Mechanical transmission of dengue virus by Aedes aegypti may influence disease transmission dynamics during outbreaks.

BACKGROUND: Dengue virus outbreaks are increasing in number and severity worldwide. Viral transmission is assumed to require a minimum time period of viral replication within the mosquito midgut. It is unknown if alternative transmission periods not requiring replication are possible. METHODS: We used a mouse model of dengue virus transmission to investigate the potential of mechanical transmission of dengue virus. We investigated minimal viral titres necessary for development of symptoms in bitten mice and used resulting parameters to inform a new model of dengue virus transmission within a susceptible population. FINDINGS: Naïve mice bitten by mosquitoes immediately after they took partial blood meals from dengue infected mice showed symptoms of dengue virus, followed by mortality. Incorporation of mechanical transmission into mathematical models of dengue virus transmission suggest that this supplemental transmission route could result in larger outbreaks which peak sooner. INTERPRETATION: The potential of dengue transmission routes independent of midgut viral replication has implications for vector control strategies that target mosquito lifespan and suggest the possibility of similar mechanical transmission routes in other disease-carrying mosquitoes. FUNDING: This study was funded by grants from the National Health Research Institutes, Taiwan (04D2-MMMOST02), the Human Frontier Science Program (RGP0033/2021), the National Institutes of Health (1R01AI143698-01A1, R01AI151004 and DP2AI152071) and the Ministry of Science and Technology, Taiwan (MOST104-2321-B-400-016).

miRNA as a Modulator of Immunotherapy and Immune Response in Melanoma.

Immune checkpoint inhibitors are a promising therapy for the treatment of cancers, including melanoma, that improved benefit clinical outcomes. However, a subset of melanoma patients do not respond or acquire resistance to immunotherapy, which limits their clinical applicability. Recent studies have explored the reasons related to the resistance of melanoma to immune checkpoint inhibitors. Of note, miRNAs are the regulators of not only cancer progression but also of the response between cancer cells and immune cells. Investigation of miRNA functions within the tumor microenvironment have suggested that miRNAs could be considered as key partners in immunotherapy. Here, we reviewed the known mechanism by which melanoma induces resistance to immunotherapy and the role of miRNAs in immune responses and the microenvironment.

Aggressive organ penetration and high vector transmissibility of epidemic dengue virus-2 Cosmopolitan genotype in a transmission mouse model.

Dengue virus (DENV) causes dengue fever and severe hemorrhagic fever in humans and is primarily transmitted by Aedes aegypti and A. albopictus mosquitoes. The incidence of DENV infection has been gradually increasing in recent years due to global urbanization and international travel. Understanding the virulence determinants in host and vector transmissibility of emerging epidemic DENV will be critical to combat potential outbreaks. The DENV serotype 2 (DENV-2), which caused a widespread outbreak in Taiwan in 2015 (TW2015), is of the Cosmopolitan genotype and is phylogenetically related to the virus strain linked to another large outbreak in Indonesia in 2015. We found that the TW2015 virus was highly virulent in type I and type II interferon-deficient mice, with robust replication in spleen, lung, and intestine. The TW2015 virus also had high transmissibility to Aedes mosquitoes and could be effectively spread in a continuous mosquitoes-mouse-mosquitoes-mouse transmission cycle. By making 16681-based mutants carrying different segments of the TW2015 virus, we identified the structural pre-membrane (prM) and envelope (E) genes as key virulence determinants in the host, with involvement in the high transmissibility of the TW2015 virus in mosquitoes. The transmission mouse model will make a useful platform for evaluation of DENV with high epidemic potential and development of new strategies against dengue outbreaks.

Type I Interferon Signaling Accelerates Liver Regeneration by Metabolic Modulation in Noninfectious Conditions.

Type I interferon (IFN-I) has a well-known function in controlling viral infections, but its contribution in hepatocyte proliferation and hepatocellular carcinoma (HCC) formation remains unclear. Mice deficient in IFN-α receptor expression in whole mice or only in hepatocytes (Ifnar-/- and IfnarΔliver) were used to investigate the role of IFN-I signaling in cell proliferation and cancer formation in the liver. Ifnar-/- mice were resistant to chemical-induced HCC formation in the absence of infection. The results show that low grade of IFN-I and interferon-stimulated gene were expressed substantially in naïve mouse liver. The low level of IFN-I activation is constantly present in mouse liver after weaning and negatively modulates forkhead box O hepatic expression. The IFN-I signaling can be partially blocked by the clearance of lipopolysaccharide. Mice lacking IFN-I signaling have lower basal proliferation activity and delayed liver regeneration processes after two-thirds partial hepatectomy. The activation of IFN-I signaling on hepatocyte controls glucose homeostasis and lipid metabolism to support proliferation potency and long-term tumorigenesis. Our results reveal a positive role of low-grade IFN-I singling to hepatocyte proliferation and HCC formation by modulating glucose homeostasis and lipid metabolism.

Uroplakins play conserved roles in egg fertilization and acquired additional urothelial functions during mammalian divergence.

Uroplakin (UP) tetraspanins and their associated proteins are major mammalian urothelial differentiation products that form unique two-dimensional crystals of 16-nm particles ("urothelial plaques") covering the apical urothelial surface. Although uroplakins are highly expressed only in mammalian urothelium and are often referred to as being urothelium specific, they are also expressed in several mouse nonurothelial cell types in stomach, kidney, prostate, epididymis, testis/sperms, and ovary/oocytes. In oocytes, uroplakins colocalize with CD9 on cell-surface and multivesicular body-derived exosomes, and the cytoplasmic tail of UPIIIa undergoes a conserved fertilization-dependent, Fyn-mediated tyrosine phosphorylation that also occurs in Xenopus laevis eggs. Uroplakin knockout and antibody blocking reduce mouse eggs' fertilization rate in in vitro fertilization assays, and UPII/IIIa double-knockout mice have a smaller litter size. Phylogenetic analyses showed that uroplakin sequences underwent significant mammal-specific changes. These results suggest that, by mediating signal transduction and modulating membrane stability that do not require two-dimensional-crystal formation, uroplakins can perform conserved and more ancestral fertilization functions in mouse and frog eggs. Uroplakins acquired the ability to form two-dimensional-crystalline plaques during mammalian divergence, enabling them to perform additional functions, including umbrella cell enlargement and the formation of permeability and mechanical barriers, to protect/modify the apical surface of the modern-day mammalian urothelium.

Establishment of a mouse model for the complete mosquito-mediated transmission cycle of Zika virus.

Zika virus (ZIKV) is primarily transmitted by Aedes mosquitoes in the subgenus Stegomyia but can also be transmitted sexually and vertically in humans. STAT1 is an important downstream factor that mediates type I and II interferon signaling. In the current study, we showed that mice with STAT1 knockout (Stat1-/-) were highly susceptible to ZIKV infection. As low as 5 plaque-forming units of ZIKV could cause viremia and death in Stat1-/- mice. ZIKV replication was initially detected in the spleen but subsequently spread to the brain with concomitant reduction of the virus in the spleen in the infected mice. Furthermore, ZIKV could be transmitted from mosquitoes to Stat1-/- mice back to mosquitoes and then to naïve Stat1-/- mice. The 50% mosquito infectious dose of viremic Stat1-/- mouse blood was close to 810 focus-forming units (ffu)/ml. Our further studies indicated that the activation of macrophages and conventional dendritic cells were likely critical for the resolution of ZIKV infection. The newly developed mouse and mosquito transmission models for ZIKV infection will be useful for the evaluation of antiviral drugs targeting the virus, vector, and host.

MicroRNA-205 targets tight junction-related proteins during urothelial cellular differentiation.

The mammalian bladder urothelium classified as basal, intermediate, and terminally differentiated umbrella cells offers one of the most effective permeability barrier functions known to exist in nature because of the formation of apical uroplakin plaques and tight junctions. To improve our understanding of urothelial differentiation, we analyzed the microRNA (miRNA) expression profiles of mouse urinary tissues and by TaqMan miRNA analysis of microdissected urothelial layers and in situ miRNA-specific hybridization to determine the dependence of these miRNAs on the differentiation stage. Our in situ hybridization studies revealed that miR-205 was enriched in the undifferentiated basal and intermediate cell layers. We then used a quantitative proteomics approach to identify miR-205 target genes in primary cultured urothelial cells subjected to antagomir-mediated knockdown of specific miRNAs. Twenty-four genes were reproducibly regulated by miR-205; eleven of them were annotated as cell junction- and tight junction-related molecules. Western blot analysis demonstrated that antagomir-induced silencing of miR-205 in primary cultured urothelial cells elevated the expression levels of Tjp1, Cgnl1, and Cdc42. Ectopic expression of miR-205 in MDCK cells inhibited the expression of tight junction proteins and the formation of tight junctions. miR-205- knockdown urothelial cells showed alterations in keratin synthesis and increases of uroplakin Ia and Ib, which are the urothelial differentiation products. These results suggest that miR-205 may contribute a role in regulation of urothelial differentiation by modulating the expression of tight junction-related molecules.

MicroRNA-196a/-196b promote cell metastasis via negative regulation of radixin in human gastric cancer.

MicroRNAs (miRNAs) play an important role to contribute carcinogenesis. The aim of the current study was to identify useful biomarkers from miRNAs. Differential miRNA profiles were analyzed using the miRNA qRT-PCR-based assay. Two of the most upregulated miRNAs were selected and validated. The miR-196a/-196b levels were significantly increased in gastric cancer (GC) tissues (n=109). Overexpression of miR-196a/-196b was significantly associated with tumor progression and poorer 5-year survival outcomes. Overexpression of miR-196a/-196b enhances GC cell migration and invasion. Further, radixin was identified as a target gene of miR-196a/-196b. Elevated miR-196a/-196b expression in GC cells led to reduced radixin protein levels and vice versa. Notably, an inverse correlation between miR-196a/-196b and radixin mRNA and protein expression was observed in GC tissues with in situ hybridization and immunohistochemistry analyses. Together, miR-196a/-196b inhibitory oligonucleotides or overexpression of the radixin may thus have therapeutic potential in suppressing GC metastasis.

A role for Epstein-Barr viral BALF1 in facilitating tumor formation and metastasis potential.

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that triggers transformation and tumorigenesis of latently infected B cells in vitro. BALF1, a Bcl-2-like EBV gene expressed in both latent and lytic stages, was recently characterized in EBV-infected cells; however, the role and function of BALF1 has remained elusive. Here, we demonstrate that BALF1 expression alters cellular morphology. Importantly, BALF1 promotes cellular transformation, with tumorigenicity assays showing larger and substantially greater numbers of tumors in BALF1 transfectant-injected mice compared to mice injected with pcDNA control transfectants. In addition, BALF1 expression increases cell survival under low-serum conditions, an effect that is attributable to suppression of apoptosis, not to promotion of cell-cycle progression. Furthermore, BALF1 transfectants exhibit markedly increased tumor metastasis in vitro and in vivo. Taken together, these findings suggest that BALF1 may be a new tumor marker for EBV diagnosis and provide a new direction for research on treatments of EBV-associated tumors.

The MYND domain-containing protein BRAM1 inhibits lymphotoxin beta receptor-mediated signaling through affecting receptor oligomerization.

MYND (myeloid-Nervy-DEAF-1) domains exist in a large number of proteins that are functionally important in development or associated with cancers. We have previously demonstrated that a MYND domain-containing protein, the bone morphogenesis protein receptor-associated molecule 1 (BRAM1), is able to interact with Epstein-Barr virus-encoded latent membrane protein 1 (LMP1), which acts as a constitutively activated tumor necrosis factor receptor (TNFR). Herein we further demonstrated that BRAM1 additionally associates with the TNFR-superfamily member, the lymphotoxin beta receptor (LTßR), and hence inhibits LTßR-mediated function. Using the yeast two-hybrid assay, we demonstrated that BRAM1 interacts with LTßR mainly through the self-association domain of LTßR (aa 336-398). The co-immunoprecipitation experiment further revealed that BRAM1 as well as MYND domain-containing proteins, MTG8 and DEAF-1, interacts with LTßR via their MYND domains. The BRAM1-LTßR interaction impedes the self-association of LTßR and the recruitment of TNFR-associated factors 2 and 3 (TRAF2 and TRAF3), leading to abolishment of LTßR-induced NF-κB signaling, JNK activation, and caspase-dependent cell death. In sum, our data demonstrate that the MYND-containing protein BRAM1 abrogates LTßR function through a protein-protein interaction. These findings may provide a direction for the treatment of dysregulation of LTßR-mediated signaling.

Relationships between health status, depression and cognitive functions of institutionalized male veterans.

The purpose of this study was to explore the relationships among health state, depression, and cognitive functions of institutionalized male veterans. A cross-sectional and correlation research design with a cluster sampling was conducted. A total of 223 veterans who were above 65 years old, with no psychiatric disorders and no organic brain lesions were recruited from two veterans' institutions in Southern Taiwan. The researcher interviewed them one-on-one using structural questionnaires, including demographic data, health status, Taiwan Geriatric Depression Scale (TGDS) and Mini-Mental State Examination (MMSE). The results of this study were as follows: (1) Veterans who were able to read, were married, had good dietary habits, avoided over-oiled and high-fat food to keep healthy, took exercise at least 30 min each time, and had static leisure activities had significant differences in cognitive functions. (2) Cognitive functions were significantly negatively correlated with age and depression, whereas positively correlated with education level, the number of children and perceived health status. (3) Depression, literacy, education level and age were the four predictors of cognitive functions, accounting for 29% of the variance. The findings of this study provide a reference to caregivers and health care professionals for home care, clinical practice and cohort study in the future.

Appearance of new tetraspanin genes during vertebrate evolution.

A detailed phylogenetic analysis of tetraspanins from 10 fully sequenced metazoan genomes and several fungal and protist genomes gives insight into their evolutionary origins and organization. Our analysis suggests that the superfamily can be divided into four large families. These four families-the CD family, CD63 family, uroplakin family, and RDS family-are further classified as consisting of several ortholog groups. The clustering of several ortholog groups together, such as the CD9/Tsp2/CD81 cluster, suggests functional relatedness of those ortholog groups. The fact that our studies are based on whole genome analysis enabled us to estimate not only the phylogenetic relationships among the tetraspanins, but also the first appearance in the tree of life of certain tetraspanin ortholog groups. Taken together, our data suggest that the tetraspanins are derived from a single (or a few) ancestral gene(s) through sequence divergence, rather than convergence, and that the majority of tetraspanins found in the human genome are vertebrate (21 instances), tetrapod (4 instances), or mammalian (6 instances) inventions.

Origin of the tetraspanin uroplakins and their co-evolution with associated proteins: implications for uroplakin structure and function.

Genome level information coupled with phylogenetic analysis of specific genes and gene families allow for a better understanding of the structure and function of their protein products. In this study, we examine the mammalian uroplakins (UPs) Ia and Ib, members of the tetraspanin superfamily, that interact with uroplakins UPII and UPIIIa/IIIb, respectively, using a phylogenetic approach of these genes from whole genome sequences. These proteins interact to form urothelial plaques that play a central role in the permeability barrier function of the apical urothelial surface of the urinary bladder. Since these plaques are found exclusively in mammalian urothelium, it is enigmatic that UP-like genomic sequences were recently found in lower vertebrates without a typical urothelium. We have cloned full-length UP-related cDNAs from frog (Xenopus laevis), chicken (Gallus gallus), and zebrafish (Danio rerio), and combined these data with sequence information from their orthologs in all the available fully sequenced and annotated animal genomes. Phylogenetic analyses of all the available uroplakin sequences, and an understanding of their distribution in several animal taxa, suggest that: (i) the UPIa/UPIb and UPII/UPIII genes evolved by gene duplication in the common ancestor of vertebrates; (ii) uroplakins can be lost in different combinations in vertebrate lineages; and (iii) there is a strong co-evolutionary relationship between UPIa and UPIb and their partners UPII and UPIIIa/IIIb, respectively. The co-evolution of the tetraspanin UPs and their associated proteins may fine-tune the structure and function of uroplakin complexes enabling them to perform diverse species- and tissue-specific functions. The structure and function of uroplakins, which are also expressed in Xenopus kidney, oocytes and fat body, are much more versatile than hitherto appreciated.

Induction of inducible nitric oxide synthase by Epstein-Barr virus B95-8-derived LMP1 in Balb/3T3 cells promotes stress-induced cell death and impairs LMP1-mediated transformation.

The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) causes cellular transformation and activation of several intracellular signaling events. In this report, we show that BLMP1 (encoded by the LMP1 gene derived from the B95-8 strain of EBV) triggers the expression of inducible nitric oxide synthase (iNOS) in Balb/3T3 fibroblasts. Intriguingly, NLMP1, a natural sequence variant of LMP1 identified in EBV-positive nasopharyngeal carcinoma biopsy, does not similarly induce iNOS expression. BLMP1-induced iNOS in Balb/3T3 cells is active to produce nitric oxide (NO), and NO production can be blocked by several iNOS inhibitors. When subjected to environmental stress, Balb/3T3 cells that produce NO lose viability more rapidly than non NO-producing cells. Blockage of NO generation by iNOS inhibitors enhances the viability of NO-producing cells under stress conditions. The activities of caspase-3 and c-Jun N-terminal kinase, two important regulators mediating stress-induced apoptosis, are significantly potentiated following heat shock treatment of BLMP1-expressing/NO-producing cells, compared to parental and NLMP1-expressing cells. Furthermore, treatment with iNOS inhibitor augmented the cloning efficiency (in culture) and tumor growth (in nude mice) of BLMP1-expressing/NO-producing cells. Collectively, the results demonstrate that BLMP1 induces iNOS expression and NO production in Balb/3T3 cells, which leads to the alteration of cell functions, including sensitivity to environmental stress, capability to colonize independent of anchorage and tumorigenicity in nude mice. Our data additionally implicate that the differential iNOS induction potential of the two LMP1 forms may represent the basis of a functional difference between the two LMP1 proteins.

Negative regulation of Epstein-Barr virus latent membrane protein 1-mediated functions by the bone morphogenetic protein receptor IA-binding protein, BRAM1.

The latent membrane protein 1 (LMP1) of Epstein-Barr virus causes cellular transformation and activates several intracellular signals, including NF-kappaB and c-Jun N-terminal kinase. Using yeast two-hybrid screening with the LMP1 C-terminal sequence as bait, we demonstrate that BRAM1 (bone morphogenetic protein receptor-associated molecule 1) is an LMP1-interacting protein. BRAM1 associates with LMP1, both in vitro and in vivo, as revealed by confocal microscopy, glutathione S-transferase pull-down, and co-immunoprecipitation assays. This association mainly involves the C-terminal half of BRAM1 comprising the MYND domain and the CTAR2 region of LMP1, which is critical in LMP1-mediated signaling pathways. We show that BRAM1 interferes with LMP1-mediated NF-kappaB activation but not the JNK signaling pathway. Because the CTAR2 region interacts with the tumor necrosis factor (TNF-alpha receptor-associated death domain protein, it is interesting to find that BRAM1 also interferes with NF-kappaB activation mediated by TNF-alpha. BRAM1 interferes LMP1-mediated and TNF-alpha-induced NF-kappaB activation by targeting IkappaBalpha molecules. Moreover, BRAM1 inhibits the resistance of LMP1-expressing cells to TNF-alpha-induced cytotoxicity. We therefore propose that the BRAM1 molecule associates with LMP1 and functions as a negative regulator of LMP1-mediated biological functions.