RelA and mitogen-activated protein kinase kinase kinases potently enhance lentiviral vector production.

The growing demands for gene therapy have encouraged development of safe and efficient lentiviral vector (LV) preparation. While much progress has been made in this field, it remains to be explored how to boost its production from producer cells. This paper reports that transient co-expression of RelA or several mitogen-activated protein kinase kinase kinases (MAP3Ks) with packaging constructs can potently enhance LV production in HEK293T producer cells. Adding in transfection a small amount of effector plasmid is sufficient to achieve 3- to 4-fold enhancement, which can further be augmented by co-expression of IκB kinase 2 or HIV Tat. It is also shown that expression of RelA or MAP3K1 can increase LV production in HEK293T/17SF cells grown in suspension. These results indicate that stimulation of intracellular signaling pathways in producer cells represents a powerful means for enhancing LV production.

Emergence of a High-Risk Klebsiella michiganensis Clone Disseminating Carbapenemase Genes.

Klebsiella michiganensis is emerging as an important human pathogen of concern especially strains with plasmid-mediated carbapenemase genes. The IncX3-bla NDM-5 plasmid has been described as the primary vector for bla NDM-5 dissemination. However, whether strains with this plasmid have any competitive edge remain largely unexplored. We characterized a bla NDM-5-producing Klebsiella michiganensis strain (KO_408) from Japan and sought to understand the driving force behind the recent dissemination of IncX3-blaNDM-5 plasmids in different bacterial hosts. Antibiotic susceptibility testing, conjugation, and whole-genome sequencing were performed for KO_408, a clinical isolate recovered from a respiratory culture. Fitness, stability, and competitive assays were performed using the IncX3-bla NDM-5 plasmid, pKO_4-NDM-5. KO_408 was ascribed to a novel sequence type, ST256, and harbored resistance genes conforming to its MDR phenotype. The bla NDM-5 gene was localized on the ~44.9 kb IncX3 plasmid (pKO_4-NDM-5), which was transferable in the conjugal assay. The acquisition of pKO_4-NDM-5 did not impose any fitness burden and showed high stability in the host cells. However, transformants with pKO_4-NDM-5 were outcompeted by their host cells and transconjugants with the IncX3-bla OXA-181 plasmid. The genetic environment of bla NDM-5 in pKO_4-NDM-5 has been previously described. pKO_4-NDM-5 showed a close phylogenetic distance with seven similar plasmids from China. KO_408 clustered with strains within the KoI phylogroup, which is closely associated with carbapenemase genes. This study highlights the emergence of a high-risk Klebsiella michiganensis clone harboring carbapenemase genes and affirms that the recent spread of IncX3-bla NDM-5 plasmids might be due to their low fitness cost and stability but not their competitive prowess.

Methodology to Detect Biological Particles Using a Biosensing Surface Integrated in Resistive Pulse Sensing.

Resistive pulse sensing (RPS) is an analytical method that can be used to individually count particles from a small sample. RPS simply monitors the physical characteristics of particles, such as size, shape, and charge density, and the integration of RPS with biosensing is an attractive theme to detect biological particles such as virus and bacteria. In this report, a methodology of biosensing on RPS was investigated. Polydopamine (PD), an adhesive component of mussels, was used as the base material to create a sensing surface. PD adheres to most materials, such as noble metals, metal oxides, semiconductors, and polymers; as a result, PD is a versatile intermediate layer for the fabrication of a biosensing surface. As an example of a biological particle, human influenza A virus (H1N1 subtype) was used to monitor translocation of particles through the pore membrane. When virus-specific ligands (6'-sialyllactose) were immobilized on the pore surface, the translocation time of the virus particles was considerably extended. The detailed translocation data suggest that the viral particles were trapped on the sensing surface by specific interactions. In addition, virus translocation processes on different pore surfaces were distinguished using machine learning. The result shows that the simple and versatile PD-based biosensor surface design was effective. This advanced RPS measurement system could be a promising analytical technique.

Simian Immunodeficiency Virus SIVgsn-99CM71 Vpu Employs Different Amino Acids To Antagonize Human and Greater Spot-Nosed Monkey BST-2.

Viral protein U (Vpu) is an accessory protein encoded by human immunodeficiency virus type 1 (HIV-1) and certain simian immunodeficiency virus (SIV) strains. Some of these viruses were reported to use Vpu to overcome restriction by BST-2 of their natural hosts. Our own recent report revealed that Vpu of SIVgsn-99CM71 (SIVgsn71) antagonizes human BST-2 through two AxxxxxxxW motifs (A22W30 and A25W33), whereas antagonizing BST-2 of its natural host, greater spot-nosed monkey (GSN), involved only the A22W30 motif. Here, we show that residues A22, A25, W30, and W33 of SIVgsn71 Vpu are all essential to antagonize human BST-2, whereas a single mutation of either A22 or W30 did not affect the ability to antagonize GSN BST-2. Similar to A18, which is located in the middle of the A14xxxxxxxW22 motif in HIV-1 NL4-3 Vpu and is essential to antagonize human BST-2, A29, located in the middle of the A25W33 motif of SIVgsn71 Vpu was found to be necessary for antagonizing human but not GSN BST-2. Further mutational analyses revealed that residues L21 and K32 of SIVgsn71 Vpu were also essential for antagonizing human BST-2. On the other hand, the ability of SIVgsn71 Vpu to target GSN BST-2 was unaffected by single amino acid substitutions but required multiple mutations to render SIVgsn71 Vpu inactive against GSN BST-2. These results suggest additional requirements for SIVgsn71 Vpu antagonizing human BST-2, implying evolution of the bst-2 gene under strong selective pressure. IMPORTANCE Genes related to survival against life-threating pathogens are important determinants of natural selection in animal evolution. For instance, BST-2, a protein showing broad-spectrum antiviral activity, shows polymorphisms entailing different phenotypes even among primate species, suggesting that the bst-2 gene of primates has been subject to strong selective pressure during evolution. At the same time, viruses readily adapt to these evolutionary changes. Thus, we found that the Vpu of an SIVgsn isolate (SIVgsn-99CM71) can target BST-2 from humans as well as from its natural host, thus potentially facilitating zoonosis. Here, we mapped residues in SIVgsn71 Vpu potentially contributing to cross-species transmission. We found that the requirements for targeting human BST-2 are distinct from and more complex than those for targeting GSN BST-2. Our results suggest that the human bst-2 gene might have evolved to acquire more restrictive phenotype than GSN bst-2 against viral proteins after being derived from their common ancestor.

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.

Determining vector competence of Aedes aegypti from Ghana in transmitting dengue virus serotypes 1 and 2.

BACKGROUND: Dengue virus (DENV) is a mosquito-borne arbovirus transmitted by Aedes mosquitoes, but is not endemic in all areas where this vector is found. For example, the relatively sparse distribution of cases in West Africa is generally attributed to the refractory nature of West African Aedes aegypti (Ae. aegypti) to DENV infection, and particularly the forest-dwelling Ae. aegypti formosus. However, recent studies have shown these mosquitoes to be competent vectors within some West African countries that have suffered outbreaks in the past, such as Senegal. There is however little information on the vector competence of the Ae. aegypti in West African countries such as Ghana with no reported outbreaks. METHODS: This study examined the vector competence of 4 Ae. aegypti colonies from urban, semi-urban, and two rural locations in Ghana in transmitting DENV serotypes 1 and 2, using a single colony from Vietnam as control. Midgut infection and virus dissemination were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR), while the presence and concentration of DENV in the saliva of infectious mosquitoes was determined by the focus forming assay. RESULTS: There were significant differences in the colonies' susceptibility to virus infection, dissemination, and transmission. All examined Ghanaian mosquitoes were refractory to infection by DENV serotype 2, while some colonies exhibited potential to transmit DENV serotype 1. None of the tested colonies were as competent as the control group colony. CONCLUSIONS: These findings give insight into the possible risk of outbreaks, particularly in the urban areas in the south of Ghana, and highlight the need for continuous surveillance to determine the transmission status and outbreak risk. This study also highlights the need to prevent importation of different DENV strains and potential invasion of new highly vector-competent Ae. aegypti strains, particularly around the ports of entry.

Emergence of oxacillinase-181 carbapenemase-producing diarrheagenic Escherichia coli in Ghana.

The emergence and spread of carbapenemase-producing bacteria are serious threats to public health. We characterized two OXA-181-producing Escherichia coli isolates from pediatric patients with diarrhea from Ghana. blaOXA-181 was localized on the self-conjugative IncX3-containing plasmid in the E. coli ST410 isolate, belonging to an emerging lineage, and an IncFIC(FII)-containing plasmid in E. coli ST940. The blaOXA-181-qnrS1 region was found on the IS26 composite transposon, which contained a 366-bp deletion in the region encoding the Rep A protein for the IncX3-containing plasmid. The IncFIC(FII) plasmid was novel and integrated with an approximately 39-kb IncX1 plasmid through conjugal transfer. Both plasmids clustered close to plasmids from Switzerland. To the best of our knowledge, this is the first report describing the presence of an IncX3 plasmid containing blaOXA-181 in strains closely related to the B4/H24RxC clade in Africa, suggesting its emergence and the need to strengthen antimicrobial resistance surveillance.

Virulence Profiles of Diarrheagenic Escherichia coli Isolated from the Western Region of Ghana.

Diarrheagenic Escherichia coli (DEC), an important agent of infectious diarrhea, is constantly evolving, making its periodic monitoring necessary. However, the DEC genotypes in Ghana remain uncharacterized. We focused on characterizing the molecular serotypes, virulence factors, multilocus sequence types, and the phylogenetic relatedness among different DEC pathotypes recovered from stool samples of pediatric patients with symptoms of diarrhea from the Western region of Ghana. We detected all five common DEC pathotypes, with the majority of the isolates being enterotoxigenic E. coli (ETEC) harboring the heat-labile enterotoxin gene. The DEC strains exhibited diverse serotypic identity with novel and previously reported outbreak strains. Sequence types (ST) ST38, ST316, and ST1722 were most prevalent, and clonal complex 10 (CC10) was the most common CC. A close evolutionary distance was observed among most of the isolates. Coli surface antigen 6 was the most prevalent (44%, n = 11) ETEC-specific colonization factor. Nearly all the isolates harbored lpfA, and the frequencies of other virulence genes such as pap and cnf1 were 7.9% and 18.4%, respectively. This study provides insights into the important and novel genotypes circulating in the Western region of Ghana that should be monitored for public health.

Establishment and characterization of a cell line from Ghanaian Aedes aegypti (Diptera: Culicidae) focusing on Aedes-borne flavivirus susceptibility.

Mosquitoes are generally considered one of the most important vectors of arboviruses, with Aedes aegypti regarded as the most important in transmission of yellow fever and dengue viruses. To investigate why there are differences in the incidence of dengue fever and Zika in different geographical areas and an absence of outbreaks in Ghana in spite of an abundance of A. aegypti mosquitoes, we established a continuous cell line from embryonic cells of A. aegypti collected in Ghana and assessed its susceptibility to dengue, yellow fever, and Zika viruses. The new cell line (designated AeAe-GH98), having an adhesive spindle-shaped web-like morphology, was serially subcultured in both VP-12 and Schneider's medium supplemented with 10% heat-inactivated fetal bovine serum. AeAe-GH98 cells were found to have a population doubling time of 1.3 d during exponential growth. The mosquito colony used to establish the cell line was confirmed to have originated from Africa using microsatellite assay. In terms of susceptibility to Aedes-borne flaviviruses, AeAe-GH98 cells were found to have different degrees of susceptibility to yellow fever, Zika, and dengue virus infection and propagation. While susceptibility of AeAe-GH98 cells to yellow fever and Zika viruses was comparable with that of C6/36 cells, susceptibility to dengue virus was significantly lower. This cell line will serve as a useful tool for determining molecular factors influencing virus-vector susceptibility in vitro.

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.

Vpu of a Simian Immunodeficiency Virus Isolated from Greater Spot-Nosed Monkey Antagonizes Human BST-2 via Two AxxxxxxxW Motifs.

BST-2/CD317/tetherin is a host transmembrane protein that potently inhibits human immunodeficiency virus type 1 (HIV-1) virion release by tethering the nascent virions to the plasma membrane. Viral protein U (Vpu) is an accessory protein encoded by HIV-1 as well as by some simian immunodeficiency viruses (SIVs) infecting wild chimpanzees, gorillas, or monkeys (SIVcpz, SIVgor, or SIVgsn/SIVmon/SIVmus, respectively). HIV-1 Vpu directly binds to and downregulates human BST-2. The antagonism is highly species specific because the amino acid sequences of BST-2 are different among animal species. Here, we show that Vpu proteins from several SIVcpz, SIVgsn, SIVmon, or SIVmus isolates fail to antagonize human BST-2. Only Vpu from an SIVgsn isolate (SIVgsn-99CM71 [SIVgsn71]) was able to antagonize human BST-2 as well as BST-2 of its natural host, greater spot-nosed monkey (GSN). This SIVgsn Vpu interacted with human BST-2, downregulated cell surface human BST-2 expression, and facilitated HIV-1 virion release in the presence of human BST-2. While the unique 14AxxxxxxxW22 motif in the transmembrane domain of HIV-1NL4-3Vpu was reported to be important for antagonizing human BST-2, we show here that two AxxxxxxxW motifs (A22W30 and A25W33) exist in SIVgsn71 Vpu. Only the A22W30 motif was needed for SIVgsn71 Vpu to antagonize GSN BST-2, suggesting that the mechanism of this antagonism resembles that of HIV-1NL4-3 Vpu against human BST-2. Interestingly, SIVgsn71 Vpu requires two AxxxxxxxW (A22W30 and A25W33) motifs to antagonize human BST-2, suggesting an as-yet-undefined way that SIVgsn71 Vpu works against human BST-2. These results imply an evolutionary impact of primate BST-2 on lentiviral Vpu.IMPORTANCE Genetic alterations conferring a selective advantage in protecting from life-threating pathogens are maintained during evolution. In fact, the amino acid sequences of BST-2 differ among primate animals and their susceptibility to viral proteins is species specific, suggesting that such genetic diversity has arisen through the evolutionarily controlled balance between the host and pathogens. The M (main) group of HIV-1 is thought to be derived from SIVcpz, which utilizes Nef, but not Vpu, to antagonize chimpanzee BST-2. SIVcpz Nef is, however, unable to antagonize human BST-2, and Vpu was consequently chosen again as an antagonist against human BST-2 in the context of HIV-1. Studies on how Vpu lost and acquired this ability, together with the distinct mechanisms by which SIVgsn71 Vpu binds to and downregulates human or GSN BST-2, may help to explain the evolution of this lentiviral protein as a result of host-pathogen interactions.

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.

CRISPR-mediated activation of endogenous BST-2/tetherin expression inhibits wild-type HIV-1 production.

The CRISPR technology not only can knock out target genes by using the RNA-guided Cas9 nuclease but also can activate their expression when a nuclease-deficient Cas9 (dCas9) is employed. Using the latter function, we here show the effect of the CRISPR-mediated pinpoint activation of endogenous expression of BST-2 (also known as tetherin), a virus restriction factor with a broad antiviral spectrum. Single-guide RNA (sgRNA) sequences targeting the BST-2 promoter were selected by promoter assays. Potential sgRNAs and dCas9 fused to the VP64 transactivation domain, along with an accessory transcriptional activator complex, were introduced into cells by lentiviral transduction. Increased expression of BST-2 mRNA in transduced cells was confirmed by real-time RT-PCR. Cells in which BST-2 expression was highly enhanced showed the effective inhibition of HIV-1 production and replication even in the presence of the viral antagonist Vpu against BST-2. These findings confirm that the physiological stoichiometry between host restriction factors and viral antagonists may determine the outcome of the battle with viruses.

Membrane-associated RING-CH (MARCH) 1 and 2 are MARCH family members that inhibit HIV-1 infection.

Membrane-associated RING-CH 8 (MARCH8) is one of 11 members of the MARCH family of RING finger E3 ubiquitin ligases and down-regulates several membrane proteins (e.g. major histocompatibility complex II [MHC-II], CD86, and transferrin receptor). We recently reported that MARCH8 also targets HIV-1 envelope glycoproteins and acts as an antiviral factor. However, it remains unclear whether other family members might have antiviral functions similar to those of MARCH8. Here we show that MARCH1 and MARCH2 are MARCH family members that reduce virion incorporation of envelope glycoproteins. Infectivity assays revealed that MARCH1 and MARCH2 dose-dependently suppress viral infection. Treatment with type I interferon enhanced endogenous expression levels of MARCH1 and MARCH2 in monocyte-derived macrophages. Expression of these proteins in virus-producing cells decreased the efficiency of viral entry and down-regulated HIV-1 envelope glycoproteins from the cell surface, resulting in reduced incorporation of envelope glycoproteins into virions, as observed in MARCH8 expression. With the demonstration that MARCH1 and MARCH2 are antiviral MARCH family members as presented here, these two proteins join a growing list of host factors that inhibit HIV-1 infection.

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.

Establishment of a quantitative and qualitative analysis and isolation method for tetracyclic iridoids from Morinda lucida Bentham leaves.

A new high performance liquid chromatography (HPLC) method has been established for quantitative and qualitative analysis of three tetracyclic iridoids: ML-2-3 (1), molucidin (2), and ML-F52 (3), which are responsible for anti-trypanosomal and anti-leishmanial activities of Morinda lucida Bentham leaves. Separation of 1-3 from dried 80% aqueous (aq.) ethanol extract was achieved on a reversed-phase cholester column packed with cholesteryl-bonded silica using an acetonitrile-0.1% aq. formic acid mobile phase system. Ultraviolet-visible (UV-VIS) spectroscopy was employed for detection of compounds, and their contents were determined by measuring absorbance at 254 nm. Depending on the above system, several factors potentially affecting the concentration of tetracyclic iridoids were evaluated resulting in several variation on plant organs, seasonality, variation between individual trees, and branch positions within the trees. Moreover, we developed a simple, quick, and effective method for tetracyclic iridoid isolation from M. lucida leaves that consisted of extraction by sonication into 80% aq. ethanol, basic hydrolysis, acid neutralization, liquid-liquid extraction into an organic solvent, and reverse phase open column chromatography. Employing this method, we have succeeded to obtain 1 as a colorless crystal yielding of 0.23%, which was 28 times higher than that of previous isolation method. Setting up methodology in this paper may be important for future in vitro and in vivo studies of tetracyclic iridoids and moreover for their applications in new drug design and development.

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.