout mice into two distinct subgroups, one of which included mouse HCC and was causatively associated with metabolic risk factors. Nine genes commonly overexpressed in human and mouse metabolic disease-associated HCC were identified; fatty acid binding protein 4 (FABP4) was remarkably enriched in intratumoral activated hepatic stellate cells (HSCs). Subclones constitutively expressing FABP4 were established from a human HSC cell line in which expression levels of inflammatory chemokines, including IL-1A and IL-6, were up-regulated through NF-κB nuclear translocation, resulting in recruitment of macrophages. An immunohistochemical validation study of 106 additional human HCC samples indicated that FABP4-positive HSCs were distributed in tumors of 38 cases, and the FABP4-high group consisted of patients with nonviral and nonalcoholic HCC (P = 0.027) and with multiple metabolic risk factors (P < 0.001) compared with the FABP4-low group. Thus, FABP4 overexpression in HSCs may contribute to hepatocarcinogenesis in patients with metabolic risk factors by modulation of inflammatory pathways.

DEPDC5 deficiency contributes to resistance to leucine starvation via p62 accumulation in hepatocellular carcinoma.

Decrease in blood concentration of branched-chain amino acids, especially leucine, is known to promote liver carcinogenesis in patients with chronic liver disease, but the mechanism is unclear. We herein established hepatocellular carcinoma (HCC) cells knocked out for DEPDC5 by using the CRISPR/Cas9 system, and elucidated that cell viability of the DEPDC5 knockout (DEPDC5-KO) cells was higher than that of the DEPDC5 wild-type (DEPDC5-WT) under leucine starvation. Considering that autophagy deficiency might be involved in acquired resistance to leucine deprivation, we observed reduction of LC3-II followed by accumulation of p62 in the DEPDC5-KO, which induced reactive oxygen species (ROS) tolerance. DEPDC5 overexpression suppressed cell proliferation and tumorigenicity in immunocompromised mice, and triggered p62 degradation with increased ROS susceptibility. In clinical specimens of HCC patients, decreased expression of DEPDC5 was positively correlated with p62 overexpression, and the progression-free (PFS) and overall survival (OS) were worse in the DEPDC5-negative cases than in the DEPDC5-positive. Moreover, multivariate analysis demonstrated DEPDC5 was an independent prognostic factor for both PFS and OS. Thus, DEPDC5 inactivation enhanced ROS resistance in HCC under the leucine-depleted conditions of chronic liver disease, contributing to poor patient outcome. It could be a potential target for cancer therapy with oxidative stress control.

A20 restores phorbol ester-induced differentiation of THP-1 cells in the absence of nuclear factor-κB activation.

A20, also referred to as tumor necrosis factor alpha (TNFα)-induced protein 3 (TNFAIP3), is an ubiquitin-editing enzyme whose expression is enhanced by NF-κB activation, and plays an important role in silencing NF-κB activity. Another well-known role for A20 is to protect cells from TNFα-induced apoptosis. Depletion of NF-κB in differentiating U937 monocytic leukemia cells is known to cause apoptotic cell death; however, much remains to be explored about the molecules that are expressed in an NF-κB-dependent manner and which support monocyte-macrophage differentiation. Using the monocytic cell line THP-1, and peripheral blood monocytes, we show here a sustained increase in A20 expression during monocyte-macrophage differentiation, which coincided with high NF-κB-dependent transcriptional activity. Depletion of NF-κB by stable expression of a super-repressor form of IκBα in THP-1 cells caused remarkable cell death during phorbol 12-myristate 13-acetate (PMA)-induced differentiation. A20 expression in these cells did not alter this NF-κB suppression, but was sufficient to protect the cells and restore the cell surface expression of a differentiation marker (CD11b) and phagocytic activity. Mutational analyses revealed that this A20 activity requires the carboxy-terminal zinc-finger domain, but not its deubiquitinase activity. Based on these findings, we conclude that A20, when ectopically expressed, can support both survival and differentiation of THP-1 cells in the absence of sustained NF-κB activity.

Overexpression of exchange protein directly activated by cAMP-1 (EPAC1) attenuates bladder cancer cell migration.

Exchange protein directly activated by cAMP (EPAC) is a mediator of a cAMP signaling pathway that is independent of protein kinase A. EPAC has two isoforms (EPAC1 and EPAC2) and is a cAMP-dependent guanine nucleotide exchange factor for the small GTPases, Rap1 and Rap2. Recent studies suggest that EPAC1 has both positive and negative influences on cancer and is involved in cell proliferation, apoptosis, migration and metastasis. We report that EPAC1 and EPAC2 expression levels were significantly lower in bladder cancer tissue than in normal bladder tissue. In addition, bladder cancer cell lines showed reduced EPAC1 mRNA expression. Furthermore, EPAC1 overexpression in bladder cancer cell lines induced morphologic changes and markedly suppressed cell migration without affecting cell viability. The overexpressed EPAC1 preferentially localized at cell-cell interfaces. In conclusion, reduced EPAC1 expression in bladder tumors and poor migration of EPAC1-overexpressing cells implicate EPAC1 as an inhibitor of bladder cancer cell migration.

N-terminally truncated POM121C inhibits HIV-1 replication.

Recent studies have identified host cell factors that regulate early stages of HIV-1 infection including viral cDNA synthesis and orientation of the HIV-1 capsid (CA) core toward the nuclear envelope, but it remains unclear how viral DNA is imported through the nuclear pore and guided to the host chromosomal DNA. Here, we demonstrate that N-terminally truncated POM121C, a component of the nuclear pore complex, blocks HIV-1 infection. This truncated protein is predominantly localized in the cytoplasm, does not bind to CA, does not affect viral cDNA synthesis, reduces the formation of 2-LTR and diminished the amount of integrated proviral DNA. Studies with an HIV-1-murine leukemia virus (MLV) chimeric virus carrying the MLV-derived Gag revealed that Gag is a determinant of this inhibition. Intriguingly, mutational studies have revealed that the blockade by N-terminally-truncated POM121C is closely linked to its binding to importin-ß/karyopherin subunit beta 1 (KPNB1). These results indicate that N-terminally-truncated POM121C inhibits HIV-1 infection after completion of reverse transcription and before integration, and suggest an important role for KPNB1 in HIV-1 replication.

Human SMOOTHENED inhibits human immunodeficiency virus type 1 infection.

Human SMOOTHENED (SMO) was identified by expression cloning as a new host factor that inhibits HIV-1 infection. Forced expression of SMO inhibited HIV-1 replication and infection with a single-round lentiviral vector, but not infection with a murine leukemia virus-based retroviral vector in human MT-4 T cells. Quantitative PCR analyses revealed that stable expression of SMO impaired formation of the integrated form of lentiviral DNA, but did not interrupt reverse transcription. This inhibition was evident in MT-4 and HUT102 human T cell lines expressing low levels of SMO mRNA, but not in SupT1 or Jurkat T cell lines expressing higher levels of SMO mRNA. Depletion of SMO mRNA in Jurkat cells facilitated HIV-1 vector infection, suggesting that endogenous SMO plays a role in limiting lentiviral infection. These results suggest that SMO inhibits HIV-1 replication after completion of reverse transcription but before integration.

Correction: EBV induces persistent NF-κB activation and contributes to survival of EBV-positive neoplastic T- or NK-cells.

[This corrects the article DOI: 10.1371/journal.pone.0174136.].

EBV induces persistent NF-κB activation and contributes to survival of EBV-positive neoplastic T- or NK-cells.

Epstein-Barr virus (EBV) has been detected in several T- and NK-cell neoplasms such as extranodal NK/T-cell lymphoma nasal type, aggressive NK-cell leukemia, EBV-positive peripheral T-cell lymphoma, systemic EBV-positive T-cell lymphoma of childhood, and chronic active EBV infection (CAEBV). However, how this virus contributes to lymphomagenesis in T or NK cells remains largely unknown. Here, we examined NF-κB activation in EBV-positive T or NK cell lines, SNT8, SNT15, SNT16, SNK6, and primary EBV-positive and clonally proliferating T/NK cells obtained from the peripheral blood of patients with CAEBV. Western blotting, electrophoretic mobility shift assays, and immunofluorescent staining revealed persistent NF-κB activation in EBV-infected cell lines and primary cells from patients. Furthermore, we investigated the role of EBV in infected T cells. We performed an in vitro infection assay using MOLT4 cells infected with EBV. The infection directly induced NF-κB activation, promoted survival, and inhibited etoposide-induced apoptosis in MOLT4 cells. The luciferase assay suggested that LMP1 mediated NF-κB activation in MOLT4 cells. IMD-0354, a specific inhibitor of NF-κB that suppresses NF-κB activation in cell lines, inhibited cell survival and induced apoptosis. These results indicate that EBV induces NF-κB-mediated survival signals in T and NK cells, and therefore, may contribute to the lymphomagenesis of these cells.

Dominant Expression of DCLK1 in Human Pancreatic Cancer Stem Cells Accelerates Tumor Invasion and Metastasis.

Patients with pancreatic cancer typically develop tumor invasion and metastasis in the early stage. These malignant behaviors might be originated from cancer stem cells (CSCs), but the responsible target is less known about invisible CSCs especially for invasion and metastasis. We previously examined the proteasome activity of CSCs and constructed a real-time visualization system for human pancreatic CSCs. In the present study, we found that CSCs were highly metastatic and dominantly localized at the invading tumor margins in a liver metastasis model. Microarray and siRNA screening assays showed that doublecortin-like kinase 1 (DCLK1) was predominantly expressed with histone modification in pancreatic CSCs with invasive and metastatic potential. Overexpression of DCLK1 led to amoeboid morphology, which promotes the migration of pancreatic cancer cells. Knockdown of DCLK1 profoundly suppressed in vivo liver metastasis of pancreatic CSCs. Clinically, DCLK1 was overexpressed in the metastatic tumors in patients with pancreatic cancer. Our studies revealed that DCLK1 is essential for the invasive and metastatic properties of CSCs and may be a promising epigenetic and therapeutic target in human pancreatic cancer.

MARCH8 inhibits HIV-1 infection by reducing virion incorporation of envelope glycoproteins.

Membrane-associated RING-CH 8 (MARCH8) is one of 11 members of the recently discovered MARCH family of RING (really interesting new gene)-finger E3 ubiquitin ligases. MARCH8 downregulates several host transmembrane proteins, including major histocompatibility complex (MHC)-II, CD86, interleukin (IL)-1 receptor accessory protein, TNF-related apoptosis-inducing ligand (TRAIL) receptor 1 and the transferrin receptor. However, its physiological roles remain largely unknown. Here we identify MARCH8 as a novel antiviral factor. The ectopic expression of MARCH8 in virus-producing cells does not affect levels of lentivirus production, but it does markedly reduce viral infectivity. MARCH8 blocks the incorporation of HIV-1 envelope glycoprotein into virus particles by downregulating it from the cell surface, probably through their interaction, resulting in a substantial reduction in the efficiency of viral entry. The inhibitory effect of MARCH8 on vesicular stomatitis virus G-glycoprotein is even more remarkable, suggesting a broad-spectrum inhibition of enveloped viruses by MARCH8. Notably, the endogenous expression of MARCH8 is high in monocyte-derived macrophages and dendritic cells, and MARCH8 knockdown or knockout in macrophages significantly increases the infectivity of virions produced by these cells. Our findings thus indicate that MARCH8 is highly expressed in terminally differentiated myeloid cells, and that it is a potent antiviral protein that targets viral envelope glycoproteins and reduces their incorporation into virions.

New anti-trypanosomal active tetracyclic iridoid isolated from Morinda lucida Benth.

Human African trypanosomiasis (HAT), commonly known as sleeping sickness has remained a serious health problem in many African countries with thousands of new infected cases annually. Chemotherapy, which is the main form of control against HAT has been characterized lately by the viewpoints of toxicity and drug resistance issues. Recently, there have been a lot of emphases on the use of medicinal plants world-wide. Morinda lucida Benth. is one of the most popular medicinal plants widely distributed in Africa and several groups have reported on its anti-protozoa activities. In this study, we have isolated one novel tetracyclic iridoid, named as molucidin, from the CHCl3 fraction of the M. lucida leaves by bioassay-guided fractionation and purification. Molucidin was structurally elucidated by (1)H and (13)C NMR including HMQC, HMBC, H-H COSY and NOESY resulting in tetracyclic iridoid skeleton, and its absolute configuration was determined. We have further demonstrated that molucidin presented a strong anti-trypanosomal activity, indicating an IC50 value of 1.27 µM. The cytotoxicity study using human normal and cancer cell lines indicated that molucidin exhibited selectivity index (SI) against two normal fibroblasts greater than 4.73. Furthermore, structure-activity relationship (SAR) study was undertaken with molucidin and oregonin, which is identical to anti-trypanosomal active components of Alnus japonica. Overlapping analysis of the lowest energy conformation of molucidin with oregonin suggested a certain similarities of aromatic rings of both oregonin and molucidin. These results contribute to the future drug design studies for HAT.