Human retroviral antisense mRNAs are retained in the nuclei of infected cells for viral persistence.

Human retroviruses, including human T cell leukemia virus type 1 (HTLV-1) and HIV type 1 (HIV-1), encode an antisense gene in the negative strand of the provirus. Besides coding for proteins, the messenger RNAs (mRNAs) of retroviral antisense genes have also been found to regulate transcription directly. Thus, it has been proposed that retroviruses likely localize their antisense mRNAs to the nucleus in order to regulate nuclear events; however, this opposes the coding function of retroviral antisense mRNAs that requires a cytoplasmic localization for protein translation. Here, we provide direct evidence that retroviral antisense mRNAs are localized predominantly in the nuclei of infected cells. The retroviral 3' LTR induces inefficient polyadenylation and nuclear retention of antisense mRNA. We further reveal that retroviral antisense RNAs retained in the nucleus associate with chromatin and have transcriptional regulatory function. While HTLV-1 antisense mRNA is recruited to the promoter of C-C chemokine receptor type 4 (CCR4) and enhances transcription from it to support the proliferation of HTLV-1-infected cells, HIV-1 antisense mRNA is recruited to the viral LTR and inhibits sense mRNA expression to maintain the latency of HIV-1 infection. In summary, retroviral antisense mRNAs are retained in nucleus, act like long noncoding RNAs instead of mRNAs, and contribute to viral persistence.

Cap Analysis of Gene Expression Clarifies Transcriptomic Divergence Within Monozygotic Twin Pairs.

Phenotypic variation is the result of gene expression based on complex interaction between genetic and environmental factors. It is well known that genetic and environmental factors influence gene expression, but our understanding of their relative importance remains limited. To obtain a hint for the understanding of their contributions, we took advantage of monozygotic twins, as they share genetic and shared environmental factors but differ in nonshared factors, such as environmental differences and stochastic factors. In this study, we performed cap analysis of gene expression on three pairs of twins and clustered each individual based on their expression profiles of annotated genes. The dendrogram of annotated gene transcripts showed a monophyletic clade for each twin pair. We also analyzed the expression of retrotransposons, such as human endogenous retroviruses (HERVs) and long interspersed nuclear elements (LINEs), given their abundance in the genome. Clustering analyses demonstrated that HERV and LINE expression diverged even within monozygotic twin pairs. Thus, HERVs and LINEs are more susceptible to nonshared factors than annotated genes. Motif analysis of differentially expressed annotated genes suggests that specificity protein/Krüppel-like factor family transcription factors are involved in the expression divergence of annotated gene influenced by nonshared factors. Collectively, our findings suggest that expressions of annotated genes and retrotransposons are differently regulated, and that the expression of retrotransposons is more susceptible to nonshared factors than annotated genes.

Identification of a Retroelement-Containing Human Transcript Induced in the Nucleus by Vaccination.

Endogenous retroelements constitute almost half of the mammalian genome. Given that more than 60% of human genomic bases are transcribed, transcripts containing these retroelements may impact various biological processes. However, the physiological roles of most retroelement-containing transcripts are yet to be revealed. Here, we profiled the expression of retroelement-containing human transcripts during vaccination and found that vaccination upregulated transcripts containing only particular retroelements, such as the MLT-int element of endogenous retroviruses. MLT-int-containing transcripts were distributed mainly in the nucleus, suggesting their unique roles in the nucleus. Furthermore, we demonstrated that MLT-int RNA suppressed interferon promoter activity in the absence of immune stimuli. Based on these lines of evidence, we speculate a model of a role of the previously unnoticed MLT-int element in preventing excess innate immune activation after elimination of immune stimuli. Our results may emphasize the importance of retroelement-containing transcripts in maintaining host immune homeostasis.

HTLV-1 bZIP Factor Impairs Anti-viral Immunity by Inducing Co-inhibitory Molecule, T Cell Immunoglobulin and ITIM Domain (TIGIT).

Human T-cell leukemia virus type 1 (HTLV-1) infects CD4+ T cells and induces proliferation of infected cells in vivo, which leads to the onset of adult T-cell leukemia (ATL) in some infected individuals. The HTLV-1 bZIP factor (HBZ) gene, which is encoded in the minus strand of HTLV-1, plays critical roles in pathogenesis. In this study, RNA-seq and ChIP-seq analyses using HBZ transduced T cells revealed that HBZ upregulates the expression and promoter acetylation levels of a co-inhibitory molecule, T cell immunoglobulin and ITIM domain (TIGIT), in addition to those of regulatory T cells related genes, Foxp3 and Ccr4. TIGIT was expressed on CD4+ T cells from HBZ-transgenic (HBZ-Tg) mice, and on ATL cells and HTLV-1 infected CD4+ T cells of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in vivo. Expression of Blimp1 and IL-10 was upregulated in TIGIT+CD4+ cells of HBZ-Tg mice compared with TIGIT-CD4+ T cells, suggesting the correlation between TIGIT expression and IL-10 production. When CD4+ T cells from HBZ-Tg mice were stimulated with TIGIT's ligand, CD155, their production of the inhibitory cytokine IL-10 was enhanced. Furthermore, dendritic cells from HBZ-Tg mice produced high levels of IL-10 after stimulation. These data suggest that HBZ alters immune system to suppressive state via TIGIT and IL-10. Importantly, TIGIT suppressed T-cell responses to another HTLV-1 virus protein, Tax, in vitro. Blocking of TIGIT and PD-1 slightly increased anti-Tax T-cell activity in some HAM/TSP patients. These results suggest that HBZ-induced TIGIT on HTLV-1 infected cells impairs T-cell responses to viral antigens. This study shows that HBZ-induced TIGIT plays a pivotal role in attenuating host immune responses and shaping a microenvironment favorable to HTLV-1.

Essential roles of the histone methyltransferase ESET in the epigenetic control of neural progenitor cells during development.

In the developing brain, neural progenitor cells switch differentiation competency by changing gene expression profiles that are governed partly by epigenetic control, such as histone modification, although the precise mechanism is unknown. Here we found that ESET (Setdb1), a histone H3 Lys9 (H3K9) methyltransferase, is highly expressed at early stages of mouse brain development but downregulated over time, and that ablation of ESET leads to decreased H3K9 trimethylation and the misregulation of genes, resulting in severe brain defects and early lethality. In the mutant brain, endogenous retrotransposons were derepressed and non-neural gene expression was activated. Furthermore, early neurogenesis was severely impaired, whereas astrocyte formation was enhanced. We conclude that there is an epigenetic role of ESET in the temporal and tissue-specific gene expression that results in proper control of brain development.

Neutrophil S100A9 supports M2 macrophage niche formation in granulomas.

Mycobacterium infection gives rise to granulomas predominantly composed of inflammatory M1-like macrophages, with bacteria-permissive M2 macrophages also detected in deep granulomas. Our histological analysis of Mycobacterium bovis bacillus Calmette-Guerin-elicited granulomas in guinea pigs revealed that S100A9-expressing neutrophils bordered a unique M2 niche within the inner circle of concentrically multilayered granulomas. We evaluated the effect of S100A9 on macrophage M2 polarization based on guinea pig studies. S100A9-deficient mouse neutrophils abrogated M2 polarization, which was critically dependent on COX-2 signaling in neutrophils. Mechanistic evidence suggested that nuclear S100A9 interacts with C/EBPß, which cooperatively activates the Cox-2 promoter and amplifies prostaglandin E2 production, followed by M2 polarization in proximal macrophages. Because the M2 populations in guinea pig granulomas were abolished via treatment with celecoxib, a selective COX-2 inhibitor, we propose the S100A9/Cox-2 axis as a major pathway driving M2 niche formation in granulomas.

Effects of activation of the LINE-1 antisense promoter on the growth of cultured cells.

Long interspersed element 1 (LINE-1, or L1) is a retrotransposon that constitutes ~ 17% of the human genome. Although ~ 6000 full-length L1s spread throughout the human genome, their biological significance remains undetermined. The L1 5' untranslated region has bidirectional promoter activity with a sense promoter driving L1 mRNA production and an antisense promoter (ASP) driving the production of L1-gene chimeric RNAs. Here, we stimulated L1 ASP activity using CRISPR-Cas9 technology to evaluate its biological impacts. Activation of the L1 ASP upregulated the expression of L1 ASP-driven ORF0 and enhanced cell growth. Furthermore, the exogenous expression of ORF0 also enhanced cell growth. These results indicate that activation of L1 ASP activity fuels cell growth at least through ORF0 expression. To our knowledge, this is the first report demonstrating the role of the L1 ASP in a biological context. Considering that L1 sequences are desilenced in various tumor cells, our results indicate that activation of the L1 ASP may be a cause of tumor growth; therefore, interfering with L1 ASP activity may be a potential strategy to suppress the growth.

A somatic role for the histone methyltransferase Setdb1 in endogenous retrovirus silencing.

Subsets of endogenous retroviruses (ERVs) are derepressed in mouse embryonic stem cells (mESCs) deficient for Setdb1, which catalyzes histone H3 lysine 9 trimethylation (H3K9me3). Most of those ERVs, including IAPs, remain silent if Setdb1 is deleted in differentiated embryonic cells; however they are derepressed when deficient for Dnmt1, suggesting that Setdb1 is dispensable for ERV silencing in somatic cells. However, H3K9me3 enrichment on ERVs is maintained in differentiated cells and is mostly diminished in mouse embryonic fibroblasts (MEFs) lacking Setdb1. Here we find that distinctive sets of ERVs are reactivated in different types of Setdb1-deficient somatic cells, including the VL30-class of ERVs in MEFs, whose derepression is dependent on cell-type-specific transcription factors (TFs). These data suggest a more general role for Setdb1 in ERV silencing, which provides an additional layer of epigenetic silencing through the H3K9me3 modification.

Suppression of cell proliferation by interferon-alpha through interleukin-1 production in adult rat dentate gyrus.

The therapeutic use of interferon-alpha (IFN-alpha), a proinflammatory cytokine, is known to cause various neuropsychiatric adverse effects. In particular, depression occurs in 30-45% of patients, frequently interrupting treatment. IFN-alpha-treated animals also show depression-like behaviors. However, mechanisms underlying the depression caused by IFN-alpha remain to be defined. Recently, a decrease in adult hippocampal neurogenesis was revealed as a possible neuropathological mechanism of depression. Therefore, we investigated the effect of subchronic IFN-alpha treatment on neurogenesis in the adult rat dentate gyrus (DG). Immediately after the administration of IFN-alpha for 1 week, a decrease in the number of 5-bromo-deoxyuridine-labeled proliferating cells was observed in the DG; however, no effect was detected on the expression of mature neuronal phenotype in the newly formed cells 3 weeks later. Also, an increase in the level of interleukin-1beta (IL-1beta), a major proinflammatory cytokine, was observed in the hippocampus following the administration of IFN-alpha. Furthermore, coadministration of an IL-1 receptor antagonist completely blocked the IFN-alpha-induced suppression of the cell-proliferative activity in the DG. Our results indicate that IFN-alpha suppresses neurogenesis in the DG, and that IL-1beta plays an essential role in the suppression. The decreased cell proliferation caused by IFN-alpha-induced IL-1beta may be responsible, at least in part, for IFN-alpha-induced depression.

Ginseng enhances contextual fear conditioning and neurogenesis in rats.

Panax Ginseng is a commonly used galenical known to have an enhancing effect on learning. Neurogenesis in the hippocampus has been shown to be necessary for hippocampus/amygdala-dependent learning tasks. To investigate the role of Ginseng in neurogenesis and learning of rats, we administered both Ginseng and BrdU for five consecutive days. As a result, Ginseng increased the number of BrdU-positive cells in the dentate gyrus in a dose-dependent manner. Further, we administered one dose of BrdU after Ginseng treatment for five consecutive days, and the number of BrdU-positive cells did not increase significantly. However, when one dose of BrdU was given 1 day before the following five consecutive days of Ginseng treatment, the number of BrdU-positive cells markedly increased in the hippocampus. Therefore, it is likely that Ginseng enhances not proliferation but survival of newly generated neurons in the hippocampus. Second, we administered both Ginseng and BrdU to rats for five consecutive days. One day after the last Ginseng and BrdU co-administration, contextual fear conditioning (CFC) was conducted. Ginseng in a dose-dependent manner increased the % freezing time and the number of BrdU-positive cells in the dentate gyrus of rats that received CFC. Thus, an increase in CFC-related neurogenesis may be one mechanism of Ginseng's properties to enhance learning ability.

A basic study on application of voice recognition input to an electronic nursing record system -evaluation of the function as an input interface-.

As computerization in the nursing field has been recently progressing, an electronic nursing record system is gradually introduced in the medical institution in Japan. Although it is expected for the electronic nursing record system to reduce the load of nursing work, the conventional keyboard operation is used for information input of the present electronic nursing record system and it has some problems concerning the input time and the operationability for common nurses who are unfamiliar with the computer operation. In the present study, we conducted a basic study on application of voice recognition input to an electronic nursing record system. The voice input is recently introduced to an electronic medical record system in a few clinics. However, so far the entered information cannot be processed because the information of the medical record must be entered as a free sentence. Therefore, we contrived a template for an electronic nursing record system and introduced it to the system for simple information entry and easy processing of the entered information in this study. Furthermore, an input experiment for evaluation of the voice input with the template was carried out by voluntary subjects for evaluation of the function as an input interface of an electronic nursing record system. The results of the experiment revealed that the input time by the voice input is obviously fast compared with that by the keyboard input and operationability of the voice input was superior to the keyboard input although all subjects had inexperience of the voice input. As a result, it was suggested our method, the voice input using the template made by us, might be useful for an input interface of an electronic nursing record system.

Development of a health management support system for patients with diabetes mellitus at home.

Recently, a patient with diabetes mellitus (DM) type 2 has been increasing in Japan. The patient should be managed not only by a specialist but also by himself focusing his attention on the improvement of his lifestyle at home. In the present study, we tried to develop a health management support system by which a diabetic patient in early stage can easily enter his daily life information, i.e. the biological information such as the data of blood sugar levels and blood pressure levels etc., the information of exercise and diet and send the information to the medical institution with a personal digital assistant (PDA). Afterwards, the patient can receive health instruction information by the physician in charge for self-care at his home with a PDA. The daily life information sent from the patient is stored in a server installed at the medical institution and analyzed. The physician can obtain the results of analysis by using a PC and send the instruction information necessary for patient management to the patient at home by using e-mail after diagnosing the patient's condition by the system. To evaluate usability of the developed patient information input system with a PDA, an experiment was conducted by corporation of 20 volunteers who were possible self management and whose age's range from 20s to 60s by questionnaire survey. As a result, almost examinees answered that lifestyle information could be easily entered by the sense like a mobile-phone and lots of positive opinions were obtained.

Stereoselective and driving-force-dependent photoinduced electron-transfer reactions of zinc myoglobin with optically active N,N'-dimethylcinchoninium and N,N'-dimethylcinchonidinium ions.

In order to understand the detailed mechanism of the stereoselective photoinduced electron-transfer (ET) reactions of zinc-substituted myoglobin (ZnMb) with optically active molecules by flash photolysis, we designed and prepared new optically active agents, such as N,N'-dimethylcinchoninium diiodide ([MCN]I2) and N,N'-dimethylcinchonidinium diiodide ([MCD]I2). The photoexcited triplet state of ZnMb, 3(ZnMb)*, was successfully quenched by [MCN]2+ and [MCD]2+ ions to form the radical pair of ZnMb cation (ZnMb.+) and reduced [MCN].+ and [MCD].+, followed by a thermal back ET reaction to the ground state. The rate constants (kq) for the ET quenching at 25 degrees C were obtained as kq(MCN)=(1.9+/-0.1)x10(6) M-1 s-1 and kq(MCD)=(3.0+/-0.2)x10(6) M-1 s-1, respectively. The ratio of kq(MCD)/kq(MCN)=1.6 indicates that the [MCD]2+ preferentially quenches 3ZnMb)*. The second-order rate constants (kb) for the thermal back ET reaction from [MCN].+ and [MCD].+ to ZnMb.+ at 25 degrees C were kb(MCN)=(0.79+/-0.04)x10(8) M-1 s-1 and kb(MCD)=(1.0+/-0.1)x10(8) M-1 s-1, respectively, and the selectivity was kq(MCD)/kq(MCN)=1.3. Both quenching and thermal back ET reactions are controlled by the ET step. In the quenching reaction, the energy differences of DeltaDeltaH ( not equal)(MCD-MCN) and DeltaDeltaS ( not equal)(MCD-MCN) at 25 degrees C were obtained as -1.1 and 0 kJ mol-1, respectively. On the other hand, DeltaDeltaH (not equal)(MCD-MCN)=11+/-2 kJ mol-1 and TDeltaDeltaS (not equal)(MCD-MCN)=-10+/-2 kJ mol-1 were given in the thermal back ET reaction. The highest stereoselectivity of 1.7 for [MCD].+ found at low temperature (10 degrees C) was due to the DeltaDeltaS ( not equal) value obtained in the thermal back ET reaction.