Sensing High 17ß-Estradiol Concentrations Using a Planar Microwave Sensor Integrated with a Microfluidic Channel.

The global issue of pollution caused by endocrine-disrupting chemicals (EDCs) has been gaining increasing attention. Among the EDCs of environmental concern, 17ß-estradiol (E2) can produce the strongest estrogenic effects when it enters the organism exogenously through various routes and has the potential to cause harm, including malfunctions of the endocrine system and development of growth and reproductive disorders in humans and animals. Additionally, in humans, supraphysiological levels of E2 have been associated with a range of E2-dependent disorders and cancers. To ensure environmental safety and prevent potential risks of E2 to human and animal health, it is crucial to develop rapid, sensitive, low cost and simple approaches for detecting E2 contamination in the environment. A planar microwave sensor for E2 sensing is presented based on the integration of a microstrip transmission line (TL) loaded with a Peano fractal geometry with a narrow slot complementary split-ring resonator (PF-NSCSRR) and a microfluidic channel. The proposed technique offers a wide linear range for detecting E2, ranging from 0.001 to 10 mM, and can achieve high sensitivity with small sample volumes and simple operation methods. The proposed microwave sensor was validated through simulations and empirical measurements within a frequency range of 0.5-3.5 GHz. The E2 solution was delivered to the sensitive area of the sensor device via a microfluidic polydimethylsiloxane (PDMS) channel with an area of 2.7 mm2 and sample value of 1.37 µL and measured by a proposed sensor. The injection of E2 into the channel resulted in changes in the transmission coefficient (S21) and resonance frequency (Fr), which can be used as an indicator of E2 levels in solution. The maximum quality factor of 114.89 and the maximum sensitivity based on S21 and Fr at a concentration of 0.01 mM were 1746.98 dB/mM and 40 GHz/mM, respectively. Upon comparing the proposed sensor with the original Peano fractal geometry with complementary split-ring (PF-CSRR) sensors without a narrow slot, several parameters were evaluated, including sensitivity, quality factor, operating frequency, active area, and sample volume. The results showed that the proposed sensor exhibited an increased sensitivity of 6.08% and had a 40.72% higher quality factor, while the operating frequency, active area, and sample volume showed decreases of 1.71%, 25%, and 28.27%, respectively. The materials under tests (MUTs) were analyzed and categorized into groups using principal component analysis (PCA) with a K-mean clustering algorithm. The proposed E2 sensor has a compact size and simple structure that can be easily fabricated with low-cost materials. With the small sample volume requirement, fast measurement with a wide dynamic range, and a simple protocol, this proposed sensor can also be applied to measure high E2 levels in environmental, human, and animal samples.

Assessment of 28-day oral exposure to Pueraria candollei var. mirifica (Fabaceae) roots on pituitary-ovarian axis function and selected metabolic parameters in ovary-intact rats.

Pueraria candollei var. mirifica (Fabaceae) root (PMR) has recently been developed as a potential selective estrogen receptor modulator (SERM) in menopausal women. Nowadays, many premenopausal women also take dietary PMR supplements, however, the exact biological effects of PMR have not been evaluated. This study included the application of the OECD guideline 407 for the assessment of 28-day oral exposure to PMR on pituitary-ovarian (PO) axis function and metabolic parameters in the premenopausal rat model. Ovary-intact adult rats were orally administrated with 10, 100, 750, 1000, and 1500 mg/kg body weight (BW)/day of PMR powder. The positive estrogenic group was given 2 mg 17ß-estradiol (E2)/kg BW/day. Serum levels of reproductive hormones, lipid and thyroid parameters, estrous cycle determination, and histomorphometric and histopathological evaluations of the anterior pituitary, ovary, uterus, vagina, mammary gland, and liver were investigated. PMR displayed neutral effects on uterine, vaginal, and body weights, and circulating E2 and prolactin levels. PMR exerted E2-like effects by i) reducing ovarian and increasing hepatic weights, ii) decreasing serum gonadotropins, iii) lowering serum lipids without altering thyroid parameters, iv) increasing the prevalence of abnormal estrous cycles with prolonged estrus, v) increasing nuclear diameter of anterior pituitary cells, vi) decreasing ovarian size and follicular numbers and increasing follicular degeneration, vii) thickening of uterine myometrium and luminal epithelium, and vaginal epithelium, and viii) induction of mammary alveolar hyperplasia and ductal secretion. Unlike E2, the appearance of very small numbers of focal microvesicular steatosis in hepatocytes demonstrated mild toxicity at high PMR doses. This is the first report that high-dose PMR exerted actions exactly like E2 on gonadotrope-ovarian axis function and histology, lipid, and thyroid parameters without affecting uterine and vaginal growth in ovary-intact rats according to OECD guidelines.

The influence of equol on the hypothalamic-pituitary-thyroid axis and hepatic lipid metabolic parameters in adult male rats.

AIMS: Equol, the principal active metabolite of soy-derived phytoestrogen daidzein, has well-known estrogenic actions. Results of several studies indicate that equol may also have anti-androgenic activities. However, mechanisms of action of equol on hypothalamic-pituitary-thyroid axis (HPTA) and hepatic lipid metabolism in adult male rats have not been determined yet. MAIN METHODS: Equol at two doses of 100 and 250mg/kgbodyweight(BW)/day was orally gavaged for 5days to groups of 4-month-old male rats. As a positive anti-androgenic control group, animals received 100mg of pure anti-androgenic drug flutamide/kgBW/day. Circulating concentrations of thyroid hormones and lipids, and expression levels of genes underlying HPTA function were determined by radioimmunoassay and TaqMan® real-time reverse transcription polymerase chain reaction, respectively. KEY FINDINGS: Flutamide significantly decreased relative prostate weight, whereas equol did not. Both equol and flutamide caused a significant increase in relative liver weights, and decreases in plasma levels of total tetraiodothyronine (T4) and triiodothyronine (T3), whereas free T4 and T3 concentrations were not reduced. Equol caused the marked down-regulation of hypothalamic thyrotropin-releasing hormone mRNA expression, whereas flutamide did not. Equol as well as flutamide significantly down-regulated the expression levels of pituitary thyrotropin beta-subunit mRNA, without altering thyrotropin secretion. Equol caused reductions in plasma levels of total cholesterol, high- and low-density lipoproteins and triglycerides, whereas flutamide exerted opposite effects. SIGNIFICANCE: This is the first study to reveal that in male rats equol did not affect HPTA function and liver lipid metabolism through the anti-androgenic pathway, however, the intrinsic estrogenic actions of equol were observed.

Lack of anti-androgenic effects of equol on reproductive neuroendocrine function in the adult male rat.

Equol (EQ), a metabolite of the soy isoflavone daidzein, has well known estrogenic properties. Data from animal studies suggested that EQ may act also as an anti-androgen. However, data regarding how EQ may affect brain functions like the regulation of neuroendocrine activity and reproductive outcomes in adult male rats are still lacking. We therefore investigated the effects of EQ on sex-steroid regulated gene expression in the brain [medial preoptic area/anterior hypothalamus (MPOA/AH) and medial basal hypothalamus/median eminence (MBH/ME)], pituitary, and prostate as a reference androgen-dependent organ. Furthermore reproductive outcomes were evaluated. The anti-androgen flutamide (FLUT) served as reference compound. Male rats (n=12 per group) were treated by gavage for 5 days with either EQ (100 or 250 mg/kgBW/day), or FLUT 100 mg/kgBW/day. All vehicle- and EQ-treated males showed successful reproductive outcomes, whereas FLUT-exposed males had severe reproductive impairments resulted in infertility. FLUT decreased relative weights of prostate, seminal vesicles and epididymides, and increased serum levels of luteinizing hormone, follicle-stimulating hormone, testosterone and 5α-dihydrotestosterone without altering prolactin levels, whereas EQ exerted opposite effects. Both EQ and FLUT decreased gonadotropin releasing hormone (GnRH) expression in the MPOA/AH. Only FLUT upregulated levels of GnRH receptor expression both in the MBH/ME and pituitary. While EQ downregulated the hypothalamic ERα and ERß expressions, but FLUT did not. In the prostate, only FLUT upregulated both ERα and AR mRNA expression levels. Taken together, our findings are the first data that EQ did not induce anti-androgenic effects on brain, prostate and male reproductive parameters, however, estrogenic neuroendocrine and reproductive effects of EQ were observed.

Effects of the natural endocrine disruptor equol on the pituitary function in adult male rats.

Equol (EQ), a potent biologically active metabolite of the soy isoflavone daidzein, interacts with estrogen receptors (ERs), however, as suggested recently, EQ may also exert anti-androgenic actions in androgen regulated tissues like prostate and seminal vesicles in adult male rats. However, data regarding a putative anti-androgenic activity of EQ on pituitary function in male individuals are still lacking. Therefore, we investigated the effects of EQ on androgen- and estrogen-regulated gene expressions in the pituitary and circulating luteinizing hormone (LH) and prolactin (PRL) levels in adult male rats. 3-Month-old male Sprague-Dawley rats (n=12 per group) were treated by gavage for 5 days with either EQ (100 and 250 mg/kg BW/day) or vehicle olive oil (1 ml/rat/day). As reference compound, the pure anti-androgenic drug flutamide (FLUT) was employed at a dose of 100 mg/kg BW/day. At day 5, animals were sacrificed. Levels of pituitary hormones and gene expression were measured by radioimmunoassays and quantitative TaqMan(®) real-time reverse transcription polymerase chain reaction, respectively. The present findings revealed that the pituitary mechanisms involved in the effects of EQ and FLUT were different due to the opposite changes in the mRNA expression levels of estrogen receptor subtype alpha (ERα)-, truncated estrogen receptor product-1 (TERP-1)- and -2 (TERP-2)-, gonadotropin releasing hormone receptor (GnRH receptor)-, beta-subunit of LH (LHß)-, and gonadotropin alpha subunit (α-subunit) genes. EQ displayed typical ER-agonistic actions as shown by the significant increases in ERα-, TERP-1/-2 mRNA expressions and serum PRL levels along with the significant reduction in serum LH levels, whereas FLUT exerted opposite effects on gonadotropin secretion and expression. Taken together, our findings are the first in vivo data that upon sub-acute oral exposure of EQ show an estrogenic effect on reproductive endocrine activity of the pituitary in adult male rats. However, EQ did not exert anti-androgenic effects on male rat pituitary function as observed at the levels of mRNA expression of androgen- and estrogen-regulated genes and circulating pituitary hormones.

Blue light signaling inactivates the mating type genes-mediated repression of asexual spore production in the higher basidiomycete Coprinopsis cinerea.

Monokaryotic mycelia of several wild-type strains of the homobasidiomycete Coprinopsis cinerea form abundant numbers of oidia both in the light and dark due to the regulation of oidia production by the A and B mating type genes. Nevertheless, little is known about whether and how the mating type loci and light signal regulate the oidiation in C. cinerea. Herein, the experimental results demonstrated that the self-compatible homokaryon AmutBmut strain, the mycelia whose nuclei carry mutations in both the A and B loci, can produce only a few oidia in the dark, whereas the formation of numerous numbers of oidia is induced by the light. The semi-compatible homokaryon AmutB, but not ABmut, has the production and behavior of oidia formation similar to those of AmutBmut. These findings indicated that in AmutBmut strain the mutation at the A locus results in repression of oidiation in the dark and the blue light alleviates this effect, whereas the mutated B genes function has no effects. Since, the oidia production relies on both A and light signal, it is possible that A locus might be linked to the blue light receptor genes. The present results demonstrated for the first time that the secondary hyphal knot formation (skn1), fruiting body maturation (mat) and basidiospore formation (bad) genes which are essential in the C. cinerea fruiting pathway are not involved in the regulation of asexual sporulation. In addition, the positive light effect on oidiation could also occur in C. cinerea dikaryons.

Evolution of the bipolar mating system of the mushroom Coprinellus disseminatus from its tetrapolar ancestors involves loss of mating-type-specific pheromone receptor function.

Mating incompatibility in mushroom fungi is controlled by the mating-type loci. In tetrapolar species, two unlinked mating-type loci exist (A and B), whereas in bipolar species there is only one locus. The A and B mating-type loci encode homeodomain transcription factors and pheromones and pheromone receptors, respectively. Most mushroom species have a tetrapolar mating system, but numerous transitions to bipolar mating systems have occurred. Here we determined the genes controlling mating type in the bipolar mushroom Coprinellus disseminatus. Through positional cloning and degenerate PCR, we sequenced both the transcription factor and pheromone receptor mating-type gene homologs from C. disseminatus. Only the transcription factor genes segregate with mating type, discounting the hypothesis of genetic linkage between the A and B mating-type loci as the causal origin of bipolar mating behavior. The mating-type locus of C. disseminatus is similar to the A mating-type locus of the model species Coprinopsis cinerea and encodes two tightly linked pairs of homeodomain transcription factor genes. When transformed into C. cinerea, the C. disseminatus A and B homologs elicited sexual reactions like native mating-type genes. Although mating type in C. disseminatus is controlled by only the transcription factor genes, cellular functions appear to be conserved for both groups of genes.

An essential gene for fruiting body initiation in the basidiomycete Coprinopsis cinerea is homologous to bacterial cyclopropane fatty acid synthase genes.

The self-compatible Coprinopsis cinerea homokaryon AmutBmut produces fruiting bodies without prior mating to another strain. Early stages of fruiting body development include the dark-dependent formation of primary hyphal knots and their light-induced transition to the more compact secondary hyphal knots. The AmutBmut UV mutant 6-031 forms primary hyphal knots, but development arrests at the transition state by a recessive defect in the cfs1 gene, isolated from a cosmid library by mutant complementation. A normal primordia phenotype was achieved when cfs1+ was embedded at both sides in at least 4.0 kb of native flanking DNA. Truncations of the flanking DNA lead to reduction in transformation frequencies and faults in primordia tissue formation, suggesting that the gene is also acting at later stages of development. The cfs1 gene encodes a protein highly similar to cyclopropane fatty acid synthases, a class of enzymes shown in prokaryotes and recently in a plant to convert membrane-bound unsaturated fatty acids into cyclopropane fatty acids. In C. cinerea 6-031, the mutant cfs1 allele carries a T-to-G transversion, leading to an amino acid substitution (Y441D) in a domain suggested to be involved in the catalytic function of the protein and/or membrane interaction.