Transcriptome Analysis by RNA Sequencing of Mouse Embryonic Stem Cells Stocked on International Space Station for 1584 Days in Frozen State after Culture on the Ground.

As a space project, in "Stem Cells" by the Japan Aerospace Exploration Agency (JAXA), frozen mouse ES cells were stored on the International Space Station (ISS) in the Minus Eighty Degree Laboratory Freezer for ISS (MELFI) for 1584 days. After taking these cells back to the ground, the cells were thawed and cultured, and their gene expressions were comprehensively analyzed using RNA sequencing in order to elucidate the early response of the cells to long-time exposure to space radiation consisting of various ionized particles. The comparisons of gene expression involved in double-stranded break (DSB) repair were examined. The expressions of most of the genes that were involved in homologous recombination (HR) and non-homologous end joining (NHEJ) were not significantly changed between the ISS-stocked cells and ground-stocked control cells. However, the transcription of Trp53inp1 (tumor protein 53 induced nuclear protein-1), Cdkn1a (p21), and Mdm2 genes increased in ISS-stocked cells as well as Fe ion-irradiated cells compared to control cells. This suggests that accumulated DNA damage caused by space radiation exposure would activate these genes, which are involved in cell cycle arrest for repair and apoptosis in a p53-dependent or -independent manner, in order to prevent cells with damaged genomes from proliferating and forming tumors.

Exploring the impact of ovariectomy on hair growth: can ovariectomized mouse serve as a model for investigating female pattern hair loss in humans?

Female pattern hair loss (FPHL), a type of hair disease common in pre- and postmenopausal women, is characterized by thinning of hair to O-type, mainly at the crown. Although a mouse model of this disease has recently been established, its details are still unknown, and thus, warrants further analysis. In this study, 3 week-old and 7- to 8 week-old C57BL/6 female mice were divided into two groups: one group underwent ovariectomy (OVX), while the other underwent sham surgery. In the 3 week-old mice, the dorsal skin was collected at seven weeks of age, while in the 7- to 8 week-old mice, it was collected at 12 and 24 weeks of age. In the former group, both the pore size of the hair follicles (HFs) and diameter of the hair shaft of telogen HFs decreased upon OVX; while in the latter group, these factors increased significantly. Notably, the thickness of the dermis and subcutis increased significantly in the OVX group. It needs to be further elucidated whether OVX mouse could serve as an ideal mouse model for FPHL, but our results upon evaluation of skin thickness indicate that it could be used to establish a novel treatment for non-hair-related diseases, such as post-menopause-related skin condition.

Low-dose irradiation of mouse embryos increases Smad-p21 pathway activity and preserves pluripotency.

PURPOSE: To study the outcomes of mouse preimplantation embryos irradiated with low doses of X-rays (≤ 1 Gy) and investigate apoptosis and pluripotency of the irradiated embryos. METHODS: Mouse embryos at the 2-cell stage were collected for in vitro culture. After reaching the 8-cell stage, embryos were irradiated with various low doses of X-rays (0-1 Gy). Blastocysts with a normal appearance were transferred into a pseudopregnant uterus. The developmental rate to blastocysts and the survival rate following embryo transfer were examined. Expression levels of p21, Smad2, Foxo1, Cdx2, Oct4, and Nanog genes were measured by RT-PCR. Apoptotic cells in mouse blastocysts were examined immunofluorescently by staining for cleaved caspase-3. RESULTS: More than 90% of non-irradiated and low-dose X-ray-irradiated preimplantation embryos developed to morphologically normal blastocysts that could be implanted and survive in the uterus. However, embryos irradiated with X-rays had more apoptotic cells in a dose-dependent manner. Expression of p21, Smad2, and Foxo1 genes in X-ray-irradiated embryos was increased significantly, while expression of Cdx2, Oct4, and Nanog genes was maintained in comparison with non-irradiated embryos. CONCLUSIONS: Although irradiated embryos contained apoptotic cells, the low doses of irradiation did not disturb development of 8-cell stage embryos to blastocysts or their survival in utero. The underlying mechanisms might involve anti-apoptotic systems, including the Smad-p21 pathway, and preservation of pluripotency.

Lower FOXO3 mRNA expression in granulosa cells is involved in unexplained infertility.

AIM: The aim of this study was to investigate whether FOXO1 and FOXO3 mRNA expression in granulosa cells is the cause of unexplained infertility. METHODS: Thirty-one patients aged <40 years (13 with unexplained infertility and 18 with male partner infertility as a control group) whose serum anti-Müllerian hormone level was >0.5 ng/µL were enrolled in the study. All patients underwent oocyte retrieval under a short protocol from June 2012 to October 2013. Real-time PCR was carried out using mRNA extracted from granulosa cells retrieved from mature follicles. We compared FOXO1 and FOXO3 mRNA expression ratios in granulosa cells between the unexplained infertility group and the male infertility group. The relation between FOXO1 and FOXO3 mRNA expression ratios in granulosa cells and assisted reproduction technology clinical outcome was also examined. RESULTS: FOXO3 mRNA expression ratio was significantly lower in the unexplained infertility group than in the male infertility group. Moreover, FOXO3 mRNA expression ratio showed a positive correlation with both the number of retrieved oocytes and serum anti-Müllerian hormone level. A positive correlation was also identified between FOXO1 mRNA expression and total dose of hMG. As well, the number of retrieved oocytes in the unexplained infertility group was statistically lower than that in the male infertility group. CONCLUSION: A lower FOXO3 mRNA expression in granulosa cells leads to poor oocyte development in patients with unexplained infertility undergoing controlled ovarian stimulation for in vitro fertilization-embryo transfer.

Analyzing the risk factors for a diminished oocyte retrieval rate under controlled ovarian stimulation.

Aim: To investigate which risk factors contribute to a lower oocyte retrieval ratio in women who are receiving controlled ovarian hyperstimulation. Methods: The authors retrospectively analyzed 329 in vitro fertilization (IVF) cycles under controlled ovarian hyperstimulation by using a gonadotropin-releasing hormone antagonist or agonist at Osaka Medical College, Japan. The patients were classified into five groups: advanced age, male infertility, severe endometriosis, tubal infertility, and unexplained infertility. The primary outcomes were the patients' age, oocyte retrieval ratio, serum basal follicle-stimulating hormone, total dose of gonadotropin, and the clinical outcome. A secondary outcome was the stepwise multivariate logistic regression analysis to assess the factors associated with the failure of oocyte retrieval. Results: The oocyte retrieval ratio declined significantly with the patient's age. The ratio of endometriosis in unsuccessful cases was significantly higher than that in successful cycles. Advanced age and endometriosis were the factors that were significantly associated with a lowered oocyte retrieval rate. Conclusion: Advanced age and endometriosis are high-risk factors that contribute to oocyte retrieval failure in infertile patients who are receiving IVF treatment.

Estradiol-mediated hepatocyte growth factor is involved in the implantation of endometriotic cells via the mesothelial-to-mesenchymal transition in the peritoneum.

The pathogenesis of endometriosis, a chronic painful gynecological disease characterized by the presence of endometrial tissue located outside of the uterus and often adhering to the peritoneum, is known to be estrogen dependent. However, the precise pathophysiology of endometriosis remains elusive. Recent studies indicate that the epithelial-to-mesenchymal transition (EMT) of human endometrial cells is important for the progression of endometriosis, and another previous study has implicated hepatocyte growth factor (HGF) in endometriosis progression. The aim of the present study was to examine the role of estradiol in the regulation of HGF production and progression of peritoneal endometriosis, focusing on the interactions between the peritoneum and endometriotic cells. Consequently, estradiol was found to promote the proliferation, invasion, and migration of immortalized human endometrial epithelial cells (hEECs) via HGF upregulation, and the estradiol-induced direct binding of estrogen receptor-α to the HGF promoter was confirmed on a chromatin immunoprecipitation (ChIP) assay. Estradiol also induced the EMT in hEECs by promoting HGF production. Furthermore, human mesothelial cells underwent the mesothelial-to-mesenchymal transition (MMT) during culture with estradiol-stimulated hEEC conditioned medium. Importantly, estradiol itself did not induce the MMT, and the estradiol-stimulated hEEC-conditioned medium in the presence of HGF antibodies reversed the MMT process. These results, which were obtained using immortalized hEECs, indicate that estradiol-induced HGF production may play a crucial role in the peritoneal implantation of human endometriotic cells by exerting proliferative and invasive effects via the EMT in hEECs and promoting the MMT in mesothelial cells.

Hepatocyte growth factor secreted by ovarian cancer cells stimulates peritoneal implantation via the mesothelial-mesenchymal transition of the peritoneum.

OBJECTIVE: A current working model for the metastatic process of ovarian carcinoma suggests that cancer cells are shed from the ovarian tumor into the peritoneal cavity and attach to the layer of mesothelial cells that line the inner surface of the peritoneum, and several studies suggest that hepatocyte growth factor (HGF) plays an important role in the dissemination of ovarian cancer. Our objectives were to evaluate the HGF expression of ovarian cancer using clinical data and assess the effect of HGF secreted from human ovarian cancer cells to human mesothelial cells. METHODS: HGF expression was immunohistochemically evaluated in 165 epithelial ovarian cancer patients arranged as tissue microarrays. HGF expression in four ovarian cancer cell lines was evaluated by using semi-quantitative polymerase chain reaction, Western blotting and enzyme-linked immunosorbent assay. The effect of ovarian cancer cell derived HGF to the human mesothelial cells was assessed by using morphologic analysis, Western blotting and cell invasion assay. The effect of HGF on ovarian cancer metastasis was assessed by using in vivo experimental model. RESULTS: The clinical data showed a significantly high correlation between the HGF expression and the cancer stage. The in vivo and in vitro experimental models revealed that HGF secreted by ovarian cancer cells induces the mesothelial-to-mesenchymal transition and stimulates the invasion of mesothelial cells. Furthermore, manipulating the HGF activity affected the degree of dissemination and ascite formation. CONCLUSIONS: We demonstrated that HGF secreted by ovarian cancer cells plays an important role in cancer peritoneal implantation.

The effect of moderate to severe endometriosis on expression of growth differentiation factor-9 mRNA in human granulosa cells under controlled ovarian hyperstimulation.

Purpose: To investigate the effect of moderate to severe endometriosis on mRNA expression of growth differentiation factor-9 (GDF-9) in the granulosa cells of mature follicles. Methods: Follicular fluid (FF) was obtained from 13 patients with moderate to severe endometriosis and 11 without endometriosis, as a control group, and GDF-9 protein levels in both were assayed by western blotting. mRNA expression by GDF-9 and LH receptor (LHR) in granulosa cells obtained from all patients in the study were investigated by StepOne Real-Time PCR. Results: Although GDF-9 in FF from patients with endometriosis was no different from that of controls, GDF-9 mRNA expression in granulosa cells of patients with endometriosis was statistically significantly lower than for the control group. The number of oocytes and high-quality embryos was positively correlated with GDF-9 mRNA expression in controls but not in patients with endometriosis Moreover, a negative correlation was identified between GDF-9 mRNA expression and serum estrogen and progesterone levels in the control group, whereas no correlation was observed for the endometriosis group. Conclusions: Moderate to severe endometriosis can significantly reduce GDF-9 mRNA expression in the granulosa cells of patients with the disease compared with those without, thus causing poor oocyte maturation and lower embryo quality.

Two new species of the macropsine leafhoppers (Hemiptera: Cicadellidae: Eurymelinae) from Japan, with description of a new subgenus.

Two new macropsine species, Celopsis yaeyamana sp. nov. and Pediopsoides (Glaphyropsis) aurantescens subgen. & sp. nov. are described and illustrated from the Ryukyu Islands of Japan. Four Celopsis species, C. membrana (Zhang), C. montaninversa (Yang & Zhang), C. trifurcata (Li, Dai & Li) and C. rhombica (Li, Dai & Li) are transferred to the new subgenus Glaphyropsis.

Taxonomic study of the genus Scaphoideus Uhler (Hemiptera, Cicadellidae, Deltocephalinae) from Japan.

The Japanese species of the genus Scaphoideus are revised. Eight species are recognized from Japan, including four new species: S. ryukyuensis sp. nov. from the Ryukyus, S. pristiophorus sp. nov. from Honshu and Kyushu, S. aurantius sp. nov. from the Ryukyus and S. brevistylus sp. nov. from Honshu, Kyushu and the Ryukyus.

Medroxyprogesterone acetate-resistant endometrial cancer cells are susceptible to ferroptosis inducers.

AIMS: Medroxyprogesterone acetate (MPA) is the most common fertility-sparing treatment in patients with early-stage endometrial cancer. If MPA treatment fails, hysterectomy is recommended. Thus, there is an urgent need for novel treatment approaches for MPA-resistant endometrial cancer patients who wish to preserve their fertility. Ferroptosis is a recently discovered type of regulated cell death caused by the excessive accumulation of reactive oxygen species (ROS), followed by aberrant lipid peroxidation. Recent studies have shown that inducing ferroptosis is a potential therapeutic strategy for cancer. However, the role of ferroptosis in endometrial cancer treatment remains to be discussed. We therefore investigated the effects of ferroptosis inducers on MPA-resistant endometrial cancer cells. MAIN METHODS: The levels of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4), the main mediators of ferroptosis, were examined. Cell viability was evaluated after treatment with the ferroptosis inducers sulfasalazine, erastin, or RSL3. The degree of intracellular oxidative stress after treatment with these drugs was evaluated by the glutathione level, ROS level, ferrous iron level, lipid peroxidation and changes in mitochondrial morphology. The effect of ferroptosis inducers in vivo was also examined. KEY FINDINGS: The expression of SLC7A11 and GPX4 in MPA-resistant ECC-1 cells decreased in comparison to parental ECC-1 cells. Sulfasalazine, erastin, and RSL3 significantly reduced cell viability and increased intracellular oxidative stress in MPA-resistant ECC-1 cells. Ferroptosis inducers also suppressed in vivo tumor growth more effectively in MPA-resistant ECC-1. SIGNIFICANCE: Treatment with ferroptosis inducers could be a novel therapeutic approach for MPA-resistant endometrial cancer.

Phylogenetic Analysis of the Genus Planaphrodes Hamilton (Hemiptera, Cicadellidae, Aphrodinae) Based on Morphological Characteristics, with Revision of Species from China, Korea and Japan.

A morphology-based phylogeny of the Holarctic leafhopper genus Planaphrodes Hamilton is reconstructed for the first time based on 39 discrete male adult morphological characters. The results support the monophyly of Planaphrodes, with the included species forming two monophyletic lineages defined mainly by the number and location of aedeagus processes. The position of Planaphrodes in the Aphrodini was resolved as follows: (Stroggylocephalus + (Anoscopus + (Planaphrodes + Aphrodes))). The fauna of Planaphrodes from China, Japan and Korea are reviewed and six species are recognized, including two new species: P. bifasciatus (Linnaeus), P. sahlbergii (Signoret), P. nigricans (Matsumura), P. laevus (Rey), P. baoxingensis sp. nov. (China: Sichuan) and P. faciems sp. nov. (China: Hubei). Acocephalus alboguttatus Kato, 1933 syn. nov. and Aphrodes daiwenicus Kuoh, 1981 syn. nov. are considered junior synonyms of Planaphrodes sahlbergii (Signoret, 1879). Planaphrodes bella Choe, 1981 is a junior synonym of Planaphrodes nigricans (Matsumura, 1912). A checklist and key to species of Planaphrodes are provided.

miR-515-5p suppresses trophoblast cell invasion and proliferation through XIAP regulation in preeclampsia.

MicroRNAs (miRNAs) are non-coding small RNA molecules that can be secreted into the circulation and which exist in remarkably stable forms. Circulating miRNAs regulate numerous biological process and are aberrantly expressed in pathological conditions. Differentially expressed circulating miRNAs have received attention as potential biomarkers for many diseases. In this study, we revealed that miR-515-5p was significantly upregulated in maternal serum from preeclampsia patients in comparison to normal pregnant women. Bioinformatics prediction and a dual-luciferase reporter gene assay revealed that miR-515-5p directly targets the X-linked inhibitor of apoptosis protein (XIAP) 3'-untranslated region. In addition, the overexpression of miR-515-5p inhibited the proliferation and invasion of HTR-8/SVneo trophoblast cells. The decreased XIAP expression and reduced epithelial-mesenchymal transition (EMT) were observed in the preeclamptic placenta. Collectively, miR-515-5p may play critical roles in the pathogenesis of preeclampsia through suppression of XIAP, and serum miR-515-5p may act as a potential biomarker for preeclampsia.

Recognition of the genera Igutettix Matsumura and Vilbasteana Anufriev (Hemiptera: Cicadellidae: Typhlocybinae), with discovery of a new related genus from Japan.

The dikraneurine genus Igutettix Matsumura and its allies are revised. The definition of Igutettix is corrected and newly proposed on the basis of the true I. pulverosus Matsumura, resurrected from the synonymy with Dikraneura oculata Lindberg. Accordingly, the genus Vilbasteana Anufriev established for the latter species is resurrected as an independent valid genus. The genera Vikabara Dworakowska and Paraafrakra Chiang, Hsu et Knight, are newly synonymized under Igutettix and Vilbasteana, respectively. Igutettix is composed of three species, I. pulverosus Matsumura sp. rev., I. ater (Dworakowska) comb. nov. and I. glossatus (Dworakowska) comb. nov. Vilbasteana comprises two species, V. oculata (Lindberg) comb. rev. and V. fulva (Chiang, Hsu et Knight) comb. nov. Furthermore, a new genus, Cornicola gen. nov. related to Igutettix is described for a new species C. mizuki sp. nov. from Japan.

miR-486-5p inhibits invasion and migration of HTR8/SVneo trophoblast cells by down-regulating ARHGAP5.

INTRODUCTION: Appropriate implantation of trophoblast cells is necessary for successful pregnancy outcome. This process requires proper migration and invasion of trophoblast cells into the maternal endometrium and the myometrium. Dysregulation of circulating microRNAs in preeclampsia has been reported in several studies. Furthermore, miR-486-5p was reportedly increased within exosomes derived from maternal circulation in preeclamptic pregnancy. However, the roles of elevated miR-486-5p in preeclampsia has not yet been clarified. METHODS: HTR8/SVneo trophoblast cells were transfected with miR-486-5p, and the ARHGAP5 expression was examined by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blotting. A reporter assay using a luciferase construct containing the ARHGAP5 3'-untranslated region (3'UTR) was performed to determine whether or not ARHGAP5 is a direct target of miR-486-5p. Changes in migration and invasion abilities were examined by a wound healing assay and invasion assay, respectively. RESULTS: The ARHGAP5 expression was significantly decreased in miR-486-5p-transfected cells according to RT-qPCR and Western blotting. A dual luciferase reporter gene assay showed that miR-486-5p acts directly on the 3'UTR of ARHGAP5 mRNA. The migration and invasion abilities were suppressed in miR-486-5p-transfected cells. Downregulation of ARHGAP5 by small interfering RNA transfection inhibited trophoblast cell migration and invasion, resembling that of miR-486-5p transfection. DISCUSSION: The migration and invasion abilities of HTR8/SVneo cells were suppressed by miR-486-5p at least partly through inhibiting the ARHGAP5 expression. These data suggest that miR-486-5p is involved in the pathogenesis of preeclampsia and that miR-486-5p is a viable potential biomarker for predicting the onset risk of preeclampsia.

Different roles of the two components of human protein O-mannosyltransferase, POMT1 and POMT2.

Protein O-mannosyltransferase 1 (POMT1) and its homolog, POMT2, are responsible for the catalysis of the first step in O-mannosyl glycan synthesis. Mutations in their genes are associated with a type of congenital muscular dystrophy called Walker-Warburg syndrome. Arg(64), Glu(78) and Arg(138) in the N-terminus region of ScPmt1p, a POMT homolog in Saccharomyces cerevisiae, are important for transferase activity. Arg(138) is also essential for complex formation with ScPmt2p. Here we examined the effects of replacing the corresponding residues in human POMT1 and POMT2 with Ala on complex formation and enzymatic activity. The human POMT1 mutants lost almost all transferase activity while the POMT2 mutants retained enzymatic activity. Neither mutant lost its ability to form complexes with the native counter component. These results indicate that ScPmtps and human POMTs have different mechanisms of complex formation. They also suggest that human POMT1 and POMT2 have discrete functions since the effect of amino acid substitutions on enzymatic activity are different.

Cholesterol-binding toxins and anti-cholesterol antibodies as structural probes for cholesterol localization.

Cholesterol is one of the major constituents of mammalian cell membranes. It plays an indispensable role in regulating the structure and function of cell membranes and affects the pathology of various diseases. In recent decades much attention has been paid to the existence of membrane microdomains, generally termed lipid "rafts", and cholesterol, along with sphingolipids, is thought to play a critical role in raft structural organization and function. Cholesterol-binding probes are likely to provide useful tools for analyzing the distribution and dynamics of membrane cholesterol, as a structural element of raft microdomains, and elsewhere within the cell. Among the probes, non-toxic derivatives of perfringolysin O, a cholesterol-binding cytolysin, bind cholesterol in a concentration-dependent fashion with a strict threshold. They selectively recognize cholesterol in cholesterol-enriched membranes, and have been used in many studies to detect microdomains in plasma and intracellular membranes. Anti-cholesterol antibodies that recognize cholesterol in domain structures have been developed in recent years. In this chapter, we describe the characteristics of these cholesterol-binding proteins and their applications to studies on membrane cholesterol localization.

Low-energy laser irradiation facilitates the velocity of tooth movement and the expressions of matrix metalloproteinase-9, cathepsin K, and alpha(v) beta(3) integrin in rats.

It has previously been reported that low-energy laser irradiation stimulated the velocity of tooth movement via the receptor activator of nuclear factor kappa B (RANK)/RANK ligand and the macrophage colony-stimulating factor/its receptor (c-Fms) systems. Matrix metalloproteinase (MMP)-9, cathepsin K, and alpha(v) beta(3) [alpha(v)beta3] integrin are essential for osteoclastogenesis; therefore, the present study was designed to examine the effects of low-energy laser irradiation on the expression of MMP-9, cathepsin K, and alpha(v)beta3 integrin during experimental tooth movement. Fifty male, 6-week-old Wistar strain rats were used in the experiment. A total force of 10g was applied to the rat molars to induce tooth movement. A Ga-Al-As diode laser was used to irradiate the area around the moving tooth and, after 7 days, the amount of tooth movement was measured. To determine the amount of tooth movement, plaster models of the maxillae were made using a silicone impression material before (day 0) and after tooth movement (days 1, 2, 3, 4, and 7). The models were scanned using a contact-type three-dimensional (3-D) measurement apparatus. Immunohistochemical staining for MMP-9, cathepsin K, and integrin subunits of alpha(v)beta3 was performed. Intergroup comparisons of the average values were conducted with a Mann-Whitney U-test for tooth movement and the number of tartrate-resistant acid phosphatase (TRAP), MMP-9, cathepsin K, and integrin subunits of alpha(v)beta3-positive cells. In the laser-irradiated group, the amount of tooth movement was significantly greater than that in the non-irradiated group at the end of the experiment (P < 0.05). Cells positively stained with TRAP, MMP-9, cathepsin K, and integrin subunits of alpha(v)beta3 were found to be significantly increased in the irradiated group on days 2-7 compared with those in the non-irradiated group (P < 0.05). These findings suggest that low-energy laser irradiation facilitates the velocity of tooth movement and MMP-9, cathepsin K, and integrin subunits of alpha(v)beta3 expression in rats.

First East Asian record of the genus Alpagut (Hemiptera: Heteroptera: Dipsocoridae), with description of a new species and a note on the metacoxal adhesive pad of Dipsocoridae.

The genus Alpagut Kiyak, 1995, is recorded from East Asia for the first time based on the description of A. masakazui sp. nov. from Japan. Habitus images and illustrations of diagnostic features, including genitalia structures, are provided. The loculus capsulae of A. masakazui sp. nov. is discussed. The presence of a metacoxal adhesive pad is reconfirmed in Dipsocoridae along with a discussion of its morphology.

New genus and species of Leptopsaltriini (Hemiptera: Cicadidae: Cicadinae) from China and Vietnam, with colour-changing behaviour reported for the first time in Cicadoidea.

One new cicada genus Versicolora gen. nov. and two new species, V. ziyongi sp. nov. from China and V. bellula sp. nov. from China and Vietnam, are described. The new genus is placed in the tribe Leptopsaltriini of the subfamily Cicadinae. The relationship of this new genus to other related taxa is discussed. Versicolora ziyongi sp. nov. camouflages itself on the bark of the host-plants and gradually changes its body colour when captured. This colour-changing behaviour is recorded for the first time in Cicadoidea, which provides innovative information for ecomorphological study of this remarkable species and other cicadas that potentially exhibit this behaviour.