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Microphysiological systems (MPSs) based on microfluidic devices are attracting attention as an alternative cell assay platform to animal experiments in drug discovery. When we use microfluidic devices for cell culture, it is possible to experiment with various culture conditions that are difficult with conventional cell culture methods, such as fabrication of microstructures for cell placement, temporal and spatial control of liquid factors and adhesive conditions, and physical stimulation by flow and expansion/contraction. MPSs, which use microfluidic technology to construct the structure and function of physiological biological tissues and organs, are being commercialized and put to practical use worldwide with the entry of venture companies and pharmaceutical companies. Although research on the practical application of MPS in Japan has lagged far behind the efforts of Western countries, the Japan Agency for Medical Research and Development (AMED) launched the MPS Development and Research Project in FY2017 and established a system for MPS commercialization through industry-government-academia collaboration. The project is characterized by the formation of a consortium involving many researchers not only from academia but also from manufacturing and pharmaceutical companies with the aim of commercializing MPS devices. By FY2021, the final year of this project, several MPSs were successfully positioned in various stages of commercialization. This paper introduces two MPSs that the author was involved in commercializing in collaboration with domestic companies within the project.

In mammalian cells, RNA polymerase II (RNAP2)-mediated transcription occurs in numerous foci in the nucleus. Transcription foci detected by RNAP2 activity have often been called transcription "factories". Recent super-resolution microscopy techniques have revealed clustering and focus formation of RNAP2 in living cells, depending on phosphorylation states of repeats in its C-terminal domain (CTD). The differentially phosphorylated initiation and processive elongation forms of RNAP2 have also been tracked using live-cell probes derived from phosphorylation-specific antibodies. Recent live cell observations have detected spatially separated initiation and elongating RNAP2 foci, suggesting the presence of transcription initiation and elongation factories.

Epstein‐Barr virus‐associated lymphoproliferative disease (EBV‐LPD) is frequently fatal. Innate immunity plays a key role in protecting against pathogens and cancers. The stimulator of interferon genes (STING) is regarded as a key adaptor protein allowing DNA sensors recognizing exogenous cytosolic DNA to activate the type I interferon signaling cascade. In terms of EBV tumorigenicity, the role of STING remains elusive. Here we showed that treatment with the STING inhibitor, C‐176, suppressed EBV‐induced transformation in peripheral blood mononuclear cells. In an EBV‐LPD mouse model, C‐176 treatment also inhibited tumor formation and prolonged survival. Treatment with B cells alone did not affect EBV transformation, but suppression of EBV‐induced transformation was observed in the presence of T cells. Even without direct B cell‐T cell contact in a transwell system, the inhibitor reduced the transformation activity, indicating that intercellular communication by humoral factors was critical to prevent EBV‐induced transformation. These findings suggest that inhibition of STING signaling pathway with C‐176 could be a new therapeutic target of EBV‐LPD., Treatment with STING inhibitor, C‐176, suppressed the tumor formation in a mouse model of EBV LPDs. Although inhibitor treatment with B cells in vitro alone did not affect EBV transformation, the suppression of EBV‐induced transformation was observed in the presence of T cells

In mammalian cell nuclei, the nuclear lamina (NL) underlies the nuclear envelope (NE) to maintain nuclear structure. The nuclear lamins, the major structural components of the NL, are involved in the protection against NE rupture induced by mechanical stress. However, the specific role of the lamins in repair of NE ruptures has not been fully determined. Our analyses using immunofluorescence and live-cell imaging revealed that the nucleoplasmic pool of lamin C rapidly accumulated at sites of NE rupture induced by laser microirradiation in mouse embryonic fibroblasts. The accumulation of lamin C at the rupture sites required both the immunoglobulin-like fold domain that binds to barrier-to-autointegration factor (BAF) and a nuclear localization signal. The accumulation of nuclear BAF and cytoplasmic cyclic GMP-AMP synthase (cGAS) at the rupture sites was in part dependent on lamin A/C. These results suggest that nucleoplasmic lamin C, BAF, and cGAS concertedly accumulate at sites of NE rupture for rapid repair.

SummaryTranscription is a dynamic process that stochastically switches between the ON and OFF states. To detect the dynamic relationship among protein clusters of RNA polymerase II (RNAPII) and coactivators, gene loci, and transcriptional activity, we inserted an MS2 repeat, a TetO repeat, and inteins with a selection marker just downstream of the transcription start site (TSS). By optimizing the individual elements, we have developed the Spliced TetO REpeAt, MS2 repeat, and INtein sandwiched reporter Gene tag (STREAMING-tag) system. Clusters of RNAPII and BRD4 were observed proximally to the TSS of Nanog when the gene was transcribed in mouse embryonic stem cells. In contrast, clusters of MED19 and MED22 Mediator subunits were constitutively located near the TSS. Thus, the STREAMING-tag system revealed the spatiotemporal relationships between transcriptional activity and protein clusters near the gene. This powerful tool is useful for quantitatively understanding dynamic transcriptional regulation in living cells.

Protein kinase A promotes beige adipogenesis downstream from β-adrenergic receptor signaling by phosphorylating proteins, including histone H3 lysine 9 (H3K9) demethylase JMJD1A. To ensure homeostasis, this process needs to be reversible however, this step is not well understood. We show that myosin phosphatase target subunit 1- protein phosphatase 1β (MYPT1-PP1β) phosphatase activity is inhibited via PKA-dependent phosphorylation, which increases phosphorylated JMJD1A and beige adipogenesis. Mechanistically, MYPT1-PP1β depletion results in JMJD1A-mediated H3K9 demethylation and activation of the Ucp1 enhancer/promoter regions. Interestingly, MYPT1-PP1β also dephosphorylates myosin light chain which regulates actomyosin tension-mediated activation of YAP/TAZ which directly stimulates Ucp1 gene expression. Pre-adipocyte specific Mypt1 deficiency increases cold tolerance with higher Ucp1 levels in subcutaneous white adipose tissues compared to control mice, confirming this regulatory mechanism in vivo. Thus, we have uncovered regulatory cross-talk involved in beige adipogenesis that coordinates epigenetic regulation with direct activation of the mechano-sensitive YAP/TAZ transcriptional co-activators.

With the remarkable scientific advances in stem cells technologies and culture systems, it is no longer a dream to construct in vitro cultured tissue/organ models that respond completely as if they were in vivo. The microphysiological system (MPS) is a symbol of the growing worldwide momentum to promote the use of in vitro methods. The development of MPS devices as a culture system itself has been almost completed both in Japan and overseas, thus, the focus from now on is on the construction of prediction systems and evaluation of their applicability in accordance with the specific requirements (context of use: CoU), for example, of the drug discovery process of pharmaceutical companies. A notable trend at this stage is the close communication between developers (companies and researchers), users, and regulatory authorities, and the organization of the European Organ on a Chip Society (EUROoCS) and the International MPS Summit involving these stakeholders has begun. It is strongly expected that academic studies on in vitro systems symbolized by MPS and their results for the evaluation and prediction of individual responses and their application in society will continue to advance, leading to the promotion of alternatives to animal experiments.

Background Viruses must adapt to the environment of their host cells to establish infection and persist. Diverse mammalian cells, including virus-infected cells, release extracellular vesicles such as exosomes containing proteins and miRNAs, and use these vesicles to mediate intercellular communication. However, the roles of exosomes in viral infection remain unclear. Results We screened viral proteins to identify those responsible for the exosome-mediated enhancement of Epstein–Barr virus (EBV) infection. We identified BGLF2 protein encapsulated in exosomes, which were released by EBV-infected cells. BGLF2 protein is a tegument protein that exists in the space between the envelope and nucleocapsid, and it is released into the cytoplasm shortly after infection. BGLF2 protein-containing exosomes enhanced viral gene expression and repressed innate immunity, thereby supporting the EBV infection. Conclusions The EBV tegument protein BGLF2 is encapsulated in exosomes and released by infected cells to facilitate the establishment of EBV infection. These findings suggest that tegument proteins support viral infection not only between the envelope and nucleocapsid, as well as in extraviral particles such as exosomes. Graphical abstract

Fractal scaling in animal behavioral activity, where similar temporal patterns appear repeatedly over a series of magnifications among time scales, governs the complex behavior of various animal species and, in humans, can be altered by neurodegenerative diseases and aging. However, the mechanism underlying fractal scaling remains unknown. Here, we culturedC. elegansin a microfluidic device for 3 days and analyzed temporal patterns ofC. elegansactivity by fractal analyses. The residence-time distribution ofC. elegansbehaviors shared a common feature with those of human and mice. Specifically, the residence-time power-law distribution of the active state changed to an exponential-like decline at a longer time scale, whereas the inactive state followed a power-law distribution. An exponential-like decline appeared with nutrient supply in wild-type animals, whereas this decline disappeared in insulin-signaling-defectivedaf-2anddaf-16mutants. The absolute value of the power-law exponent of the inactive state distribution increased with nutrient supply in wild-type animals, whereas the value decreased indaf-2anddaf-16mutants. We conclude that insulin signaling differentially affects mechanisms that determine the residence time in active and inactive states inC. elegansbehavior. In humans, diabetes mellitus, which is caused by defects in insulin signaling, is associated with mood disorders that affect daily behavioral activities. We hypothesize that comorbid behavioral defects in patients with diabetes may be attributed to altered fractal scaling of human behavior.

While the long-term administration of antipsychotics is known to cause dopamine supersensitivity psychosis (DSP), recent studies revealed that DSP helps form the foundation of treatment resistance. Electroconvulsive shock (ES) is one of the more effective treatments for treatment-resistant schizophrenia. The objective of this study was to examine whether repeated ES can release rats from dopamine supersensitivity states such as striatal dopamine D2 receptor (DRD2) up-regulation and voluntary hyperlocomotion following chronic administration of haloperidol (HAL). HAL (0.75 mg/kg/day) was administered for 14 days via mini-pumps implanted in rats, and DRD2 density and voluntary locomotion were measured one day after drug cessation to confirm the development of dopamine supersensitivity. The rats with or without dopamine supersensitivity received repeated ES or sham treatments, and then DRD2 density was assessed and a voluntary locomotion test was performed. Chronic treatment with HAL led to the up-regulation of striatal DRD2 and hyperlocomotion in the rats one day after drug cessation. We thus confirmed that these rats experienced a dopamine supersensitivity state. Moreover, after repeated ES, locomotor activity and DRD2 density in the DSP model rats fell to the control level, while an ES sham operation had no effect on the dopamine supersensitivity state. The present study suggests that repeated ES could release DSP model rats from dopamine supersensitivity states. ES may be helpful for patients with DSP.

Background DNA methylation is a significant epigenetic modification that is evolutionarily conserved in various species and often serves as a repressive mark for transcription. DNA methylation levels and patterns are regulated by a balance of opposing enzyme functions, DNA methyltransferases, DNMT1/3A/3B and methylcytosine dioxygenases, TET1/2/3. In mice, the TET enzyme converts DNA cytosine methylation (5mC) to 5-hydroxymethylcytosine (5hmC) at the beginning of fertilisation and gastrulation and initiates a global loss of 5mC, while the 5mC level is increased on the onset of cell differentiation during early embryonic development. Objective Global loss and gain of DNA methylation may be differently regulated in diverged species. Methods Chicken B-cell lymphoma DT40 cells were used as an avian model to compare differences in the overall regulation of DNA modification with mammals. Results We found that DNA methylation is maintained at high levels in DT40 cells through compact chromatin formation, which inhibits TET-mediated demethylation. Human and mouse chromosomes introduced into DT40 cells by cell fusion lost the majority of 5mC, except for human subtelomeric repeats. Conclusion Our attempt to elucidate the differences in the epigenetic regulatory mechanisms between birds and mammals explored the evidence that they share a common chromatin-based regulation of TET–DNA access, while chicken DNMT1 is involved in different target sequence recognition systems, suggesting that factors inducing DNMT–DNA association have already diverged.

For in vivo functional analysis of a protein of interest (POI), multiple transgenic strains with a POI that harbors different tags are needed but generation of these strains is still labor-intensive work. To overcome this, we have developed a versatile Drosophila toolkit with a genetically encoded single-chain variable fragment for the HA epitope tag: ‘HA Frankenbody’. This system allows various analyses of HA-tagged POI in live tissues by simply crossing an HA Frankenbody fly with an HA-tagged POI fly. Strikingly, the GFP-mCherry tandem fluorescent-tagged HA Frankenbody revealed a block in autophagic flux and an accumulation of enlarged autolysosomes in the last instar larval and prepupal fat body. Mechanistically, lysosomal activity was downregulated at this stage, and endocytosis, but not autophagy, was indispensable for the swelling of lysosomes. Furthermore, forced activation of lysosomes by fat body-targeted overexpression of Mitf, the single MiTF/TFE family gene in Drosophila, suppressed the lysosomal swelling and resulted in pupal lethality. Collectively, we propose that downregulated lysosomal function in the fat body plays a role in the metamorphosis of Drosophila.

Parenchymal hepatocytes are responsible for most of the metabolic functions of the liver, but exhibit distinct functional properties depending on their localization within the hepatic lobule. Cytochrome P450 oxidases represent a family of drug-metabolizing enzymes, which are expressed predominantly in hepatocytes localized in the centrilobular area (zone 3). The present study describes a unique transgenic mouse strain that distinguishes zone 3 hepatocytes from periportal zone 1 hepatocytes by the intensity of EGFP fluorescence. Both zone 1 and zone 3 hepatocytes isolated from these mice showed the same zone-specific gene expression patterns as in liver tissue in vivo. Experiments using primary cultures of hepatocytes indicated that a combination of low oxygen concentration and activation of Wnt/β-catenin signaling maintained the expression of zone 3-specific P450 drug-metabolizing enzymes, which was characterized by their susceptibility to acetaminophen-induced mitochondrial dysfunction. These zone-specific hepatocytes provide a useful system in the research area of liver pathophysiology and drug development.

Epstein‐Barr virus (EBV) is a well‐established tumor virus that has been implicated in a wide range of immunodeficiency‐associated lymphoproliferative disorders (LPDs). Although rituximab, a CD20 mAb, has proven effective against EBV‐associated LPDs, prolonged use of this drug could lead to resistance due to the selective expansion of CD20− cells. We have previously shown that cyclin‐dependent kinase (CDK) inhibitors are able to specifically suppress the expression of viral late genes, particularly those encoding structural proteins; however, the therapeutic effect of CDK inhibitors against EBV‐associated LPDs is not clear. In this study, we examined whether CDK inhibitors confer a therapeutic effect against LPDs in vivo. Treatment with alsterpaullone, an inhibitor of the CDK2 complex, resulted in a survival benefit and suppressed tumor invasion in a mouse model of LPDs. Inhibition of CDK efficiently induced G1 cell cycle arrest and apoptosis in EBV‐positive B cells. These results suggest that alsterpaullone suppresses cell cycle progression, resulting in the antitumor effect observed in vivo., Epstein‐Barr virus (EBV) is a well‐established tumor virus that has been implicated in a wide range of immunodeficiency‐associated lymphoproliferative disorders (LPDs). In this study, we examined whether cyclin‐dependent kinase (CDK) inhibitors confer a therapeutic effect against LPDs in vivo. Treatment with alsterpaullone, an inhibitor of the CDK2 complex, resulted in a survival benefit and suppressed tumor invasion in a mouse model of LPDs. Inhibition of CDK efficiently induced G1 cell cycle arrest and apoptosis in EBV‐positive B cells.

Enhancer activation is essential for cell-type specific gene expression during cellular differentiation, however, how enhancers transition from a hypoacetylated “primed” state to a hyperacetylated-active state is incompletely understood. Here, we show SET domain-containing 5 (SETD5) forms a complex with NCoR-HDAC3 co-repressor that prevents histone acetylation of enhancers for two master adipogenic regulatory genes Cebpa and Pparg early during adipogenesis. The loss of SETD5 from the complex is followed by enhancer hyperacetylation. SETD5 protein levels were transiently increased and rapidly degraded prior to enhancer activation providing a mechanism for the loss of SETD5 during the transition. We show that induction of the CDC20 co-activator of the ubiquitin ligase leads to APC/C mediated degradation of SETD5 during the transition and this operates as a molecular switch that facilitates adipogenesis., How enhancers transition from a hypoacetylated primed state to a hyperacetylated-active state in response to differentiation stimuli is incompletely understood. Here the authors show that SETD5 forms a complex with NCoR-HDAC3 co-repressor to prevent histone acetylation of master adipogenic gene enhancers, while SETD5 degradation triggers enhancer hyperacetylation and transition to active state.

Posttranslational histone modifications are associated with the regulation of genome function. Some modifications are quite stable to maintain epigenome states of chromatin, and others can exhibit dynamic changes in response to internal and external stimuli. To track the local and global changes in histone modifications, multiplexed imaging in living cells is beneficial. Among live cell probes for detecting histone modifications, genetically encoded modification-specific intracellular antibodies, or mintbodies, are convenient and suitable tools for this purpose. We here describe the mintbody-based methods to monitor the changes in histone modification levels induced by histone methyltransferase and deacetylase inhibitors. By measuring the nuclear to cytoplasmic intensity ratios of mintbodies in living cells, changes in histone H4 lysine 20 methylation states and the increase in histone H3 acetylation were detected.

Background The excessive blockade of dopamine D2 receptors (DRD2s) with long-term antipsychotic treatment is known to induce a dopamine supersensitivity state (DSS). The mechanism of DSS is speculated to be a compensatory up-regulation of DRD2s, but an excess blockade of DRD2s can also cause glutamatergic neuronal damage. Herein, we investigated whether antipsychotic-induced neuronal damage plays a role in the development of DSS. Methods Haloperidol (HAL; 0.75 mg/kg/day for 14 days) or vehicle was administered to rats via an osmotic mini-pump. Haloperidol-treated rats were divided into groups of DSS rats and non-DSS rats based on their voluntary locomotion data. We then determined the tissue levels of glutamate transporter-1 (GLT-1)/glutamine synthetase (GS) and heat shock protein-70 (HSP-70) in the rats' brain regions. Results The levels of HSP-70 in the striatum and CA-3 region of the DSS rats were significantly higher than those of the control and non-DSS rats, whereas the dentate gyrus HSP-70 levels in both the DSS and non-DSS rats were increased versus the controls. The levels of GLT-1/GS in the CA-3 and nucleus accumbens were increased in the DSS rats. Conclusions These results suggest that the DSS rats experienced striatal neuronal damage and indicate that a HAL-induced upregulation of HSP-70 and the GLT-1/GS system in the CA3 may be involved in the development of DSS. It remains unknown why the non-DSS rats did not suffer neuronal damage. In view of the need for therapeutic strategies for treatment-resistant schizophrenia, dopamine supersensitivity psychosis, and tardive dyskinesia, further investigations of our findings are warranted.

Formation of processes in podocytes is regarded as the hallmark of maturity and normal physical condition for the cell. There are many accumulated findings about molecular mechanisms that cause retraction of podocyte processes; however, there is little knowledge of the positive mechanisms that promote process formation in vitro, and most previous reports about this topic have been limited to low-density cultures. Here, we found that process formation can be induced in 100% confluent cultures of conditionally immortalized podocytes in mouse, rat, and human species by combining serum depletion and Y-27632 ROCK inhibitor supplementation on the scaffold of laminin-521(L521). We noted the cytoskeletal reorganization of the radial extension pattern of vimentin filaments and downregulation of actin stress fiber formation under that condition. We also found that additional standard amount of serum, depletion of ROCK inhibitor, or slight mismatch of the scaffold as laminin-511(L511) hinder process formation. These findings suggest that the combination of reduced serum, podocyte-specific scaffold, and intracellular signaling to reduce the overexpression of ROCK are required factors for process formation.

Posttranslational histone modifications are critical for the regulation of genome function. The levels of histone modifications oscillate during the cell cycle. Most modifications are diluted after DNA replication and then their levels are restored during the rest of the cell cycle with different kinetics depending on the modification. Some modifications, like histone H4 Lys20 monomethylation (H4K20me1), exhibit cell cycle-dependent dynamic changes. To track histone modifications in living cells, we have developed genetically encoded probes termed modification specific intracellular antibodies, or "mintbodies." As mintbodies shuttle between the cytoplasm and nucleus by diffusion, their nuclear concentration depends on the target modification level. By measuring the nuclear to cytoplasmic intensity ratio of H4K20me1-specific mintbody, we have monitored the increase of H4K20me1 in the G2 phase. Here we describe how the mintbody-based methods can be applied to track a specific chromosome, such as the inactive X chromosome (Xi), on which genes are repressed through histone H3 Lys27 trimethylation (H3K27me3). When H3K27me3-specific mintbodies are expressed in cells that harbor Xi, the mintbodies are concentrated on Xi and the dynamic behavior of Xi can be tracked using a confocal microscope. After acquiring 3D time-lapse images, an image analysis allows measuring the volume, shape and H3K27me3 level of Xi during the cell cycle.

The lysine methyltransferase SETDB1, an enzyme responsible for methylation of histone H3 at lysine 9, plays a key role in H3K9 tri-methylation-dependent silencing of endogenous retroviruses and developmental genes. Recent studies have shown that ubiquitination of human SETDB1 complements its catalytic activity and the silencing of endogenous retroviruses in human embryonic stem cells. However, it is not known whether SETDB1 ubiquitination is essential for its other major role in epigenetic silencing of developmental gene programs. We previously showed that SETDB1 contributes to the formation of H3K4/H3K9me3 bivalent chromatin domains that keep adipogenic Cebpa and Pparg genes in a poised state for activation and restricts the differentiation potential of pre-adipocytes. Here, we show that ubiquitin-resistant K885A mutant of SETDB1 represses adipogenic genes and inhibits pre-adipocyte differentiation similar to wild-type SETDB1. We show this was due to a compensation mechanism for H3K9me3 chromatin modifications on the Cebpa locus by other H3K9 methyltransferases Suv39H1 and Suv39H2. In contrast, the K885A mutant did not repress other SETDB1 target genes such as Tril and Gas6 suggesting SETDB1 represses its target genes by two mechanisms; one that requires its ubiquitination and another that still requires SETDB1 but not its enzyme activity.

Mutations in the factor-induced-gene 4 (FIG 4) gene are associated with multiple disorders, including Charcot-Marie-Tooth disease (CMT), epilepsy with polymicrogyria, Yunis-Varón syndrome and amyotrophic lateral sclerosis. The wide spectrum of disorders associated with FIG 4 may be related to the dysregulated epigenetics. Using Gene Expression Omnibus, we found that HDAC1 binds to the FIG 4 gene locus in the genome of human CD4+ T cells. Rpd3 is a well-known Drosophila homolog of human HDAC1. We previously established Drosophila models targeting Drosophila FIG 4 (dFIG 4) that exhibited defective locomotive ability, abnormal synapse morphology at neuromuscular junctions, enlarged vacuoles in the fat body and aberrant compound eye morphology. Genetic crossing experiments followed by physiological and immunocytochemical analyses revealed that Rpd3 mutations suppressed these defects induced by dFIG 4 knockdown. This demonstrated Rpd3 to be an important epigenetic regulator of dFIG 4, suggesting that the inhibition of HDAC1 represses the pathogenesis of FIG 4-associated disorders, including CMT. Defects in epigenetic regulators, such as HDAC1, may also explain the diverse symptoms of FIG 4-associated disorders.

Recent developments in therapeutic strategies have improved the prognosis of head and neck squamous cell carcinoma (HNSCC). Nevertheless, 5-year survival rate remains only 40%, necessitating new therapeutic agents. Oncolytic virotherapy entails use of replication-competent viruses to selectively kill cancer cells. We aimed to explore the potential of HF10 as an oncolytic virus against human or mouse HNSCC cell lines, and primary-cultured HNSCC cells. HF10 replicated well in all the HNSCC cells, in which it induced cytopathic effects and cell killing. Next, we investigated the oncolytic effects of HF10 in ear tumor models with human or mouse tumor cells. We detected HF10-infected cells within the ear tumors based on their expression of green fluorescent protein. HF10 injection suppressed ear tumor growth and prolonged overall survival. In the syngeneic model, HF10 infection induced tumor necrosis with infiltration of CD8-positive cells. Moreover, the splenocytes of HF10-treated mice released antitumor cytokines, IL-2, IL-12, IFN-alpha, IFN-beta, IFN-gamma, and TNF-alpha, after stimulation with tumor cells in vitro. The HF10-treated mice that survived their original tumor burdens rejected tumor cells upon re-challenge. These results suggested that HF10 killed HNSCC cells and induced antitumoral immunity, thereby establishing it as a promising agent for the treatment of HNSCC patients.

Epstein-Barr virus (EBV) infection is highly prevalent in humans and is implicated in various diseases, including cancer1,2. Chronic active EBV infection (CAEBV) is an intractable disease classified as a lymphoproliferative disorder in the 2016 World Health Organization lymphoma classification1,2. CAEBV is characterized by EBV-infected T/natural killer (NK) cells and recurrent/persistent infectious mononucleosis-like symptoms3. Here, we show that CAEBV originates from an EBV-infected lymphoid progenitor that acquires DDX3X and other mutations, causing clonal evolution comprising multiple cell lineages. Conspicuously, the EBV genome in CAEBV patients harboured frequent intragenic deletions (27/77) that were also common in various EBV-associated neoplastic disorders (28/61), including extranodal NK/T-cell lymphoma and EBV-positive diffuse large B-cell lymphoma, but were not detected in infectious mononucleosis or post-transplant lymphoproliferative disorders (0/47), which suggests a unique role of these mutations in neoplastic proliferation of EBV-infected cells. These deletions frequently affected BamHI A rightward transcript microRNA clusters (31 cases) and several genes that are essential for producing viral particles (20 cases). The deletions observed in our study are thought to reactivate the lytic cycle by upregulating the expression of two immediate early genes, BZLF1 and BRLF14-7, while averting viral production and subsequent cell lysis. In fact, the deletion of one of the essential genes, BALF5, resulted in upregulation of the lytic cycle and the promotion of lymphomagenesis in a xenograft model. Our findings highlight a pathogenic link between intragenic EBV deletions and EBV-associated neoplastic proliferations.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective loss of motor neurons. We have previously shown that autophagosome-like vesicular structures are progressively accumulated in the spinal axons of an ALS mouse model, overexpressing human Cu/Zn superoxide dismutase (SOD1) mutant, prior to the onset of motor symptoms. This suggests that axonal transport perturbation can be an early sign of neuronal dysfunction. However, the exact causal relationship between axonal transport deficits and neurodegeneration is not fully understood. To clarify whether axonal transport of organelles even in neurons at early developmental stages was affected by overexpression of mutant SOD1, we conducted a microfluidic device-based high-throughput quantitative analysis of the axonal transport of acidic vesicles and mitochondria in primary cultured cortical neurons established from SOD1

Examining intestine–liver interactions is important for achieving the desired physiological drug absorption and metabolism response in in vitro drug tests. Multi-organ microphysiological systems (MPSs) constitute promising tools for evaluating inter-organ interactions in vitro. For coculture on MPSs, normal cells are challenging to use because they require complex maintenance and careful handling. Herein, we demonstrated the potential of coculturing normal cells on MPSs in the evaluation of intestine–liver interactions. To this end, we cocultured human-induced pluripotent stem cell-derived intestinal cells and fresh human hepatocytes which were isolated from PXB mice with medium circulation in a pneumatic-pressure-driven MPS with pipette-friendly liquid-handling options. The cytochrome activity, albumin production, and liver-specific gene expressions in human hepatocytes freshly isolated from a PXB mouse were significantly upregulated via coculture with hiPS-intestinal cells. Our normal cell coculture shows the effects of the interactions between the intestine and liver that may occur in vivo. This study is the first to demonstrate the coculturing of hiPS-intestinal cells and fresh human hepatocytes on an MPS for examining pure inter-organ interactions. Normal-cell coculture using the multi-organ MPS could be pursued to explore unknown physiological mechanisms of inter-organ interactions in vitro and investigate the physiological response of new drugs.

The fine-scale dynamics from euchromatin (EC) to facultative heterochromatin (fHC) has remained largely unclear. Here, we focus on Xist and its silencing initiator Tsix as a paradigm of transcription-mediated conversion from EC to fHC. In mouse epiblast stem cells, induction of Tsix recapitulates the conversion at the Xist promoter. Investigating the dynamics reveals that the conversion proceeds in a stepwise manner. Initially, a transient opened chromatin structure is observed. In the second step, gene silencing is initiated and dependent on Tsix, which is reversible and accompanied by simultaneous changes in multiple histone modifications. At the last step, maintenance of silencing becomes independent of Tsix and irreversible, which correlates with occupation of the −1 position of the transcription start site by a nucleosome and initiation of DNA methylation introduction. This study highlights the hierarchy of multiple chromatin events upon stepwise gene silencing establishment., Cell Reports, 34 (13), ISSN:2666-3864, ISSN:2211-1247

Summary During X chromosome inactivation (XCI), the inactive X chromosome (Xi) is recruited to the nuclear lamina at the nuclear periphery. Beside X chromosome reactivation resulting in a highly penetrant aging-like hematopoietic malignancy, little is known about XCI in aged hematopoietic stem cells (HSCs). Here, we demonstrate that LaminA/C defines a distinct repressive nuclear compartment for XCI in young HSCs, and its reduction in aged HSCs correlates with an impairment in the overall control of XCI. Integrated omics analyses reveal higher variation in gene expression, global hypomethylation, and significantly increased chromatin accessibility on the X chromosome (Chr X) in aged HSCs. In summary, our data support the role of LaminA/C in the establishment of a special repressive compartment for XCI in HSCs, which is impaired upon aging., Graphical abstract, Highlights • Xist lncRNA expression is reduced in HSCs upon aging • The inactive Chr X is distant from the repressive nuclear periphery in aged HSCs • The inactive Chr X is located at the LaminA/C pole in young HSCs • Chromatin accessibility on the X chromosome is increased in aged HSCs, In this article, Mulaw, Florian and colleagues show that LaminA/C reduction in aged HSCs correlates with an impairment in X chromosome inactivation (XCI) in aged hematopoietic stem cells (HSCs). Consistently, integrated omics analyses reveal higher variation in gene expression, global hypomethylation, and significantly increased chromatin accessibility on the X chromosome (Chr X) in aged stem cells.

© 2021 EMBO. This is an open access article under the terms of the Creative Commons Attribution License,which permits use, distribution and reproduction in any medium, provided the original work is properly cited., During X chromosome inactivation (XCI), in female placental mammals, gene silencing is initiated by the Xist long non-coding RNA. Xist accumulation at the X leads to enrichment of specific chromatin marks, including PRC2-dependent H3K27me3 and SETD8-dependent H4K20me1. However, the dynamics of this process in relation to Xist RNA accumulation remains unknown as is the involvement of H4K20me1 in initiating gene silencing. To follow XCI dynamics in living cells, we developed a genetically encoded, H3K27me3-specific intracellular antibody or H3K27me3-mintbody. By combining live-cell imaging of H3K27me3, H4K20me1, the X chromosome and Xist RNA, with ChIP-seq analysis we uncover concurrent accumulation of both marks during XCI, albeit with distinct genomic distributions. Furthermore, using a Xist B and C repeat mutant, which still shows gene silencing on the X but not H3K27me3 deposition, we also find a complete lack of H4K20me1 enrichment. This demonstrates that H4K20me1 is dispensable for the initiation of gene silencing, although it may have a role in the chromatin compaction that characterises facultative heterochromatin., This work was supported by Fundação para a Ciência e Tecnologia (S.T.d.R), project grants PTDC/BIA‐ MOL/29320/2017 IC&DT (A. C. R. & S.T.d.R), CEECUIND/01234/207 (S.T.d.R), and SFRH/BD/137099/2018 (A.C.R.), by an ERC Advanced Investigator award ERC‐ADG‐2014 671027 attributed to E.H., Sir Henry Wellcome Postdoctoral Fellowship (J.J.Z.), Japan Society for the Promotion of Science KAKENHI grants (JP17KK0143 and JP20K06484 to Y.S., JP19H04970, JP19H03158 and JP20H05393 to K.M., JP17K17719 to T.H., JP18H05534 to H.Ku, JP18H05527 and JP20H00456 to Y.O., JP17H01417 and JP18H05527 to H.Ki), and Japan Science and Technology Agency (JST) CREST JPMJCR16G1 to T.K., H.Ku, Y.O. and H.Ki, PREST JPMJPR2026 to K.M., and ERATO JPMJER1901 to H.Ku. J.J.Z. is supported by core funding of The Novo Nordisk Foundation Center for Stem Cell Biology (Novo Nordisk Foundation grant number NNF17CC0027852). Open Access funding enabled and organized by Projekt DEAL.

Background Some individuals with schizophrenia present with a dopamine supersensitivity state (DSS) induced by a long-term administration of excessive antipsychotics; this is recognized as dopamine supersensitivity psychosis (DSP). The mechanisms underlying DSP are not established. Here, we investigated dopamine signaling in DSS rats. Methods Haloperidol (HAL; 0.75 mg/kg/day for 14 days) or vehicle was administered to rats via an osmotic mini-pump. We then screened DSS rats from HAL-treated rats by a voluntary locomotion test. The striatal levels of dopamine (DA) and its metabolites 3,4-hydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were determined, as were the levels of protein kinase v-akt murine thymoma viral oncogene homolog (AKT), glycogen synthase kinase-3 (GSK-3), and phosphorylated GSK-3 in the striatal regions. Results In the DSS rats, the DA, DOPAC, and HVA levels were significantly decreased. In a western blot analysis, the DSS rats exhibited a significant decrease in GSK-3α/β and an increase in the pGSK-3β/GSK-3β ratio, whereas AKT was not changed. Conclusions Our results indicated that the DSS rats had hypofunction of the basal dopamine release and AKT/GSK-3 signaling even at 7 days after the antipsychotic was discontinued. Protracted reductions in pre- and post-dopamine D2 receptor signaling might cause prolonged DSS.

In vitro spermatogenesis (IVS) using air–liquid interphase organ culture method is possible with mouse testis tissues. The same method, however, has been hardly applicable to animals other than mice, only producing no or limited progression of spermatogenesis. In the present study, we challenged IVS of rats with modifications of culture medium, by supplementing chemical substances, including hormones, antioxidants, and lysophospholipids. In addition, reducing oxygen tension by placing tissues in an incubator of lower oxygen concentration and/or applying silicone cover ceiling on top of the tissue were effective for improving the spermatogenic efficiency. Through these modifications of the culture condition, rat spermatogenesis up to round spermatids was maintained over 70 days in the cultured tissue. Present results demonstrated a significant progress in rat IVS, revealing conditions commonly favorable for mice and rats as well as finding rat-specific optimizations. This is an important step towards successful IVS in many animal species, including humans.

The kinetic properties of transcriptional bursting were comprehensively analyzed in mouse embryonic stem cells., Transcriptional bursting is the stochastic activation and inactivation of promoters, contributing to cell-to-cell heterogeneity in gene expression. However, the mechanism underlying the regulation of transcriptional bursting kinetics (burst size and frequency) in mammalian cells remains elusive. In this study, we performed single-cell RNA sequencing to analyze the intrinsic noise and mRNA levels for elucidating the transcriptional bursting kinetics in mouse embryonic stem cells. Informatics analyses and functional assays revealed that transcriptional bursting kinetics was regulated by a combination of promoter- and gene body–binding proteins, including the polycomb repressive complex 2 and transcription elongation factors. Furthermore, large-scale CRISPR-Cas9–based screening identified that the Akt/MAPK signaling pathway regulated bursting kinetics by modulating transcription elongation efficiency. These results uncovered the key molecular mechanisms underlying transcriptional bursting and cell-to-cell gene expression noise in mammalian cells.

Organ culture experiments can be hampered by central degeneration or necrosis due to the inadequate permeation of oxygen and nutrients, which deteriorates the function and growth of cultured tissues. In the current study, we aimed to overcome this limitation of organ culture through spreading the tissue two dimensionally on an agarose gel stand and molding into a disc shape by placing a ceiling of polydimethylsiloxane (PDMS) chip, which is highly oxygen permeable. By this, every part of the tissue can receive a sufficient supply of oxygen through PDMS as well as nutrients through the agarose gel below. This method not only prevented central necrosis of tissues, but also supported the tissue growth over time. In addition, such growth, as volume enlargement, could be easily measured. Under these conditions, we examined the effect of several factors on the growth of neonatal mouse testis, and found that follicle stimulating hormone (FSH) and insulin significantly promoted the growth. These results are in good agreement with previous in vivo reports. Notably, the growth achieved over 7 days in our in vitro system is almost comparable to, about 80% of, that observed in vivo. Thus, we successfully monitored the promotion of tissue growth beyond the limits of the conventional organ culture method. This extremely simple method could offer a unique platform to evaluate the growth as well as functional properties of organs, not only the testis but also others as well.

Kidney organoid is an emerging topic of importance for research in kidney development and regeneration. Conventional culture systems for kidney organoids reported thus far use culture media containing serum, which may compromise our understanding and the potential clinical applicability of the organoid system. In our present study, we tested two serum-free culture conditions and compared their suitability for the maintenance and growth of kidney organoids in culture. One of the serum-free culture conditions was the combination of keratinocytes serum free medium (KSFM) with knockout serum replacement (KSR) (KSFM + KSR), and the other was the combination of knockout DMEM/F12 (KD/F12) and KSR (KD/F12 + KSR). With cell aggregates derived from E12.5 mouse embryonic kidneys, we found that KD/F12 + KSR was superior to KSFM + KSR in promoting the growth of the aggregate with expansion of Six2+ nephron progenitor cells (NPC) and elaborated ureteric branching morphogenesis. With KD/F12 + KSR, we found that lower concentrations of KSR at 5–10% were superior to a higher concentration (20%) in promoting the growth of aggregates without affecting the expression levels of NPC marker genes. We also found that NPC in aggregates retained their differentiation potential to develop nephron tubules through mesenchyme-to-epithelial transition (MET), after being maintained in culture under these conditions for up to 7 days. In conclusion, we have identified a defined serum-free culture condition suitable for the maintenance and growth of kidney organoids that retain the differentiation potential to develop nephron structures. This defined serum-free culture condition may serve as a useful platform for further investigation of kidney organoids in vitro.

A microbioreactor immobilized with a synthase-type mutant enzyme, Endo-M-N175Q (glycosynthase) of endo-β-N-acetylglucosaminidase derived from Mucor hiemalis (Endo-M), was constructed and used for glycoconjugate synthesis. The transglycosylation was performed with a reaction mixture containing an oxazoline derivative of sialo complex-type glycoside (SG), which was prepared from a sialo complex-type glycopeptide SGP derived from hen egg yolk, as a glycosyl donor and N-Fmoc-N-acetylglucosaminyl- l -asparagine [Fmoc-Asn(GlcNAc)-OH] as an acceptor. The reaction mixture was injected into a glycosynthase microbioreactor at a constant flow rate. Highly efficient and nearly stoichiometric transglycosylation occurred in the microbioreactor, and the transglycosylation product was eluted from the other end of the reactor. The glycosynthase microbioreactor was stable and could be used repeatedly for a long time.

Daiokanzoto (DKT) exerts its laxative effect via colonic inflammation caused by sennoside A in Daio (rhubarb). Previously, we showed that the laxative effect of sennoside A is related to decreased aquaporin-3 (AQP3) expression in mucosal epithelial cells due to colonic inflammation. We also found that a combination of glycyrrhizin, an ingredient in Kanzo (glycyrrhiza), and sennoside A attenuates the inflammatory response induced by sennoside A and reduces its laxative effect. These findings indicate that DKT may be a long-term treatment for chronic constipation, but there is no evidence supporting this hypothesis. In this study, we analyzed the laxative effect of repeated DKT administration, focusing on AQP3 expression in the colon. After rats were treated for 7 days, decreased AQP3 expression and the onset of diarrhea were observed in the DKT group, but were not seen in the Daio group either. Although the relative abundance of gut microbiota after repeated DKT administration was similar to that after control treatment, Daio reduced Lactobacillaceae, Bifidobacteriaceae, and Bacteroidaceae levels and markedly increased Lachnospiraceae levels. In this study, we show that DKT has a sustained laxative effect, even upon repeated use, probably because it maintains decreased AQP3 expression and gut microbiota homeostasis. This outcome therefore indicates that DKT can be used as a long-term treatment for chronic constipation.

In eukaryotes, DNA is packed inside the cell nucleus in the form of chromatin, which consists of DNA, proteins such as histones, and RNA. Euchromatin, which is permissive for transcription, is spatially organized into transcriptionally inactive domains interspersed with pockets of transcriptional activity. While transcription and RNA have been implicated in euchromatin organization, it remains unclear how their interplay forms and maintains transcription pockets. Here we combine theory and experiment to analyze the dynamics of euchromatin organization as pluripotent zebrafish cells exit mitosis and begin transcription. We show that accumulation of RNA induces formation of transcription pockets which displace transcriptionally inactive chromatin. We propose that the accumulating RNA recruits RNA-binding proteins that together tend to separate from transcriptionally inactive euchromatin. Full phase separation is prevented because RNA remains tethered to transcribed euchromatin through RNA polymerases. Instead, smaller scale microphases emerge that do not grow further and form the typical pattern of euchromatin organization.

The human herpes virus, Epstein-Barr virus (EBV), is a known oncogenic virus and plays important roles in life-threatening T/NK-cell lymphoproliferative disorders (T/NK-cell LPD) such as hypersensitivity to mosquito bite (HMB), chronic active EBV infection (CAEBV), and NK/T-cell lymphoma/leukemia. During the clinical courses of HMB and CAEBV, patients frequently develop malignant lymphomas and the diseases passively progress sequentially. In the present study, gene expression of CD16(-)CD56(+)-, EBV(+) HMB, CAEBV, NK-lymphoma, and NK-leukemia cell lines, which were established from patients, was analyzed using oligonucleotide microarrays and compared to that of CD56brightCD16dim/- NK cells from healthy donors. Principal components analysis showed that CAEBV and NK-lymphoma cells were relatively closely located, indicating that they had similar expression profiles. Unsupervised hierarchal clustering analyses of microarray data and gene ontology analysis revealed specific gene clusters and identified several candidate genes responsible for disease that can be used to discriminate each category of NK-LPD and NK-cell lymphoma/leukemia.

The complement C4 is a thioester-containing protein, and a histidine (H) residue catalyzes the cleavage of the thioester to allow covalent binding to carbohydrates on target cells. Some mammalian and teleost species possess an additional isotype where the catalytic H is replaced by an aspartic acid (D), which binds preferentially to proteins. We found the two C4 isotypes in many other jawed vertebrates, including sharks and birds/reptiles. Phylogenetic analysis suggested that C4 gene duplication occurred in the early days of the jawed vertebrate evolution. The D-type C4 of bony fish except for mammals formed a cluster, termed D-lineage. The D-lineage genes were located in a syntenic region outside MHC, and evolved conservatively. Mammals lost the D-lineage before speciation, but D-type C4 was regenerated by recent gene duplication in some mammalian species or groups. Dual C4 molecules with different substrate specificities would have contributed to development of the antibody-dependent classical pathway.

The octopus arm contains a tridimensional array of muscles with a massive sensory-motor system. We herein provide the first evidence for the existence of serotonin (5-HT) in the octopus arm nervous system and investigated its distribution using immunohistochemistry. 5-HT-like immunoreactive (5-HT-lir) nerve cell bodies were exclusively localized in the cellular layer of the axial nerve cord. Those cell bodies emitted 5-HT-lir nerve fibers in the direction of the sucker, the intramuscular nerves cords, the ganglion of the sucker, and the intrinsic musculature. Others 5-HT-lir nerve fibers were observed in various tissues, including the cerebrobrachial tract, the skin, and the blood vessels. 5-HT was detected by high-performance liquid chromatography in various regions of the octopus arm at levels matching the density of 5-HT-lir staining. The absence of 5-HT-lir interconnections between the cerebrobrachial tract and the other components of the axial nerve cord suggests that two types of 5-HT-lir innervation exist in the arm. One type, which originates from the brain, may innervate the periphery through the cerebrobrachial tract. Another type, which originates in the cellular layer of the axial nerve cord, may form an intrinsic network in the arm. In addition, 5-HT-lir fibers likely emitted from the neuropil of the axial nerve cord were found to project into cells showing staining for peripheral choline acetyltransferase, a marker of sensory cells of the sucker. Taken together, these observations suggest that intrinsic 5-HT-lir innervation may participate in the sensory transmission in the octopus arm.

// Shotaro Ando 1 , Jun-ichi Kawada 1 , Takahiro Watanabe 2 , Michio Suzuki 1 , Yoshitaka Sato 2 , Yuka Torii 1 , Masato Asai 3 , Fumi Goshima 2 , Takayuki Murata 2 , Norio Shimizu 4 , Yoshinori Ito 1 , Hiroshi Kimura 2 1 Departments of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan 2 Departments of Virology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan 3 Departments of Pathology, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya 466-8550, Japan 4 Center of Stem Cell and Regenerative Medicine, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo 101-0062, Japan Correspondence to: Jun-ichi Kawada, email: kawadaj@med.nagoya-u.ac.jp Keywords: tofacitinib, EBV, lymphoma, cell-cycle arrest Received: June 13, 2016 Accepted: October 03, 2016 Published: October 08, 2016 ABSTRACT Epstein-Barr virus (EBV) infects not only B cells, but also T cells and natural killer (NK) cells, and is associated with T or NK cell lymphoma. These lymphoid malignancies are refractory to conventional chemotherapy. We examined the activation of the JAK3/STAT5 pathway in EBV-positive and -negative B, T and NK cell lines and in cell samples from patients with EBV-associated T cell lymphoma. We then evaluated the antitumor effects of the selective JAK3 inhibitor, tofacitinib, against these cell lines in vitro and in a murine xenograft model. We found that all EBV-positive T and NK cell lines and patient samples tested displayed activation of the JAK3/STAT5 pathway. Treatment of these cell lines with tofacitinib reduced the levels of phospho-STAT5, suppressed proliferation, induced G1 cell-cycle arrest and decreased EBV LMP1 and EBNA1 expression. An EBV-negative NK cell line was also sensitive to tofacitinib, whereas an EBV-infected NK cell line was more sensitive to tofacitinib than its parental line. Tofacitinib significantly inhibited the growth of established tumors in NOG mice. These findings suggest that tofacitinib may represent a useful therapeutic agent for patients with EBV-associated T and NK cell lymphoma.

Histone post-translational modifications are key gene expression regulators, but their rapid dynamics during development remain difficult to capture. We applied a Fab-based live endogenous modification labeling technique to monitor the changes in histone modification levels during zygotic genome activation (ZGA) in living zebrafish embryos. Among various histone modifications, H3 Lys27 acetylation (H3K27ac) exhibited most drastic changes, accumulating in two nuclear foci in the 64- to 1k-cell-stage embryos. The elongating form of RNA polymerase II, which is phosphorylated at Ser2 in heptad repeats within the C-terminal domain (RNAP2 Ser2ph), and miR-430 transcripts were also concentrated in foci closely associated with H3K27ac. When treated with α-amanitin to inhibit transcription or JQ-1 to inhibit binding of acetyl-reader proteins, H3K27ac foci still appeared but RNAP2 Ser2ph and miR-430 morpholino were not concentrated in foci, suggesting that H3K27ac precedes active transcription during ZGA. We anticipate that the method presented here could be applied to a variety of developmental processes in any model and non-model organisms., Summary: FabLEM, an endogenous labeling technique that uses modification-specific antigen-binding fragments, is used to examine changes in histone modification levels and transcription during zygotic genome activation in live zebrafish embryos.

Sleep apnea syndrome (SAS) is a very common disease involving intermittent hypoxia (IH), recurrent symptoms of deoxygenation during sleep, strong daytime sleepiness, and significant loss of quality of life. A number of epidemiological researches have shown that SAS is an important risk factor for insulin resistance and type 2 diabetes mellitus (DM), which is associated with SAS regardless of age, gender, or body habitus. IH, hallmark of SAS, plays an important role in the pathogenesis of SAS and experimental studies with animal and cellular models indicate that IH leads to attenuation of glucose-induced insulin secretion from pancreatic β cells and to enhancement of insulin resistance in peripheral tissues and cells, such as liver (hepatocytes), adipose tissue (adipocytes), and skeletal muscles (myocytes). In this review, we focus on IH-induced dysfunction in glucose metabolism and its underlying molecular mechanisms in several cells and tissues related to glucose homeostasis.

Epstein&ndash, Barr virus (EBV) is a ubiquitous virus that causes infectious mononucleosis and several types of cancer, such as Burkitt lymphoma, T/NK-cell lymphoma, and nasopharyngeal carcinoma. As a herpesvirus, it encodes more than 80 genes, many of which have not been characterized. EBV BamHI S rightward reading frame 1 (BSRF1) encodes a tegument protein that, unlike its homologs herpes simplex virus unique long 51 (UL51) and human cytomegalovirus UL71, has not been extensively investigated. To examine the role of BSRF1, we prepared knockout and revertant strains using the bacterial artificial chromosome system. Unexpectedly, the disruption of the gene had little or no effect on EBV lytic replication and the transformation of primary B cells. However, the knockdown of BSRF1 in B95-8 cells decreased progeny production. An immunofluorescence assay revealed that BSRF1 localized to the Golgi apparatus in the cytoplasm, as did its homologs. BSRF1 also associated with BamHI G leftward reading frame 3.5 (BGLF3.5), BamHI B rightward reading frame 2 (BBRF2), and BamHI A leftward reading frame 1 (BALF1), and BALF1 was incorporated into the tegument fraction with BSRF1. Taken together, our results indicate that BSRF1 plays a role in secondary envelopment or virion egress in the cytoplasm, as do its homolog genes.

Clinical studies have demonstrated that allopregnanolone (3α5α-tetrahydroprogesterone, ALLO) has antidepressant-like effects on patients with depression. Previous studies have shown alteration of the astroglial glutamate transporter-1 (GLT-1) and glutamine synthetase (GS) in depression, and ALLO is known to modulate glutamate release. The present study aimed to investigate whether astroglial GLT-1 and GS are indeed involved in the antidepressant-like effects of ALLO in learned helplessness (LH) rats, a validated animal model of depression. The results of this study showed that bilateral microinjection of ALLO into the lateral ventricles could normalize the levels of GLT-1 and GS in the nucleus accumbens (NAc) and of GS in the hippocampal CA1 region of LH rats. These results suggest a certain connection between the antidepressant-like effects of ALLO and the astroglial GLT-1/GS system of the NAc in LH rats.

Desi-type chickpeas, which have long been used as a natural treatment for diabetes, have been reported to lower visceral adiposity, dyslipidemia and insulin resistance induced by a chronic high-fat diet in rats. In this study, in order to examine the effects of chickpeas of this type in an in vitro system, we used the 3T3-L1 mouse cell line, a subclone of Swiss 3T3 cells, which can differentiate into cells with an adipocyte-like phenotype, and we used ethanol extracts of chickpeas (ECP) instead of chickpeas. Treatment of the 3T3-L1 cells with ECP led to a decrease in the lipid content in the cells. The desaturation index, defined as monounsaturated fatty acids (MUFAs)/saturated fatty acids (SFAs), was also decreased by ECP due to an increase in the cellular content of SFAs and a decrease in the content of MUFAs. The decrease in this index may reflect a decreased reaction from SFA to MUFA, which is essential for fat storage. To confirm this hypothesis, we conducted a western blot analysis, which revealed a reduction in the amount of stearoyl-CoA desaturase 1 (SCD1), a key enzyme catalyzing the reaction from SFA to MUFA. We observed simultaneous inactivations of enzymes participating in lipogenesis, i.e., liver kinase B1 (LKB1), acetyl-CoA carboxylase (ACC) and AMPK, by phosphorylation, which may lead to the suppression of reactions from acetyl-CoA to SFA via malonyl-CoA in lipogenesis. We also investigated whether lipolysis is affected by ECP. The amount of carnitine palmitoyltransferase 1 (CPT1), an enzyme important for the oxidation of fatty acids, was increased by ECP treatment. ECP also led to an increase in uncoupling protein 2 (UCP2), reported as a key protein for the oxidation of fatty acids. All of these results obtained regarding lipogenesis and fatty acid metabolism in our in vitro system are consistent with the results previously shown in rats. We also examined the effects on SCD1 and lipid contents of ethanol extracts of Kabuli-type chickpeas, which are used worldwide. The effects were similar, but of much lesser magnitude compared to those of ECP described above. Thus, Desi-type chickpeas may prove to be effective for the treatment of diabetes, as they can alter the lipid content, thus reducing fat storage.

The inactive X chromosome (Xi) harbors characteristic epigenetic features, including the enrichment of histone H3 lysine 27 trimethylation (H3K27me3) and H4 lysine 20 monomethylation (H4K20me1) as well as a lack of histone acetylation. Recently, these modifications have been visualized not only in fixed specimen, but also in living cells via probes derived from modification-specific antibodies. The probes include fluorescently labeled antigen binding fragments (Fabs), which can be loaded into cells, as well as genetically encoded single-chain variable fragments tagged with the green fluorescent protein. We refer to the latter as modification specific intracellular antibodies, or "mintbodies" for short. By using Fabs or mintbodies to target Xi-specific modifications, the dynamics of Xi in living cells can be visualized.

Chromatin plays a crucial role in gene regulation, and chromatin immunoprecipitation followed by sequencing (ChIP-seq) has been the standard technique for examining protein-DNA interactions across the whole genome. However, it is difficult to obtain epigenomic information from limited numbers of cells by ChIP-seq because of sample loss during chromatin preparation and inefficient immunoprecipitation. In this study, we established an immunoprecipitation-free epigenomic profiling method named chromatin integration labelling (ChIL), which enables the amplification of genomic sequences closely associated with the target molecules before cell lysis. Using ChIL followed by sequencing (ChIL-seq), we reliably detected the distributions of histone modifications and DNA-binding factors in 100-1,000 cells. In addition, ChIL-seq successfully detected genomic regions associated with histone marks at the single-cell level. Thus, ChIL-seq offers an alternative method to ChIP-seq for epigenomic profiling using small numbers of cells, in particular, those attached to culture plates and after immunofluorescence.

Epstein-Barr virus (EBV), which is associated with B-cell proliferative disorders, also transforms T- or natural killer (NK)-lineage cells and has been connected with various T- or NK (T/NK)-cell malignancies, such as extranodal NK/T-cell lymphoma-nasal type and aggressive NK-cell leukemia. Chronic active EBV (CAEBV) disease , which occurs most often in children and young adults in East Asia, is an EBV-associated T-/NK-cell lymphoproliferative disease. Patients with CAEBV often progress to overt lymphoma or leukemia over a long-term clinical course. EBV's transforming capacity in B cells is well characterized, but the molecular pathogenesis of clonal expansion caused by EBV in T/NK cells has not yet been clarified. In the primary infection, EBV infects B cells and epithelial cells and may also infect some T/NK cells. In some individuals, because of poor presentation by specific human leukocyte antigens or the genetic background, EBV-infected T/NK cells evade host immunity and survive. Occasionally, with the help of viral oncogenes, EBV-associated T/NK lymphoproliferative diseases, such as CAEBV, may develop. The subsequent accumulation of genetic mutations and/or epigenetic modifications in driver genes, such as DDX3X and TP53, may lead to overt lymphoma and leukemia. Activation-induced cytidine deaminase and the APOBEC3 family, driven by EBV infection, may induce chromosomal recombination and somatic mutations.

Emerging evidence from recent studies has unraveled the roles of long noncoding RNAs (lncRNAs) in the function of various tissues. However, little is known about the roles of lncRNAs in kidney development. In our present study, we aimed to identify functional lncRNAs in one of the three lineages of kidney progenitor cells, i.e., metanephric mesenchymal (MM) cells. We conducted comprehensive analyses of the chromatin signature and transcriptome by RNA-seq and ChIP-seq. We found seventeen lncRNAs that were expressed specifically in MM cells with an active chromatin signature, while remaining silenced in a bivalent chromatin state in non-MM cells. Out of these MM specific lncRNAs, we identified a lncRNA, Gm29418, in a distal enhancer region of Six2, a key regulatory gene of MM cells. We further identified three transcript variants of Gm29418 by Rapid Amplification of cDNA Ends (RACE), and confirmed that the transcription-start-sites (TSSs) of these variants were consistent with the result of Cap Analysis Gene Expression (CAGE). In support of the enhancer-like function of Gm29418 on Six2 expression, we found that knock-down of Gm29418 by two independent anti-sense locked nucleic acid (LNA) phosphorothioate gapmers suppressed Six2 mRNA expression levels in MM cells. We also found that over-expression of Gm29418 led to an increase in Six2 mRNA expression levels in a mouse MM cell line. In conclusion, we identified a lncRNA, Gm29418, in nephron progenitor cells that has an enhancer-like function on a key regulatory gene, Six2.

In our previous study, we produced a microfluidic device (MFD) which successfully maintained spermatogenesis for over 6 months in mouse testis tissues loaded in the device. In the present study, we developed a new MFD, a monolayer device (ML-D) with a barrier structure consisting of pillars and slits, which is simpler in design and easier to make. This ML-D was also effective for inducing mouse spermatogenesis and maintained it for a longer period than the conventional culture method. In addition, we devised a way of introducing sample tissue into the device during its production, just before bonding the upper layer of polydimethylsiloxane (PDMS) and bottom glass slide. The tissue can obtain nutrients horizontally from the medium running beside it and oxygen vertically from above through PDMS. In addition, the glass slide set at the bottom improved the visibility of the sample tissue with an inverted microscope. When we took photos of cultured tissue of the Acr-Gfp transgenic mouse testis in ML-D sequentially every day, morphological changes of the acrosome during spermiogenesis were successfully recorded. The ML-D is simple in design and useful for culturing testis tissue for inducing and maintaining spermatogenesis with clearer visibility. Due to the new method of sample loading, tissues other than testis should also be applicable.