Physiological comfort evaluation under different airflow directions in a heating environment.

BACKGROUND: Indoor airflow and thermal comfort are difficult to assess through subjective evaluations because airflow sensations can differ based on various factors, such as personal characteristics, interests, preferences, and the current state of mind. Thus, subjective evaluations should be combined with objective assessments, such as physiological measurements. This study evaluated airflow and thermal comfort through physiological measurements, including skin temperature, electroencephalography, respiration, and electrocardiography, in addition to subjective evaluations. METHODS: Twenty participants entered a test room at 30 °C after staying in an acclimation room at 18 °C for 20 min. They were exposed to indirect and direct airflow toward their faces and performed four tasks under each condition: resting, counting to 10 s following time alerts, counting to 10 s in the mind, and mental calculation. The mean speed of the air directed to the participants' faces was 0.123 m/s and 0.225 m/s in the indirect and direct conditions, respectively. RESULTS: The gamma and beta bands of electroencephalograms taken at the left-temporal (T3) and left-parietal (P7) sites showed significantly lower amplitudes under the indirect condition (gamma, T3: p = 0.034, P7: p = 0.030; beta, T3: p = 0.051, P7: p = 0.028). Similarly, the variability of respiration was lower under the indirect condition (p < 0.010). The amplitudes of gamma and beta waves showed significant correlations with anxiousness levels (gamma, T3: r = 0.41; beta, T3: r = 0.35). CONCLUSIONS: Our results suggest that indirect heating airflow causes lower mental stress and fatigue than those induced by direct flow, which is equivalent to more comfort. The results of this study suggest that physiological measurements can be used for the evaluation of unconscious indoor comfort, which cannot be detected by subjective evaluations alone.

Physiological and subjective comfort evaluation under different airflow directions in a cooling environment.

Indoor comfort is influenced by airflow direction, but subjective evaluations can differ. This study evaluates the airflow comfort with subjective assessments and physiological measurements, including skin temperature, electroencephalograms, and electrocardiograms. Nineteen participants entered a test room at 20°C after staying in a room at 32°C for acclimation. They were exposed to indirect and direct airflow conditions to their faces and performed four tasks under each condition: resting, counting to 10 s following time alerts, counting to 10 s in mind, and mental calculation. Subjective assessments showed relatively higher thermal sensation and pleasantness under indirect airflow. The psychological time calculated from counting behaviors was longer under indirect airflow, indicating suppression of negative emotions. The face temperatures significantly declined during experiments under direct airflow. The beta and gamma bands of electroencephalograms were inhibited under the indirect condition, and these amplitudes were negatively correlated with pleasant feelings. Electrocardiogram parameters indicated that sympathetic nervous activity was predominant during counting, following alerts and mental calculation in indirect airflow. This study supports the comfort of indirect airflow based on reliable evidence.

Mouse polycomb group gene Cbx2 promotes osteoblastic but suppresses adipogenic differentiation in postnatal long bones.

A set of key developmental genes is essential for skeletal growth from multipotent progenitor cells at weaning. Polycomb group proteins, which regulate such genes contributes to the cell lineage commitment and subsequent differentiation via epigenetic chromatin modification and remodeling. However, it is unclear which cell lineage and gene sets are targeted by polycomb proteins during skeletal growth. We now report that mice deficient in a polycomb group gene Cbx2cterm/cterm exhibited skeletal hypoplasia in the tibia, femur, and cranium. Long bone cavities in these mice contained fewer multipotent mesenchymal stromal cells. RNA-sequencing of bone marrow cells showed downregulation and upregulation of osteoblastic and adipogenic genes, respectively. Furthermore, the expression levels of genes specifically expressed in B-cell precursors were decreased. Forced expression of Cbx2 in Cbx2cterm/cterm bone marrow stromal cell recovered fibroblastic colony formation and suppressed adipogenic differentiation. Collectively, our results suggest that Cbx2 controls the maintenance and adipogenic differentiation of mesenchymal stromal cells in the bone marrow.

Ad4BP/SF-1 regulates cholesterol synthesis to boost the production of steroids.

Housekeeping metabolic pathways such as glycolysis are active in all cell types. In addition, many types of cells are equipped with cell-specific metabolic pathways. To properly perform their functions, housekeeping and cell-specific metabolic pathways must function cooperatively. However, the regulatory mechanisms that couple metabolic pathways remain largely unknown. Recently, we showed that the steroidogenic cell-specific nuclear receptor Ad4BP/SF-1, which regulates steroidogenic genes, also regulates housekeeping glycolytic genes. Here, we identify cholesterogenic genes as the targets of Ad4BP/SF-1. Further, we reveal that Ad4BP/SF-1 regulates Hummr, a candidate mediator of cholesterol transport from endoplasmic reticula to mitochondria. Given that cholesterol is the starting material for steroidogenesis and is synthesized from acetyl-CoA, which partly originates from glucose, our results suggest that multiple biological processes involved in synthesizing steroid hormones are governed by Ad4BP/SF-1. To our knowledge, this study provides the first example where housekeeping and cell-specific metabolism are coordinated at the transcriptional level.

Mechanistic evaluation and transcriptional signature of a glutathione S-transferase omega 1 inhibitor.

Glutathione S-transferase omega 1 (GSTO1) is an atypical GST isoform that is overexpressed in several cancers and has been implicated in drug resistance. Currently, no small-molecule drug targeting GSTO1 is under clinical development. Here we show that silencing of GSTO1 with siRNA significantly impairs cancer cell viability, validating GSTO1 as a potential new target in oncology. We report on the development and characterization of a series of chloroacetamide-containing potent GSTO1 inhibitors. Co-crystal structures of GSTO1 with our inhibitors demonstrate covalent binding to the active site cysteine. These potent GSTO1 inhibitors suppress cancer cell growth, enhance the cytotoxic effects of cisplatin and inhibit tumour growth in colon cancer models as single agent. Bru-seq-based transcription profiling unravelled novel roles for GSTO1 in cholesterol metabolism, oxidative and endoplasmic stress responses, cytoskeleton and cell migration. Our findings demonstrate the therapeutic utility of GSTO1 inhibitors as anticancer agents and identify the novel cellular pathways under GSTO1 regulation in colorectal cancer.

Fetal Leydig Cells Persist as an Androgen-Independent Subpopulation in the Postnatal Testis.

Two distinct types of Leydig cells emerge during the development of eutherian mammals. Fetal Leydig cells (FLCs) appear shortly after gonadal sex differentiation, and play a crucial role in masculinization of male fetuses. Meanwhile, adult Leydig cells (ALCs) emerge after birth and induce the secondary male-specific sexual maturation by producing testosterone. Previous histological studies suggested that FLCs regress completely soon after birth. Furthermore, gene disruption studies indicated that androgen signaling is dispensable for FLC differentiation but indispensable for postnatal ALC differentiation. Here, we performed lineage tracing of FLCs using a FLC enhancer of the Ad4BP/SF-1 (Nr5a1) gene and found that FLCs persist in the adult testis. Given that postnatal FLCs expressed androgen receptor (AR) as well as LH receptor (LuR), the effects of AR disruption on FLCs and ALCs were analyzed by crossing AR knockout (KO) mice with FLC-specific enhanced green fluorescent protein (EGFP) mice. Moreover, to eliminate the influence of elevated LH levels in ARKO mice, LuRKO mice and AR/LuR double-KO mice were analyzed. The proportion of ALCs to postnatal FLCs was decreased in ARKO mice, and the effect was augmented in the double-KO mice, suggesting that androgen signaling plays important roles in ALCs, but not in FLCs. Finally, ARKO was achieved in an FLC-specific manner (FLCARKO mice), but the FLC number and gene expression pattern appeared unaffected. These findings support the conclusion that FLCs persist as an androgen-independent Leydig subpopulation in the postnatal testis.

Heterogeneity of ovarian theca and interstitial gland cells in mice.

It has been established that two developmentally and functionally distinct cell types emerge within the mammalian testis and adrenal gland throughout life. Fetal and adult types of steroidogenic cells (i.e., testicular Leydig cells and adrenocortical cells) develop in the prenatal and postnatal period, respectively. Although the ovary synthesizes steroids postnatally, the presence of fetal-type steroidogenic cells has not been described. We had previously established transgenic mouse lines in which fetal Leydig cells were labeled with an EGFP reporter gene by the FLE (fetal Leydig enhancer) of the Ad4BP/SF-1 (Nr5a1) gene. In the present study, we examined the reporter gene expression in females and found that the reporter gene is turned on in postnatal ovaries. A comparison of the expressions of the EGFP and marker genes revealed that EGFP is expressed in not all but rather a proportion of steroidogenic theca and in interstitial gland cells in the ovary. This finding was further supported by experiments using BAC transgenic mice in which reporter gene expression recapitulated endogenous Ad4BP/SF-1 gene expression. In conclusion, our observations from this study strongly suggest that ovarian theca and interstitial gland cells in mice consist of at least two cell types.

Glycolytic genes are targets of the nuclear receptor Ad4BP/SF-1.

Genetic deficiencies in transcription factors can lead to the loss of certain types of cells and tissue. The steroidogenic tissue-specific nuclear receptor Ad4BP/SF-1 (NR5A1) is one such gene, because mice in which this gene is disrupted fail to develop the adrenal gland and gonads. However, the specific role of Ad4BP/SF-1 in these biological events remains unclear. Here we use chromatin immunoprecipitation sequencing to show that nearly all genes in the glycolytic pathway are regulated by Ad4BP/SF-1. Suppression of Ad4BP/SF-1 by small interfering RNA reduces production of the energy carriers ATP and nicotinamide adenine dinucleotide phosphate, as well as lowers expression of genes involved in glucose metabolism. Together, these observations may explain tissue dysgenesis as a result of Ad4BP/SF-1 gene disruption in vivo. Considering the function of estrogen-related receptor α, the present study raises the possibility that certain types of nuclear receptors regulate sets of genes involved in metabolic pathways to generate energy carriers.

Fragment-based discovery of 8-hydroxyquinoline inhibitors of the HIV-1 integrase-lens epithelium-derived growth factor/p75 (IN-LEDGF/p75) interaction.

On the basis of an initial molecular modeling study suggesting the favorable binding of the "privileged" fragment 8-hydroxyquinoline with HIV-1 integrase (IN) at the IN-lens epithelium-derived growth factor/p75 (LEDGF/p75) interface , we developed a set of modified 8-hydroxyquinoline fragments demonstrating micromolar IC50 values for inhibition of the IN-LEDGF/p75 interaction, but significant cytotoxicity was associated with these initial compounds. Diverse modifications at the C5 and C7 carbons of the 8-hydroxyquinoline core improved potency, but reduction of diversity to only modifications at the C5 position ultimately yielded potent inhibitors with low cytotoxicity. Two of these particular compounds, 5-((p-tolylamino)methyl)quinolin-8-ol and 5-(((3,4-dimethylphenyl)amino)methyl)quinolin-8-ol, inhibited viral replication in MT-4 cells with low micromolar EC50. This is the first study providing evidence for 8-hydroxyquinolines as novel inhibitors of the IN-LEDGF/p75 interaction. Our lead compounds are druglike, have low molecular weights, and are amenable to various substitutions suitable for enhancing their potency and selectivity.

Discovery of novel inhibitors of LEDGF/p75-IN protein-protein interactions.

Human lens epithelium-derived growth factor (LEDGF)/p75 plays an important role in the HIV life cycle by stimulating integrase (IN)-led viral DNA integration into cellular chromosomes. Mechanistic studies show the majority of IN inhibitors chelate magnesium ions in the catalytic active site, a region topologically distant from the LEDGF/p75 binding site. Compounds disrupting the formation of LEDGF/p75 and IN complexes serve as a novel mechanistic approach different from current antiretroviral therapies. We previously built pharmacophore models mimicking LEDGF/p75 residues and identified four classes of LEDGF/p75-IN inhibitors. Substructure and similarity searches yielded additional LEDGF/p75-IN inhibitors containing an acylhydrazone moiety. The most potent of the acylhydrazones inhibited LEDGF/p75-IN interaction with an IC(50) value of 400nM. We explored structure-activity relationships (SAR) and identified new acylhydrazones, hydrazines, and diazenes as lead molecules for further optimization. Two lead LEDGF/p75-IN inhibitors showed antiviral activity.

Contribution of Leydig and Sertoli cells to testosterone production in mouse fetal testes.

Testosterone is a final product of androgenic hormone biosynthesis, and Leydig cells are known to be the primary source of androgens. In the mammalian testis, two distinct populations of Leydig cells, the fetal and the adult Leydig cells, develop sequentially, and these two cell types differ both morphologically and functionally. It is well known that the adult Leydig cells maintain male reproductive function by producing testosterone. However, it has been controversial whether fetal Leydig cells can produce testosterone, and the synthetic pathway of testosterone in the fetal testis is not fully understood. In the present study, we generated transgenic mice in which enhanced green fluorescence protein was expressed under the control of a fetal Leydig cell-specific enhancer of the Ad4BP/SF-1 (Nr5a1) gene. The transgene construct was prepared by mutating the LIM homeodomain transcription factor (LHX9)-binding sequence in the promoter, which abolished promoter activity in the undifferentiated testicular cells. These transgenic mice were used to collect highly pure fetal Leydig cells. Gene expression and steroidogenic enzyme activities in the fetal Leydig cells as well as in the fetal Sertoli cells and adult Leydig cells were analyzed. Our results revealed that the fetal Leydig cells synthesize only androstenedione because they lack expression of Hsd17b3, and fetal Sertoli cells convert androstenedione to testosterone, whereas adult Leydig cells synthesize testosterone by themselves. The current study demonstrated that both Leydig and Sertoli cells are required for testosterone synthesis in the mouse fetal testis.

Tracing the emergence of a novel sex-determining gene in medaka, Oryzias luzonensis.

Three sex-determining (SD) genes, SRY (mammals), Dmy (medaka), and DM-W (Xenopus laevis), have been identified to date in vertebrates. However, how and why a new sex-determining gene appears remains unknown, as do the switching mechanisms of the master sex-determining gene. Here, we used positional cloning to search for the sex-determining gene in Oryzias luzonensis and found that GsdfY (gonadal soma derived growth factor on the Y chromosome) has replaced Dmy as the master sex-determining gene in this species. We found that GsdfY showed high expression specifically in males during sex differentiation. Furthermore, the presence of a genomic fragment that included GsdfY converts XX individuals into fertile XX males. Luciferase assays demonstrated that the upstream sequence of GsdfY contributes to the male-specific high expression. Gsdf is downstream of Dmy in the sex-determining cascade of O. latipes, suggesting that emergence of the Dmy-independent Gsdf allele led to the appearance of this novel sex-determining gene in O. luzonensis.

Identification of an enhancer in the Ad4BP/SF-1 gene specific for fetal Leydig cells.

Adrenal 4 binding protein/steroidogenic factor 1 (Ad4BP/SF-1) (Nr5a1) is a nuclear receptor essential for reproductive tissue development and endocrine regulation. This factor is expressed in steroidogenic tissues (e.g. adrenal glands and gonads), and expression of this factor is tightly regulated in a tissue and cell type-specific manner. Our previous studies have identified tissue and cell type-specific enhancers in the introns of the Ad4BP/SF-1 gene in fetal adrenal glands, ventromedial hypothalamus, and pituitary gonadotrope. Characterization of the enhancers had provided new insights into tissue and cell development. However, these studies have failed to identify any gonad-specific enhancer. Here, we identified a fetal Leydig cell-specific enhancer in the upstream region of the mouse Ad4BP/SF-1 gene using transgenic mouse assays. Alignment of the upstream regions among vertebrate animal species demonstrated that the enhancer consisted of three conserved regions, whereby the most highly conserved region contained an Ad4BP/SF-1 binding sequence and an E-box. Mutation of each sequence abolished the enhancer activity and led to a loss of reporter gene expression. These results suggested that Ad4BP/SF-1 gene expression in the fetal Leydig cell is regulated by a yet unidentified E-box binding protein(s) and by an autoregulatory loop formed by Ad4BP/SF-1. Although fetal Leydig cells have been thought to play crucial roles for masculinization of various fetal tissues through androgen production, other functions have remained elusive. Our identification of a fetal Leydig cell-specific enhancer in the Ad4BP/SF-1 gene would be a powerful tool to address these gaps in the knowledge base.

The medaka sex-determining gene DMY acquired a novel temporal expression pattern after duplication of DMRT1.

The male sex-determining gene, DMY, of the medaka is considered to have arisen via gene duplication of DMRT1. In the medaka, both genes are expressed in Sertoli cell lineage cells, but their temporal expression patterns are quite different. DMY expression starts just before the sex-determining period, whereas DMRT1 expression occurs during the testicular differentiation period. To evaluate the alterations to the expression patterns of the DMRT1 genes after duplication, we analyzed the morphological gonadal sex differentiation processes and expression patterns of DMRT1 in Oryzias luzonensis and Oryzias mekongensis, which are closely related to the medaka but do not have DMY. Male-specific upregulation of DMRT1 in these two species occurred during the testicular differentiation period, similar to the case for DMRT1 in the medaka. These findings suggest that DMY acquired a novel temporal expression pattern after duplication and that this event played a critical role in the evolutionary process of this gene.

The Y chromosome that lost the male-determining function behaves as an X chromosome in the medaka fish, Oryzias latipes.

The medaka, Oryzias latipes, has an XX/XY sex-determination system, and a Y-linked DM-domain gene, DMY, is the sex-determining gene in this species. Since DMY appears to have arisen from a duplicated copy of the autosomal DMRT1 gene approximately 10 million years ago, the medaka Y chromosome is considered to be one of the youngest male-determining chromosomes in vertebrates. In the screening process of sex-reversal mutants from wild populations, we found a population that contained a number of XY females. PCR, direct sequencing, and RT-PCR analyses revealed two different null DMY mutations in this population. One mutation caused loss of expression during the sex-determining period, while the other comprised a large deletion in putative functional domains. YY females with the mutant-type DMY genes on their Y chromosomes were fully fertile, indicating that the X and Y chromosomes were functionally the same except for the male-determining function. In addition, we investigated the frequencies of the sex chromosome types in this population over four successive generations. The Y chromosomes bearing the mutant-type DMY genes were detected every year with no significant differences in their frequencies. These results demonstrate that aberrant Y chromosomes behaving as X chromosomes have been maintained in this population.

Three isolations of Chaetomium globosum from erythematous epilation of canine skin.

Chaetomium globosum is commonly found in natural environments worldwide and is known to be a causative agent for emerging fungal infections. The present study describes a case of erythematous epilation of a dog caused by C. globosum. A mixed-breed young dog, a 4-months-old male, weighing 7.25 kg, showed depilation, scales, and dermatitis with slightly itchiness on his skin. The main symptom was an erythematous epilation on the left subocular skin 7.5 cm in diameter, accompanied by elephantiasis-like hyperplasia and scales. Similar lesions were observed on the skin on both sides of the ear lobes, the heels, tail, and left angulus oris. The scales from the crusted lesion were cultured on chrolamphenicole-added potato dextrose agar plates at the first visit, as well as followed by ambulatory practices. The isolates at the first visit, 1 and 3 weeks after treatment, were identified as C. globosum by mycological study and the D1/D2 domain of the large subunit rRNA gene sequence. The patient dog was treated by ketoconzole both orally and externally. The lesions were cured, showing new hair growth 9 weeks later. In addition, the susceptibilities to antifungal agents for the present C. globosum isolate were as follows: amphotericin B, 4.0 microg/ml; 5-FC 64.0 microg/ml; itraconazole, 0.5 microg/ml; miconazole, 1.0 microg/ml; fulconazole, 16.0 microg/ml; ketoconazole, 0.25 microg/ml; and micafungin, 16.0 microg/ml.

Wild-derived XY sex-reversal mutants in the Medaka, Oryzias latipes.

The medaka, Oryzias latipes, has an XX/XY sex-determination mechanism. A Y-linked DM domain gene, DMY, has been isolated by positional cloning as a sex-determining gene in this species. Previously, we found 23 XY sex-reversed females from 11 localities by examining the genotypic sex of wild-caught medaka. Genetic analyses revealed that all these females had Y-linked gene mutations. Here, we aimed to clarify the cause of this sex reversal. To achieve this, we screened for mutations in the amino acid coding sequence of DMY and examined DMY expression at 0 days after hatching (dah) using densitometric semiquantitative RT-PCR. We found that the mutants could be classified into two groups. One contained mutations in the amino acid coding sequence of DMY, while the other had reduced DMY expression at 0 dah although the DMY coding sequence was normal. For the latter, histological analyses indicated that YwOurYwOur (YwOur, Y chromosome derived from an Oura XY female) individuals with the lowest DMY expression among the tested mutants were expected to develop into females at 0 dah. These results suggest that early testis development requires DMY expression above a threshold level. Mutants with reduced DMY expression may prove valuable for identifying DMY regulatory elements.

Field survey of sex-reversals in the medaka, Oryzias latipes: genotypic sexing of wild populations.

The medaka, Oryzias latipes, has an XX/XY sex determination mechanism. A Y-linked DM domain gene, DMY, has been isolated by positional cloning as a prime candidate for the sex-determining gene. Furthermore, the crucial role of DMY during male development was established by studying two wild-derived XY female mutants. In this study, to find new DMY and sex-determination related gene mutations, we conducted a broad survey of the genotypic sex (DMY-negative or DMY-positive) of wild fish. We examined 2274 wild-caught fish from 40 localities throughout Japan, and 730 fish from 69 wild stocks from Japan, Korea, China, and Taiwan. The phenotypic sex type agreed with the genotypic sex of most fish, while 26 DMY-positive (XY) females and 15 DMY-negative (XX) males were found from 13 and 8 localities, respectively. Sixteen XY sex-reversals from 11 localities were mated with XY males of inbred strains, and the genotypic and phenotypic sexes of the F(1) progeny were analyzed. All these XY sex-reversals produced XY females in the F(1) generation, and all F(1) XY females had the maternal Y chromosome. These results show that DMY is a common sex-determining gene in wild populations of O. latipes and that all XY sex-reversals investigated had a DMY or DMY-linked gene mutation.