The 2009 pandemic H1N1 influenza A virus spread quickly worldwide in 2009. Since most of the fatal cases were reported in developing countries, rapid and accurate diagnosis methods that are usable in poorly equipped laboratories are necessary. In this study, a mobile detection system for the 2009 H1N1 influenza A virus was developed using a reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) kit with a disposable pocket-warmer as a heating device (designated as pwRT-LAMP). The pwRT-LAMP can detect as few as 100 copies of the virus--which is nearly as sensitive as real-time reverse-transcription polymerase chain reaction (RT-PCR)--and does not cross-react with RNA of seasonal influenza viruses. To evaluate the usefulness of the pwRT-LAMP system, nasal swab samples were collected from 56 patients with flu-like symptoms and were tested. Real-time RT-PCR confirmed that the 2009 H1N1 influenza A virus was present in 27 of the 56 samples. Of these 27 positive samples, QuickVue Influenza A+B immunochromatography detected the virus in only 11 samples (11/27; 40.7%), whereas the pwRT-LAMP system detected the virus in 26 of the 56 samples (26/27 of the positive samples; 96.3%). These findings indicate that the mobile pwRT-LAMP system is an accurate diagnostic system for the 2009 H1N1 influenza A virus, and has great potential utility in diagnosing future influenza pandemics.

Red yeast, Xanthophyllomyces dendrorhous (Phaffia rhodozyma) is the only yeast known to produce astaxanthin, an anti-oxidant isoprenoid (carotenoid) that is widely used in the aquaculture, food, pharmaceutical and cosmetic industries. Recently, the potential of this microorganism as a platform cell factory for isoprenoid production has been recognized because of high flux through its native terpene pathway. Addition of mevalonate, the common precursor for isoprenoid biosynthesis, has been shown to be critical to enhance the astaxanthin content in X. dendrorhous. However, addition of mevalonate is unrealistic during industrial isoprenoid production because it is an unstable and costly chemical. Therefore, up-regulating the intracellular mevalonate supply by enhancing the mevalonate synthetic pathway though genetic engineering is a promising strategy to improve isoprenoid production in X. dendrorhous. However, a system to strongly express multiple genes has been poorly developed for X. dendrorhous.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignancy with poor prognosis and its incidence is increasing worldwide. Recently, several types of cells have been considered as the origin of ICC, namely cholangiocytes, liver progenitor cells, and hepatocytes. Here, we have established a novel mouse model of ICC by liver-specific Kras activation and Pten deletion. An activating mutation of Kras in combination with deletion of Pten was introduced in embryonic hepatic bipotential progenitor cells (so-called hepatoblasts) and mature hepatocytes using the Cre-loxP system. As a result, liver-specific Kras activation and homozygous Pten deletion cooperated to induce ICCs exclusively. In contrast, Kras activation in combination with heterozygous Pten deletion induced both ICCs and HCCs, whereas Kras activation alone resulted in HCCs but not ICCs. Furthermore, a cell-lineage visualization system using tamoxifen-inducible Cre-loxP demonstrated that the ICCs did not originate from hepatocytes but from cholangiocytes. Our data suggest that mice carrying liver-specific Kras activation in combination with homozygous Pten deletion should be useful for the investigation of therapeutic strategies for human ICC.

The aberrant expression of squamous lineage markers in pancreatic ductal adenocarcinoma (PDA) has been correlated with poor clinical outcomes. However, the functional role of this putative transdifferentiation event in PDA pathogenesis remains unclear. Here, we show that expression of the transcription factor TP63 (ΔNp63) is sufficient to install and sustain the enhancer landscape and transcriptional signature of the squamous lineage in human PDA cells. We also demonstrate that TP63-driven enhancer reprogramming promotes aggressive tumor phenotypes, including enhanced cell motility and invasion, and an accelerated growth of primary PDA tumors and metastases in vivo. This process ultimately leads to a powerful addiction of squamous PDA cells to continuous TP63 expression. Our study demonstrates the functional significance of squamous transdifferentiation in PDA and reveals TP63-based reprogramming as an experimental tool for investigating mechanisms and vulnerabilities linked to this aberrant cell fate transition.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by dense stromal reaction (desmoplasia). We have previously reported that mice with conditional KrasG12D mutation and knockout of TGF-β receptor type II (Tgfbr2), PKF mice, develop PDAC with desmoplasia modulated by CXC chemokines that are produced by PDAC cells through tumor-stromal interaction. In this study, we further discovered that PDAC and cancer-associated fibroblast (CAF) accelerated each other's invasion and migration through the CXC chemokines-receptor (CXCLs-CXCR2) axis. Heterozygous knockout of Cxcr2 in PKF mice (PKF2h mice) prolonged survival and inhibited both tumor angiogenesis and PDAC microinvasion. Infiltration of neutrophils, myeloid-derived suppressor cells (MDSCs), and arginase-1+ M2-like tumor-associated macrophages (TAMs) significantly decreased in the tumors of PKF2h mice, whereas inducible nitric oxide synthase (iNOS)+ M1-like TAMs and apoptotic tumor cells markedly increased, which indicated that blockade of the CXCLs-CXCR2 axis resulted in a shift of immune-inflammatory microenvironment. These results suggest that blocking of the CXCLs-CXCR2 axis in tumor-stromal interactions could be a therapeutic approach against PDAC progression.

The APOBEC3 deaminases are potent inhibitors of virus replication and barriers to cross-species transmission. For simian immunodeficiency virus (SIV) to transmit to a new primate host, as happened multiple times to seed the ongoing HIV-1 epidemic, the viral infectivity factor (Vif) must be capable of neutralizing the APOBEC3 enzymes of the new host. Although much is known about current interactions of HIV-1 Vif and human APOBEC3s, the evolutionary changes in SIV Vif required for transmission from chimpanzees to gorillas and ultimately to humans are poorly understood. Here, we demonstrate that gorilla APOBEC3G is a factor with the potential to hamper SIV transmission from chimpanzees to gorillas. Gain-of-function experiments using SIVcpzPtt Vif revealed that this barrier could be overcome by a single Vif acidic amino acid substitution (M16E). Moreover, degradation of gorilla APOBEC3F is induced by Vif through a mechanism that is distinct from that of human APOBEC3F. Thus, our findings identify virus adaptations in gorillas that preceded and may have facilitated transmission to humans.

Peribiliary glands (PBGs), clusters of epithelial cells residing in the submucosal compartment of extrahepatic bile ducts, have been suggested as biliary epithelial stem/progenitor cell niche; however, evidence to support this claim is limited because of a lack of PBG-specific markers. We therefore sought to identify PBG-specific markers to investigate the potential role of PBGs as stem/progenitor cell niches, as well as an origin of cancer.

Pancreatic ductal adenocarcinoma (PDA) is a deadly cancer characterized by complex metabolic adaptations that promote survival in a severely hypoxic and nutrient-limited tumor microenvironment (TME). Modeling microenvironmental influences in cell culture has been challenging, and technical limitations have hampered the comprehensive study of tumor-specific metabolism in vivo. To systematically interrogate metabolic vulnerabilities in PDA, we employed parallel CRISPR-Cas9 screens using in vivo and in vitro systems. This work revealed striking overlap of in vivo metabolic dependencies with those in vitro. Moreover, we identified that intercellular nutrient sharing can mask dependencies in pooled screens, highlighting a limitation of this approach to study tumor metabolism. Furthermore, metabolic dependencies were similar between 2D and 3D culture, although 3D culture may better model vulnerabilities that influence certain oncogenic signaling pathways. Lastly, our work demonstrates the power of genetic screening approaches to define in vivo metabolic dependencies and pathways that may have therapeutic utility.

Immune evasion is a major obstacle for cancer treatment. Common mechanisms of evasion include impaired antigen presentation caused by mutations or loss of heterozygosity of the major histocompatibility complex class I (MHC-I), which has been implicated in resistance to immune checkpoint blockade (ICB) therapy1-3. However, in pancreatic ductal adenocarcinoma (PDAC), which is resistant to most therapies including ICB4, mutations that cause loss of MHC-I are rarely found5 despite the frequent downregulation of MHC-I expression6-8. Here we show that, in PDAC, MHC-I molecules are selectively targeted for lysosomal degradation by an autophagy-dependent mechanism that involves the autophagy cargo receptor NBR1. PDAC cells display reduced expression of MHC-I at the cell surface and instead demonstrate predominant localization within autophagosomes and lysosomes. Notably, inhibition of autophagy restores surface levels of MHC-I and leads to improved antigen presentation, enhanced anti-tumour T cell responses and reduced tumour growth in syngeneic host mice. Accordingly, the anti-tumour effects of autophagy inhibition are reversed by depleting CD8+ T cells or reducing surface expression of MHC-I. Inhibition of autophagy, either genetically or pharmacologically with chloroquine, synergizes with dual ICB therapy (anti-PD1 and anti-CTLA4 antibodies), and leads to an enhanced anti-tumour immune response. Our findings demonstrate a role for enhanced autophagy or lysosome function in immune evasion by selective targeting of MHC-I molecules for degradation, and provide a rationale for the combination of autophagy inhibition and dual ICB therapy as a therapeutic strategy against PDAC.

As the hosts of lentiviruses, almost 40 species of felids (family Felidae) are distributed around the world, and more than 20 feline species test positive for feline immunodeficiency virus (FIV), a lineage of lentiviruses. These observations suggest that FIVs globally infected a variety of feline species through multiple cross-species transmission events during a million-year history. Cellular restriction factors potentially inhibit lentiviral replication and limit cross-species lentiviral transmission, and cellular APOBEC3 deaminases are known as a potent restriction factor. In contrast, lentiviruses have evolutionary-acquired viral infectivity factor (Vif) to neutralize the APOBEC3-mediated antiviral effect. Because the APOBEC3-Vif interaction is strictly specific for viruses and their hosts, a comprehensive investigation focusing on Vif-APOBEC3 interplay can provide clues that will elucidate the roles of this virus-host interplay on cross-species transmission of lentiviruses. Here, we performed a comprehensive investigation with 144 patterns of a round robin test using 18 feline APOBEC3Z3 genes, an antiviral APOBEC3 gene in felid, and 8 FIV Vifs and derived a matrix showing the interplay between feline APOBEC3Z3 and FIV Vif. We particularly focused on the interplay between the APOBEC3Z3 of three felids (domestic cat, ocelot, and Asian golden cat) and an FIV Vif (strain Petaluma), and revealed that residues 65 and 66 of the APOBEC3Z3 protein of multiple felids are responsible for the counteraction triggered by FIV Petaluma Vif. Altogether, our findings can be a clue to elucidate not only the scenarios of the cross-species transmissions of FIVs in felids but also the evolutionary interaction between mammals and lentiviruses. IMPORTANCE Most of the emergences of new virus infections originate from the cross-species transmission of viruses. The fact that some virus infections are strictly specific for the host species indicates that certain "species barriers" in the hosts restrict cross-species jump of viruses, while viruses have evolutionary acquired their own "arms" to overcome/antagonize/neutralize these hurdles. Therefore, understanding of the molecular mechanism leading to successful cross-species viral transmission is crucial for considering the menus of the emergence of novel pathogenic viruses. In the field of retrovirology, APOBEC3-Vif interaction is a well-studied example of the battles between hosts and viruses. Here, we determined the sequences of 11 novel feline APOBEC3Z3 genes and demonstrated that all 18 different feline APOBEC3Z3 proteins tested exhibit anti-feline immunodeficiency virus (FIV) activity. Our comprehensive investigation focusing on the interplay between feline APOBEC3 and FIV Vif can be a clue to elucidate the scenarios of the cross-species transmissions of FIVs in felids.

Following the Zika virus (ZIKV) outbreak in the Americas, ZIKV was causally associated with microcephaly and a range of neurological and developmental symptoms, termed congenital Zika syndrome (CZS). The viruses responsible for this outbreak belonged to the Asian lineage of ZIKV. However, in vitro and in vivo studies assessing the pathogenesis of African-lineage ZIKV demonstrated that African-lineage isolates often replicated to high titers and caused more-severe pathology than Asian-lineage isolates. To date, the pathogenesis of African-lineage ZIKV in a translational model, particularly during pregnancy, has not been rigorously characterized. Here, we infected four pregnant rhesus macaques with a low-passage-number strain of African-lineage ZIKV and compared its pathogenesis to those for a cohort of four pregnant rhesus macaques infected with an Asian-lineage isolate and a cohort of mock-inoculated controls. The viral replication kinetics for the two experimental groups were not significantly different, and both groups developed robust neutralizing antibody titers above levels considered to be protective. There was no evidence of significant fetal head growth restriction or gross fetal harm at delivery (1 to 1.5 weeks prior to full term) in either group. However, a significantly higher burden of ZIKV viral RNA (vRNA) was found in the maternal-fetal interface tissues of the macaques exposed to an African-lineage isolate. Our findings suggest that ZIKV of any genetic lineage poses a threat to pregnant individuals and their infants. IMPORTANCE ZIKV was first identified in 1947 in Africa, but most of our knowledge of ZIKV is based on studies of the distinct Asian genetic lineage, which caused the outbreak in the Americas in 2015 to 2016. In its most recent update, the WHO stated that improved understanding of African-lineage ZIKV pathogenesis during pregnancy must be a priority. The recent detection of African-lineage isolates in Brazil underscores the need to understand the impact of these viruses. Here, we provide the first comprehensive assessment of African-lineage ZIKV infection during pregnancy in a translational nonhuman primate model. We show that African-lineage isolates replicate with kinetics similar to those of Asian-lineage isolates and can infect the placenta. However, there was no evidence of more-severe outcomes with African-lineage isolates. Our results highlight both the threat that African-lineage ZIKV poses to pregnant individuals and their infants and the need for epidemiological and translational in vivo studies with African-lineage ZIKV.

Chromatin architecture governs cell lineages by regulating the specific gene expression; however, its role in the diversity of cancer development remains unknown. Among pancreatic cancers, pancreatic ductal adenocarcinoma (PDAC) and intraductal papillary mucinous neoplasms (IPMN) with an associated invasive carcinoma (IPMNinv) arise from 2 distinct precursors, and their fundamental differences remain obscure. Here, we aimed to assess the difference of chromatin architecture regulating the transcriptional signatures or biological features in pancreatic cancers.

The development of novel fertilization treatments, including in vitro fertilization and intracytoplasmic injection, has made pregnancy possible regardless of the level of activity of the spermatozoa; however, the etiology of male-factor infertility is poorly understood. Multiple studies, primarily through the use of transgenic animals, have contributed to a list of candidate genes that may affect male infertility in humans. We examined single nucleotide polymorphisms (SNPs) as a cause of male infertility in an analysis of spermatogenesis-specific genes.

Countermeasures against Zika virus (ZIKV), including vaccines, are frequently tested in nonhuman primates (NHP). Macaque models are important for understanding how ZIKV infections impact human pregnancy due to similarities in placental development. The lack of consistent adverse pregnancy outcomes in ZIKV-affected pregnancies poses a challenge in macaque studies where group sizes are often small (4-8 animals). Studies in small animal models suggest that African-lineage Zika viruses can cause more frequent and severe fetal outcomes. No adverse outcomes were observed in macaques exposed to 1x104 PFU (low dose) of African-lineage ZIKV at gestational day (GD) 45. Here, we exposed eight pregnant rhesus macaques to 1x108 PFU (high dose) of African-lineage ZIKV at GD 45 to test the hypothesis that adverse pregnancy outcomes are dose-dependent. Three of eight pregnancies ended prematurely with fetal death. ZIKV was detected in both fetal and placental tissues from all cases of early fetal loss. Further refinements of this exposure system (e.g., varying the dose and timing of infection) could lead to an even more consistent, unambiguous fetal loss phenotype for assessing ZIKV countermeasures in pregnancy. These data demonstrate that high-dose exposure to African-lineage ZIKV causes pregnancy loss in macaques and also suggest that ZIKV-induced first trimester pregnancy loss could be strain-specific.

The enteric neurotransmitter acetylcholine governs important intestinal epithelial secretory and immune functions through its actions on epithelial muscarinic Gq-coupled receptors such as M3R. Its role in the regulation of intestinal stem cell function and differentiation, however, has not been clarified. Here, we find that nonselective muscarinic receptor antagonism in mice as well as epithelial-specific ablation of M3R induces a selective expansion of DCLK1-positive tuft cells, suggesting a model of feedback inhibition. Cholinergic blockade reduces Lgr5-positive intestinal stem cell tracing and cell number. In contrast, Prox1-positive endocrine cells appear as primary sensors of cholinergic blockade inducing the expansion of tuft cells, which adopt an enteroendocrine phenotype and contribute to increased mucosal levels of acetylcholine. This compensatory mechanism is lost with acute irradiation injury, resulting in a paucity of tuft cells and acetylcholine production. Thus, enteroendocrine tuft cells appear essential to maintain epithelial homeostasis following modifications of the cholinergic intestinal niche.

The molecular subtypes of pancreatic cancer (PC), either classical/progenitor-like or basal/squamous-like, are currently a major topic of research because of their direct association with clinical outcomes. Some transcription factors (TFs) have been reported to be associated with these subtypes. However, the mechanisms by which these molecular signatures of PCs are established remain unknown. Epigenetic regulatory processes, supported by dynamic changes in the chromatin structure, are essential for transcriptional profiles. Previously, we reported the importance of open chromatin profiles in the biological features and transcriptional status of PCs. Here, we aimed to analyze the relationships between three-dimensional (3D) genome structures and the molecular subtypes of human PCs using Hi-C analysis. We observed a correlation of the specific elements of 3D genome modules, including compartments, topologically associating domains, and enhancer-promoter loops, with the expression of related genes. We focused on HNF1B, a TF that is implicated in the progenitor subtype. Forced expression of HNF1B in squamous-type PC organoids induced the upregulation and downregulation of genes associated with progenitor and squamous subtypes, respectively. Long-range genomic interactions induced by HNF1B were accompanied by compartment modulation and H3K27ac redistribution. We also found that these HNF1B-induced changes in subtype-related gene expression required an intrinsically disordered region, suggesting a possible involvement of phase separation in compartment modulation. Thus, mapping of 3D structural changes induced by TFs, such as HNF1B, may become a useful resource for further understanding the molecular features of PCs.

A highly aggressive subset of pancreatic ductal adenocarcinomas undergo trans-differentiation into the squamous lineage during disease progression. Here, we investigated whether squamous trans-differentiation of human and mouse pancreatic cancer cells can influence the phenotype of non-neoplastic cells in the tumor microenvironment. Conditioned media experiments revealed that squamous pancreatic cancer cells secrete factors that recruit neutrophils and convert pancreatic stellate cells into cancer-associated fibroblasts (CAFs) that express inflammatory cytokines at high levels. We use gain- and loss-of-function approaches to show that squamous-subtype pancreatic tumor models become enriched with neutrophils and inflammatory CAFs in a p63-dependent manner. These effects occur, at least in part, through p63-mediated activation of enhancers at pro-inflammatory cytokine loci, which includes IL1A and CXCL1 as key targets. Taken together, our findings reveal enhanced tissue inflammation as a consequence of squamous trans-differentiation in pancreatic cancer, thus highlighting an instructive role of tumor cell lineage in reprogramming the stromal microenvironment.

The increased incidence of obesity in the global population has increased the risk of several chronic inflammation-related diseases, including non-alcoholic steatohepatitis (NASH)-hepatocellular carcinoma (HCC). The progression from NASH to HCC involves a virus-independent liver carcinogenic mechanism; however, we currently lack effective treatment and prevention strategies. Several reports have suggested that fecal volatile organic compounds (VOCs) are strongly associated with NASH-HCC; therefore, we explored the biomarkers involved in its pathogenesis and progression. Fecal samples collected from control and NASH-HCC model STAM mice were subjected to headspace autosampler gas chromatography-electron ionization-mass spectrometry. Non-target profiling analysis identified diacetyl (2,3-butandione) as a fecal VOC that characterizes STAM mice. Although fecal diacetyl levels were correlated with the HCC in STAM mice, diacetyl is known as a cytotoxic/tissue-damaging compound rather than genotoxic or mutagenic; therefore, we examined the effect of bioactivity associated with NASH progression. We observed that diacetyl induced several pro-inflammatory molecules, including tumor necrosis factor-α, cyclooxygenase-2, monocyte chemoattractant protein-1, and transforming growth factor-β, in mouse macrophage RAW264.7 and Kupffer KPU5 cells. Additionally, we observed that diacetyl induced α-smooth muscle actin, one of the hallmarks of fibrosis, in an ex vivo cultured hepatic section, but not in in vitro hepatic stellate TWNT-1 cells. These results suggest that diacetyl would be a potential biomarker of fecal VOC in STAM mice, and its ability to trigger the macrophage-derived inflammation and fibrosis may partly contribute to NASH-HCC carcinogenesis.

Mild cognitive impairment (MCI) is an early stage that can result in dementia. MCI can be reversed, and diagnosis at an early stage is crucial to control the progression to dementia. Dementia is currently diagnosed based on interviews and screening tests; however, novel biomarkers must be identified to allow early MCI detection. Therefore, the present study aimed to identify novel biomarkers in the form of blood microRNAs (miRNAs/miRs) for the diagnosis of MCI or early dementia. Blood samples were collected from C57BL/6NJcl male mice at four time points, including 4-week-old (4W), 8-week-old (8W), 36-week-old (36W) and 58-week-old (58W), and serum was isolated. Body weight and blood total cholesterol levels were increased, and blood alkaline phosphatase was decreased with aging. The 8W mice exhibited the highest cognitive ability in the Morris water maze test, whereas the 58W mice demonstrated decreased cognitive ability. The serum RNA concentrations of the 4W, 8W, 36W and 58W mice demonstrated no significant differences. Furthermore, small RNA levels were detected in the serum of all mice. miRNA microarray analysis revealed a >1.5-fold increase in the serum expression of two miRNAs (miR-21a-5p and miR-92a-3p) and a >1.5-fold decrease in the serum expression of two other miRNAs (miR-6769b-5p and miR-709) in 58W mice compared with those in 8W mice. In the future, we aim to further analyze aged mice to discover novel MCI biomarkers.

Cholangiocarcinoma is a life-threatening disease with a poor prognosis. Although genome analysis unraveled some genetic mutation profiles in cholangiocarcinoma, it remains unknown whether such genetic abnormalities relate to the effects of anticancer drugs. Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) are exclusively found in almost 20% of intrahepatic cholangiocarcinoma (ICC). Recently, the anticancer effects of BET inhibitors including JQ1 have been shown in various tumors. In the present study, we report that the antigrowth effect of JQ1 differs among ICC cells and IDH1 mutation sensitizes ICC cells to JQ1. RBE cells harboring IDH1 mutation was more sensitive to JQ1 than HuCCT1 or HuH28 cells with wild-type IDH1. JQ1 induced apoptosis only in RBE cells through the upregulation of proapoptotic genes BAX and BIM. We found that the antigrowth effect was not attributed to downregulation of the MYC gene as a well-known target of JQ1 in various cancer cells. Notably, the forced expression of mutant IDH1 successfully sensitized HuCCT1 cells to JQ1. In addition, AGI-5198, a selective inhibitor of mutant IDH1 partially reversed the decrease in viability after JQ1 treatment and also suppressed the JQ1-induced apoptosis in RBE cells. These data suggest that IDH1 mutation contributed to the growth inhibitory effect of JQ1 in RBE cells. Furthermore, given that the effect of mutant IDH1 was not recapitulated in glioblastoma cells, the enhancement of JQ1 sensitivity by IDH1 mutation seems to be specific for ICC cells. Our findings propose a new stratified therapeutic strategy based on IDH1 mutation in ICC.