The clinical application of cell transplantation for severe heart failure is a promising strategy to improve impaired cardiac function. Recently, an array of cell types, including bone marrow cells, endothelial progenitors, mesenchymal stem cells, resident cardiac stem cells, and embryonic stem cells, have become important candidates for cell sources for cardiac repair. In the present study, we focused on the placenta as a cell source. Cells from the chorionic plate in the fetal portion of the human placenta were obtained after delivery by the primary culture method, and the cells generated in this study had the Y sex chromosome, indicating that the cells were derived from the fetus. The cells potentially expressed 'working' cardiomyocyte-specific genes such as cardiac myosin heavy chain 7beta, atrial myosin light chain, cardiac alpha-actin by gene chip analysis, and Csx/Nkx2.5, GATA4 by RT-PCR, cardiac troponin-I and connexin 43 by immunohistochemistry. These cells were able to differentiate into cardiomyocytes. Cardiac troponin-I and connexin 43 displayed a discontinuous pattern of localization at intercellular contact sites after cardiomyogenic differentiation, suggesting that the chorionic mesoderm contained a large number of cells with cardiomyogenic potential. The cells began spontaneously beating 3 days after co-cultivation with murine fetal cardiomyocytes and the frequency of beating cells reached a maximum on day 10. The contraction of the cardiomyocytes was rhythmical and synchronous, suggesting the presence of electrical communication between the cells. Placenta-derived human fetal cells may be useful for patients who cannot supply bone marrow cells but want to receive stem cell-based cardiac therapy.

Biosynthesis of sakuranetin, a flavonoid anti-fungal phytoalexin that occurs in rice, is highly dependent on jasmonic acid (JA) signalling and induced by a variety of environmental stimuli. We previously identified OsNOMT, which encodes naringenin 7-O-methyltransferase (NOMT); NOMT is a key enzyme for sakuranetin production. Although OsNOMT expression is induced by JA treatment, the regulation mechanism that activates the biosynthetic pathway of sakuranetin has not yet been elucidated. In this study, we show that JA-inducible basic helix-loop-helix transcriptional factor OsMYC2 drastically enhances the activity of the OsNOMT promoter and is essential for JA-inducible sakuranetin production. In addition, we identified 2 collaborators of OsMYC2, OsMYC2-like protein 1 and 2 (OsMYL1 and OsMYL2) that further activated the OsNOMT promoter in synergy with OsMYC2. Physical interaction of OsMYC2 with OsMYL1 and OsMYL2 further supported the idea that these interactions lead to the enhancement of the transactivation activity of OsMYC2. Our results indicate that JA signalling via OsMYC2 is reinforced by OsMYL1 and OsMYL2, resulting in the inductive production of sakuranetin during defence responses in rice.

Gonadotropin-releasing hormone (GnRH) is a reproductive neuropeptide, which controls vertebrate reproduction. In most vertebrates, there are more than two GnRH orthologs in the brain. In cichlid fish, the Nile tilapia (Oreochromis niloticus), GnRH1 is the primary hypophysiotropic hormone, while GnRH2 and GnRH3 are non-hypophysiotropic but neuromodulatory in function. Hypophysiotropic GnRH neurons are thought to inter-communicate, while it remains unknown if hypophysiotropic and non-hypophysiotropic GnRH systems communicate with each other. In the present study, we examined interrelationship between three GnRH types using specific antibodies raised against their respective GnRH associated peptide (GAP) sequence. Double-immunofluorescence labeling coupled with confocal microscopy revealed that in sexually mature males, GnRH-GAP1-immunoreactive (-ir) processes are in proximities of GnRH-GAP3-ir cell somata in the terminal nerve, while GnRH-GAP1-ir cell somata were also accompanied by GnRH-GAP3-ir processes in the preoptic area. However, such interaction was not seen in immature males. Further, there was no interaction between GnRH-GAP2 and GnRH-GAP1 or GnRH-GAP3 neurons. Single cell gene expression analysis revealed co-expression of multiple GnRH receptor genes (gnrhr1 and gnrhr2) in three GnRH-GAP cell types. In mature males, high levels of gnrhr2 mRNA were expressed in GnRH-GAP1-ir cells. In immature males, gnrhr1 and gnrhr2 mRNAs are highly expressed in GnRH-GAP3-ir cells. These results suggest heterologous interactions between the three GnRH-GAP cell types and their potential functional interaction during different reproductive stages.

Jasmonic acid (JA) plays central roles in various events in plants, especially defence against pathogens and insects. The basic helix-loop-helix (bHLH) transcription factor MYC2 has attracted attention as a master regulator of JA signalling in dicotyledonous plants. However, how MYC2 functions in monocotyledonous plants, including agriculturally important crops such as cultivated rice, has been poorly understood. To elucidate the comprehensive effects of rice MYC2 (OsMYC2) on the JA-inducible transcriptional modifications, we performed RNA-sequencing by using OsMYC2-knockdown plants (osmyc2RNAi). In osmyc2RNAi, JA-inducible expression of many defence-related genes, for example chitinases and proteinase inhibitors, was compromised. Decrease in JA-dependent activation of the biosynthetic pathways of specialised metabolites, especially defence compounds, was also evident in the osmyc2RNAi line. Furthermore, a substantial change was noted in the expression of distinct types of transcription factors, such as MYB-type factors, likely depicting the importance of OsMYC2 in not only defence responses but also other morphogenetic events. Our findings provide fundamental information to understand the overall functions of MYC2 in JA signalling in monocotyledonous plants, which might yield agricultural benefits.

Stress impairs the hypothalamic-pituitary-gonadal (HPG) axis, probably through its influence on the hypothalamic-pituitary-adrenal (= interrenals in the teleost, HPI) axis leading to reproductive failures. In this study, we investigated the response of hypothalamic neuropeptides, gonadotropin-inhibitory hormone (GnIH), a component of the HPG axis, and corticotropin-releasing hormone (CRH) a component of the HPI axis, to acute social defeat stress in the socially hierarchical male Nile tilapia (Oreochromis niloticus). Localization of GnIH cell bodies, GnIH neuronal processes, and numbers of GnIH cells in the brain during acute social defeat stress was studied using immunohistochemistry. Furthermore, mRNA levels of GnIH and CRH in the brain together with GnIH receptor, gpr147, and adrenocorticotropic hormone (ACTH) in the pituitary were quantified in control and socially defeated fish. Our results show, the number of GnIH-immunoreactive cell bodies and GnIH mRNA levels in the brain and the levels of gpr147 mRNA in the pituitary significantly increased in socially defeated fish. However, CRH and ACTH mRNA levels did not change during social defeat stress. Further, we found glucocorticoid type 2b receptor mRNA in laser captured immunostained GnIH cells. These results show that acute social defeat stress activates GnIH biosynthesis through glucocorticoid receptors type 2b signalling but does not change the CRH and ACTH mRNA expression in the tilapia, which could lead to temporary reproductive dysfunction.

G-protein coupled receptor 139 (GPR139) is an evolutionarily conserved orphan receptor, predominantly expressing in the habenula of vertebrate species. The habenula has recently been implicated in aversive response and its associated learning. Here, we tested the hypothesis that GPR139 signalling in the habenula may play a role in fear learning in the zebrafish. We examined the effect of intraperitoneal injections of a human GPR139-selective agonist (JNJ-63533054) on alarm substance-induced fear learning using conditioned place avoidance paradigm, where an aversive stimulus is paired with one compartment, while its absence is associated with the other compartment of the apparatus. The results indicate that fish treated with 1 µg/g body weight of GPR139 agonist displayed no difference in locomotor activity and alarm substance-induced fear response. However, avoidance to fear-conditioned compartment was diminished, which suggests that the agonist blocks the consolidation of contextual fear memory. On the other hand, fish treated with 0.1 µg/g body weight of GPR139 agonist spent a significantly longer time in the unconditioned neutral compartment as compared to the conditioned (punished and unpunished) compartments. These results suggest that activation of GPR139 signalling in the habenula may be involved in fear learning and the decision-making process in the zebrafish.

Substance P (SP) and neurokinin A (NKA), encoded by TAC1/Tac1 gene are members of the tachykinin family, which exert their neuromodulatory roles in vertebrate reproduction. In mammals, SP and NKA have been shown to regulate gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion via kisspeptin neurons. On the other hand, the role of SP/NKA in the regulation of reproduction in non-mammalian vertebrates is not well known. In the present study, we first localized expression of tac1 mRNA in the brain of male and female zebrafish, Danio rerio. Next, using an antibody against zebrafish tachykinin1 (Tac1), we examined the neural association of SP/NKA neural processes with GnRH3 neurons, and with kisspeptin (kiss2) neurons, in the brains of male and female zebrafish. In situ hybridization showed an apparent male-dominant tac1 expression in the ventral telencephalic area, the anterior and posterior parts of the parvocellular preoptic nucleus, and the suprachiasmatic nucleus. On the other hand, there was female-dominant tac1 expression in the ventral periventricular hypothalamus. Confocal images of double-labeled zebrafish Tac1 and GnRH3 showed associations between Tac1-immunoreactive processes and GnRH3 neurons in the ventral telencephalic area. In contrast, there was no apparent proximity of Tac1 processes to kiss2 mRNA-expressing neurons in the hypothalamus. Lastly, to elucidate possible direct action of SP/NKA on GnRH3 or Kiss2 neurons, expression of SP/NKA receptor, tacr1a mRNA was examined in regions containing GnRH3 or Kiss2 neurons by in situ hybridization. Expression of tacr1a mRNA was seen in several brain regions including the olfactory bulb, preoptic area and hypothalamus, where GnRH3 and Kiss2 cells are present. These results suggest that unlike in mammals, Tac1 may be involved in male reproductive functions via direct action on GnRH3 neurons but independent of kisspeptin in the zebrafish.

Tissue adhesion between plant species occurs both naturally and artificially. Parasitic plants establish intimate relationship with host plants by adhering tissues at roots or stems. Plant grafting, on the other hand, is a widely used technique in agriculture to adhere tissues of two stems. Here we found that the model Orobanchaceae parasitic plant Phtheirospermum japonicum can be grafted on to interfamily species. To understand molecular basis of tissue adhesion between distant plant species, we conducted comparative transcriptome analyses on both infection and grafting by P. japonicum on Arabidopsis. Despite different organs, we identified the shared gene expression profile, where cell proliferation- and cell wall modification-related genes are up-regulated. Among genes commonly induced in tissue adhesion between distant species, we showed a gene encoding a secreted type of β-1,4-glucanase plays an important role for plant parasitism. Our data provide insights into the molecular commonality between parasitism and grafting in plants.

The habenula is an evolutionarily conserved brain structure, which has recently been implicated in fear memory. In the zebrafish, kisspeptin (Kiss1) is predominantly expressed in the habenula, which has been implicated as a modulator of fear response. Hence, in the present study, we questioned whether Kiss1 has a role in fear memory and morphine-induced fear memory impairment using an odorant cue (alarm substances, AS)-induced fear avoidance paradigm in adult zebrafish, whereby the fear-conditioned memory can be assessed by a change of basal place preference (= avoidance) of fish due to AS-induced fear experience. Subsequently, to examine the possible role of Kiss1 neurons-serotonergic pathway, kiss1 mRNA and serotonin levels were measured. AS exposure triggered fear episodes and fear-conditioned place avoidance. Morphine treatment followed by AS exposure, significantly impaired fear memory with increased time-spent in AS-paired compartment. However, fish administered with Kiss1 (10-21 mol/fish) after morphine treatment had significantly lower kiss1 mRNA levels but retained fear memory. In addition, the total brain serotonin levels were significantly increased in AS- and Kiss1-treated groups as compared to control and morphine treated group. These results suggest that habenular Kiss1 might be involved in consolidation or retrieval of fear memory through the serotonin system.

Most vertebrates possess at least two gonadotropin-releasing hormone (GnRH) neuron types. To understand the physiological significance of the multiple GnRH systems in the brain, we examined three GnRH neuron type-specific transcriptomes using single-cell microarray analyses in the medaka (Oryzias latipes). A microarray profile of the three GnRH neuron types revealed five genes that are uniquely expressed in specific GnRH neuron types. GnRH1 neurons expressed three genes that are homologous to functionally characterised genes, GnRH2 neurons uniquely expressed one unnamed gene, and GnRH3 neurons uniquely expressed one known gene. These genes may be involved in the modulation or maintenance of each GnRH neuron type.

The LHb expression is up-regulated during puberty in female zebrafish. However, the molecular mechanism underlying how LHb expression is regulated during puberty remains largely unknown. In this study, we found that the mRNA expression levels of lhb, fshb and cyp19a1b were up-regulated along with the puberty onset in zebrafish. Among the three nuclear estrogen receptors (nERs), the esr2b is the only type whose expression is significantly up-regulated during puberty onset in the pituitary. However, in situ hybridization results revealed that lhb mRNA was colocalized with esr1 and esr2a but not esr2b. Exposure to estradiol (E2) significantly stimulates LHb expression in both wild-type and kiss1-/-;kiss2-/-;gnrh3-/- triple knockout pubertal zebrafish. Moreover, exposure of cultured pituitary cells to E2 increased the LHb expression, indicating that the estrogenic effect on LHb expression could be acted at the pituitary level. Finally, we cloned and analyzed the promoter of lhb by luciferase assay. Our results indicated that the E2 responsive regions of lhb promoter for ERα and ERβ2 are identical, suggesting that ERα and ERβ2 could bind to the same half ERE region of the promoter of lhb, exhibiting a classical ERE-dependent pathway. In summary, we demonstrate that E2 could directly act on the pituitary level to stimulate LHb transcription during puberty in zebrafish.

The ARID1A gene encodes a protein that is part of the large adenosine triphosphate (ATP)-dependent chromatin remodeling complex SWI/SNF and is frequently mutated in human pancreatic ductal adenocarcinomas (PDACs). We investigated the functions of ARID1A during formation of PDACs in mice.

Tumor stem cells (TSCs), capable of self-renewal and continuous production of progeny cells, could be potential therapeutic targets. We have recently reported that chromatin remodeling regulator Brg1 is required for maintenance of murine intestinal TSCs and stemness feature of human colorectal cancer (CRC) cells by inhibiting apoptosis. However, it is still unclear how BRG1 suppression changes the underlying intracellular mechanisms of human CRC cells. We found that Brg1 suppression resulted in upregulation of the JNK signaling pathway in human CRC cells and murine intestinal TSCs. Simultaneous suppression of BRG1 and the JNK pathway, either by pharmacological inhibition or silencing of c-JUN, resulted in even stronger inhibition of the expansion of human CRC cells compared to Brg1 suppression alone. Consistently, high c-JUN expression correlated with worse prognosis for survival in human CRC patients with low BRG1 expression. Therefore, the JNK pathway plays a critical role for expansion and stemness of human CRC cells in the context of BRG1 suppression, and thus a combined blockade of BRG1 and the JNK pathway could be a novel therapeutic approach against human CRC.

Mesenchymal activation, characterized by dense stromal infiltration of immune and mesenchymal cells, fuels the aggressiveness of colorectal cancers (CRC), driving progression and metastasis. Targetable molecules in the tumor microenvironment (TME) need to be identified to improve the outcome in CRC patients with this aggressive phenotype. This study reports a positive link between high thrombospondin-1 (THBS1) expression and mesenchymal characteristics, immunosuppression, and unfavorable CRC prognosis. Bone marrow-derived monocyte-like cells recruited by CXCL12 are the primary source of THBS1, which contributes to the development of metastasis by inducing cytotoxic T-cell exhaustion and impairing vascularization. Furthermore, in orthotopically generated CRC models in male mice, THBS1 loss in the TME renders tumors partially sensitive to immune checkpoint inhibitors and anti-cancer drugs. Our study establishes THBS1 as a potential biomarker for identifying mesenchymal CRC and as a critical suppressor of antitumor immunity that contributes to the progression of this malignancy with a poor prognosis.

The rat homologue of a mitochondrial ATP-dependent protease Lon was cloned from cultured astrocytes exposed to hypoxia. Expression of Lon was enhanced in vitro by hypoxia or ER stress, and in vivo by brain ischemia. These observations suggested that changes in nuclear gene expression (Lon) triggered by ER stress had the potential to impact important mitochondrial processes such as assembly and/or degradation of cytochrome c oxidase (COX). In fact, steady-state levels of nuclear-encoded COX IV and V were reduced, and mitochondrial-encoded subunit II was rapidly degraded under ER stress. Treatment of cells with cycloheximide caused a similar imbalance in the accumulation of COX subunits, and enhanced mRNA for Lon and Yme1, the latter another mitochondrial ATP-dependent protease. Furthermore, induction of Lon or GRP75/mtHSP70 by ER stress was inhibited in PERK (-/-) cells. Transfection studies revealed that overexpression of wild-type or proteolytically inactive Lon promoted assembly of COX II into a COX I-containing complex, and partially prevented mitochondrial dysfunction caused by brefeldin A or hypoxia. These observations demonstrated that suppression of protein synthesis due to ER stress has a complex effect on the synthesis of mitochondrial-associated proteins, both COX subunits and ATP-dependent proteases and/or chaperones contributing to assembly of the COX complex.

In vertebrates, gonadotropin-releasing hormone (GnRH) and gonadotropin-inhibitory hormone (GnIH), respectively, regulate reproduction in positive and negative manners. GnIH belongs to the LPXRFa family of peptides previously identified in mammalian and nonmammalian vertebrates. Studying the detailed distribution of LPXRFa as well as its receptor (LPXRFa-R) in the brain and pituitary is important for understanding their multiple action sites and potential functions. However, the distribution of LPXRFa and LPXRFa-R has not been studied in teleost species, partially because of the lack of fish-specific antibodies. Therefore, in the present study, we generated specific antibodies against LPXRFa and its receptor from Nile tilapia (Oreochromis niloticus), and examined their distributions in the brain and pituitary by immunohistochemistry. Tilapia LPXRFa-immunoreactive neurons lie in the posterior ventricular nucleus of the caudal preoptic area, whereas LPXRFa-R-immunoreactive cells are distributed widely. Double immunofluorescence showed that neither LPXRFa-immunoreactive fibers nor LPXRFa-R is closely associated or coexpressed with GnRH1, GnRH3, or kisspeptin (Kiss2) neurons. In the pituitary, LPXRFa fibers are closely associated with gonadotropic endocrine cells [expressing luteinizing hormone (LH) and follicle-stimulating hormone (FSH)], with adrenocorticomelanotropic cells [corticotropin (ACTH) and α-melanotropin (α-MSH)], and with somatolactin endocrine cells. In contrast, LPXRFa-R are expressed only in LH, ACTH, and α-MSH cells. These results suggest that LPXRFa and LPXRFa-R signaling acts directly on the pituitary cells independent from GnRH or kisspeptin and could play multiple roles in reproductive and nonreproductive functions in teleosts. J. Comp. Neurol. 524:2753-2775, 2016. © 2016 Wiley Periodicals, Inc.

We evaluated the yields of Oryza sativa L. 'Nipponbare' rice lines expressing a gene encoding an A20/AN1 domain stress-associated protein, AlSAP, from the halophyte grass Aeluropus littoralis under the control of different promoters. Three independent field trials were conducted, with drought imposed at the reproductive stage. In all trials, the two transgenic lines, RN5 and RN6, consistently out-performed non-transgenic (NT) and wild-type (WT) controls, providing 50-90% increases in grain yield (GY). Enhancement of tillering and panicle fertility contributed to this improved GY under drought. In contrast with physiological records collected during previous greenhouse dry-down experiments, where drought was imposed at the early tillering stage, we did not observe significant differences in photosynthetic parameters, leaf water potential, or accumulation of antioxidants in flag leaves of AlSAP-lines subjected to drought at flowering. However, AlSAP expression alleviated leaf rolling and leaf drying induced by drought, resulting in increased accumulation of green biomass. Therefore, the observed enhanced performance of the AlSAP-lines subjected to drought at the reproductive stage can be tentatively ascribed to a primed status of the transgenic plants, resulting from a higher accumulation of biomass during vegetative growth, allowing reserve remobilization and maintenance of productive tillering and grain filling. Under irrigated conditions, the overall performance of AlSAP-lines was comparable with, or even significantly better than, the NT and WT controls. Thus, AlSAP expression inflicted no penalty on rice yields under optimal growth conditions. Our results support the use of AlSAP transgenics to reduce rice GY losses under drought conditions.

Metallothionein (MT) is a small cysteine-rich heavy metal-binding protein involved in metal homeostasis, detoxification and free radical-scavenging. MT is ubiquitously expressed in several tissues, but its role in the central nervous system is not well understood. In this study, we identified two MT homologous genes (mt2 and smtb) in the zebrafish. Digoxigenin-in situ hybridization showed the expression of mt2 and smtb genes in the ventricular layers in the telencephalon, diencephalon, mesencephalon and rhombencephalon, most of which are cell proliferating regions in the brain of zebrafish. Cellular characteristics of MT genes expressing cells were examined by double-labelling with markers for neurons (HuC/D) and astrocytes (glial fibrillary acidic protein, GFAP and S100 protein) and cell proliferation marker (PCNA). mt2 and smtb mRNAs are expressed in neurons and not in astrocytes, and they were co-localized with PCNA. These results suggest that mt2 and smtb may play an important role in neurogenesis and neuroprotection.

Kisspeptin has recently been recognized as a critical regulator of reproductive function in vertebrates. During the sexual development, kisspeptin neurons receive sex steroids feedback to trigger gonadotropin-releasing hormone (GnRH) neurons. In teleosts, a positive correlation has been found between the thyroid status and the reproductive status. However, the role of thyroid hormone in the regulation of kisspeptin system remains unknown. We cloned and characterized a gene encoding kisspeptin (kiss2) in a cichlid fish, the Nile tilapia (Oreochromis niloticus). Expression of kiss2 mRNA in the brain was analyzed by in situ hybridization. The effect of thyroid hormone (triiodothyronine, T3) and hypothyroidism with methimazole (MMI) on kiss2 and the three GnRH types (gnrh1, gnrh2, and gnrh3) mRNA expression was analyzed by real-time PCR. Expression of thyroid hormone receptor mRNAs were analyzed in laser-captured kisspeptin and GnRH neurons by RT-PCR. The kiss2 mRNA expressing cells were seen in the nucleus of the lateral recess in the hypothalamus. Intraperitoneal administration of T3 (5 μg/g body weight) to sexually mature male tilapia significantly increased kiss2 and gnrh1 mRNA levels at 24 h post injection (P < 0.001), while the treatment with an anti-thyroid, MMI (100 ppm for 6 days) significantly reduced kiss2 and gnrh1 mRNA levels (P < 0.05). gnrh2, gnrh3, and thyrotropin-releasing hormone mRNA levels were insensitive to the thyroid hormone manipulations. Furthermore, RT-PCR showed expression of thyroid hormone receptor mRNAs in laser-captured GnRH neurons but not in kiss2 neurons. This study shows that GnRH1 may be directly regulated through thyroid hormone, while the regulation of Kiss2 by T3 is more likely to be indirect.

Diabetes mellitus (DM) has deleterious influence on cardiac performance independent of coronary artery disease and hypertension. The objective of the present study was to investigate the role of the renin-angiotensin-aldosterone system, especially angiotensin II type 1a receptor (AT1aR) and mineralocorticoid receptor (MR) signaling, in left ventricular (LV) dysfunction induced by diabetes mellitus (DM).