Rapid removal of detergent in glycolipids using ionic liquids.

Detergent removal in glycolipid after sample preparation, such as enzymatic reaction or isolation of detergent-resistant membrane microdomain, is indispensable for further structural characterization. We previously established the rapid and effective method of detergent removal in glycolipid samples from glass test tube using 1,2-dichloroethane (DCE) washing. However, the use of DCE has several drawbacks, such as environmental risks, harmful effects (potentially carcinogenic), and high vaporability and flammability. To solve the issue, we used ionic liquids to remove detergents from glycolipid samples, and found 1-butyl-3-methylimidazolium iodide was a suitable alternative for DCE.

Single Nucleotide Polymorphisms of HAAO and IRX6 Genes as Risk Factors for Hypospadias.

PURPOSE: We evaluated the association of hypospadias and 17 susceptibility loci previously identified by a European genome-wide association study in a cohort of Japanese patients. We also examined the expression of candidate genes in male mouse embryos to determine the possible underlying mechanisms of this disease. MATERIALS AND METHODS: We enrolled 169 Japanese patients (mean age at surgery 3.7 years) who underwent repair of hypospadias. Genotyping of 17 single nucleotide polymorphisms was performed using a multiplex polymerase chain reaction invader assay. We also performed in situ hybridization to determine whether candidate genes were expressed in the male genital tubercle during embryonic development of the external genitalia in mice. RESULTS: Single nucleotide polymorphism rs3816183 of HAAO was significantly associated with susceptibility to hypospadias in general (p = 0.0019) and to anterior/middle hypospadias (p = 0.0283) and posterior hypospadias (p = 0.0226), while single nucleotide polymorphism rs6499755 of IRX6 showed an association with susceptibility to anterior/middle hypospadias (p = 0.0472). In mouse embryos there was no significant upregulation of Haao expression in the developing male external genitalia. Irx3 and Irx5, which are linked to Irx6 within the IrxB cluster, were expressed in the mesenchyme remote from the urethral plate epithelium during the critical embryonic period for masculinization. Irx6 was expressed in the ectodermal epithelium, demonstrating prominent dorsal ectodermal expression without expression in the ventral ectoderm adjacent to the urethral plate during the same period. CONCLUSIONS: Genetic variations of HAAO and IRX6 influence susceptibility to hypospadias in the Japanese population. Further research is needed to clarify the mechanism by which variations in these genes contribute to the pathogenesis of hypospadias.

Regulation of masculinization: androgen signalling for external genitalia development.

The biology of masculinization is fundamentally important for understanding the embryonic developmental processes that are involved in the development of the male reproductive tract, external genitalia, and also the tumorigenesis of prostate cancer. The molecular mechanisms of masculinization are of interest to many researchers and clinicians involved in varied fields, including molecular developmental biology, cancer research, endocrinology, and urology. Androgen signalling is mediated by the nuclear androgen receptor, which has fundamental roles in masculinization during development. Various modes of androgen signalling, including 5α-dihydrotestosterone-induced regulation of mesenchymal cell proliferation, have been observed in masculinization. Such regulation is essential for regulating urogenital tissue development, including external genitalia development. Androgen-induced genes, such as MAFB, which belongs to the activator protein 1 (AP-1) superfamily of genes, have essential roles in male urethral formation, and disruption of its signalling can interfere with urethral formation, which often results in hypospadias. Another AP-1 superfamily gene, ATF3, could be responsible for some instances of hypospadias in humans. These androgen-dependent signals and downstream events are crucial for not only developmental processes but also processes of diseases such as hypospadias and prostate cancer.

Recurrent triple-negative breast cancer (TNBC) tissues contain a higher amount of phosphatidylcholine (32:1) than non-recurrent TNBC tissues.

Triple-negative breast cancer (TNBC) is one of the breast cancer subtype that displays a high risk of early recurrence and short overall survival. Improvement of the prognosis of patients with TNBC requires identifying a predictive factor of recurrence, which would make it possible to provide beneficial personalized treatment. However, no clinically reliable predictive factor is currently known. In this study, we investigated the predictive factor of recurrence in TNBC using matrix-assisted laser desorption/ionization-imaging mass spectrometry for lipid profiling of breast cancer specimens obtained from three and six patients with recurrent and non-recurrent TNBC, respectively. The signal for phosphatidylcholine (PC) (32:1) at m/z 732.5 was significantly higher in the recurrence group compared to the non-recurrence group (P = 0.024). PC (32:1) was more abundant in the cancer epithelial area than it was in the surrounding stroma, suggesting that abnormal lipid metabolism was associated with malignant transformation. Our results indicate PC (32:1) as a candidate predictive factor of TNBC recurrence. A future prospective study investigating whether personalized therapy based on PC (32:1) intensity improves the prognosis of patients with TNBC is recommended.

Effect of different surgical procedures on the accuracy of prediction of the plasma concentration of fentanyl: comparison between mastectomy and laparoscopic prostatectomy.

BACKGROUND: The accuracy of simulation-predicted fentanyl concentration in different types of surgical procedure is not fully understood. We wished to estimate the effect of different types of surgical procedure on the accuracy of such simulations. FINDINGS: Fifty patients who had undergone elective mastectomy or laparoscopic prostatectomy (American Society of Anesthesiologists physical status = I-II) were enrolled. Anesthesia was maintained throughout surgery with sevoflurane and a bolus infusion of fentanyl. A maintenance infusion was administered with 8 mL/kg/h Ringer's acetate solution from the start of anesthesia to completion of blood sampling. An infusion to compensate for blood loss was administered (one to two volumes of hydroxyethyl starch). A blood sample was drawn every 30 min during anesthesia.We measured the plasma concentration of fentanyl in 358 samples from 50 patients. The plasma concentration of fentanyl was correlated significantly with the simulated predicted fentanyl concentration (r = 0.734, P < 0.01) but 36.0% of all samples had a difference greater than ±0.5 ng/mL. Approximately 0.3 ng/mL of a fixed bias was shown throughout mastectomy. During laparoscopic prostatectomy, the fixed bias gradually became negative from ≈0.3 to -0.3 ng/mL as the sampling stage proceeded. CONCLUSIONS: The predicted concentration of fentanyl was significantly correlated with the plasma concentration of fentanyl (r = 0.734). However, there were different patterns of a fixed bias between mastectomy and laparoscopic prostatectomy groups. We should pay attention to this tendency among different surgical procedures. TRIAL REGISTRATION: UMIN000005110.

Selective improvement of peptides imaging on tissue by supercritical fluid wash of lipids for matrix-assisted laser desorption/ionization mass spectrometry.

There is a high analytical demand for improving the detection sensitivity for various peptides in matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) because exhaustive distribution analyses of various peptides could help to reveal the function of peptides in vivo. To improve the sensitivity of peptide detection, we used supercritical fluid of CO2 (scCO2) as washing solvent for a pretreatment to remove lipids. We evaluated whether our wash method using scCO2 with an entrainer improved the detection of peptides and suppressed lipid detection in MALDI-IMS. Our analysis revealed that the signal intensities of peptides such as m/z 3339.8, 3530.9, 4233.3, 4936.7, and 4963.7 were increased in scCO2-washed samples. The greatest improvement in the signal-to-noise ratio (S/N) was found at m/z 4963.7, which was identified as thymosin ß4, with the S/N reaching almost 190-fold higher than the control. Additionally, all of the improved signals were associated with the morphologic structure. Our method allows us to analyze the distribution of molecules, especially in the region of m/z 3000-5200. For these improvements, the polarity difference between scCO2 and the matrix solution used was considered as a key. A wider variety of molecules can be analyzed in the future due to this improvement of the detection sensitivity by optimizing the polarity of scCO2 with various entrainers. Graphical Abstract Mass spectra of m/z 4900-5000 obtained from a scCO2-washed tissue (upper, blue) and a control tissue (lower, red). Ion distribution of the signals at m/z 4936.7 and m/z 4963.7 specifically ditected from scCO2-washed samples.

Androgen Regulates Mafb Expression Through its 3'UTR During Mouse Urethral Masculinization.

External genitalia are prominent organs showing hormone-dependent sexual differentiation. Androgen is an essential regulator of masculinization of the genital tubercle, which is the anlage of external genitalia. We have previously shown that v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) is an androgen-inducible regulator of embryonic urethral masculinization in mice. However, it remains unclear how androgen regulates Mafb expression. The current study suggests that the Mafb 3' untranslated region (UTR) is an essential region for its regulation by androgen. We identified 2 functional androgen response elements (AREs) in Mafb 3'UTR. Androgen receptor is bound to such AREs in 3'UTR during urethral masculinization. In addition to 3'UTR, Mafb 5'UTR also showed androgen responsiveness. Moreover, we also demonstrated that ß-catenin, one of genital tubercle masculinization factors, may be an additional regulator of Mafb expression during urethral masculinization. This study provides insights to elucidate mechanisms of gene regulation through AREs present in Mafb 3'UTR for a better understanding of the processes of urethral masculinization.

Long-term morbidity of IgA nephropathy in children evaluated with newly proposed remission criteria in Japan.

BACKGROUND: The long-term outcome of pediatric IgA nephropathy (IgAN) is unclear. Objective IgAN remission criteria were proposed by the Japanese Society of Nephrology in 2013. METHODS: Children with newly developed IgAN followed for >5 years were analyzed. They were divided into two groups based on histological findings at initial kidney biopsy: the focal mesangial proliferation group (Focal group) and diffuse mesangial proliferation group (Diffuse group). The primary outcome was the remission rate according to the newly proposed IgAN remission criteria. RESULTS: The patients comprised 53 children (31 boys; mean age at IgAN onset, 10.0 years). The Focal and Diffuse groups comprised 21 and 32 patients, respectively. No significant differences in patient characteristics were found between the groups except for steroid administration. The median follow-up period from onset was 9.9 years. Sixteen patients in the Diffuse group and 10 in the Focal group had not achieved remission at the last observation. Patient conditions 2 years after the initial treatment were almost identical to those at the last observation. Multivariate analysis revealed that proteinuria, particularly <0.5 g/g Cr at 2 years, was significantly associated with remission at the last observation regardless of proteinuria status at the start of treatment. CONCLUSIONS: Pediatric IgAN has a prolonged course that is longer than expected regardless of severity at diagnosis. Patient conditions 2 years after initial treatment predicted their conditions at the last observation. Although the final renal function of these patients is presently unclear, children with IgAN should be followed beyond adolescence and further into adulthood.

Sexually dimorphic expression of Mafb regulates masculinization of the embryonic urethral formation.

Masculinization of external genitalia is an essential process in the formation of the male reproductive system. Prominent characteristics of this masculinization are the organ size and the sexual differentiation of the urethra. Although androgen is a pivotal inducer of the masculinization, the regulatory mechanism under the control of androgen is still unknown. Here, we address this longstanding question about how androgen induces masculinization of the embryonic external genitalia through the identification of the v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog B (Mafb) gene. Mafb is expressed prominently in the mesenchyme of male genital tubercle (GT), the anlage of external genitalia. MAFB expression is rarely detected in the mesenchyme of female GTs. However, exposure to exogenous androgen induces its mesenchymal expression in female GTs. Furthermore, MAFB expression is prominently down-regulated in male GTs of androgen receptor (Ar) KO mice, indicating that AR signaling is necessary for its expression. It is revealed that Mafb KO male GTs exhibit defective embryonic urethral formation, giving insight into the common human congenital anomaly hypospadias. However, the size of Mafb KO male GTs is similar with that of wild-type males. Moreover, androgen treatment fails to induce urethral masculinization of the GTs in Mafb KO mice. The current results provide evidence that Mafb is an androgen-inducible, sexually dimorphic regulator of embryonic urethral masculinization.

Serum concentration of fentanyl during conversion from intravenous to transdermal administration to patients with chronic cancer pain.

BACKGROUND: To the best of our knowledge, there have been no reports on the pharmacokinetics and pharmacodynamics during the conversion from continuous intravenous infusion (CII) to transdermal fentanyl administration. The primary objective of the present study was to clarify the pharmacokinetic characteristics during this conversion. A secondary objective was to identify an association between serum albumin and the absorption of fentanyl from the transdermal patch. METHODS: A prospective study was conducted from February 2010 to August 2011 that enrolled 19 patients with chronic cancer pain. Patients were classified into 2 study groups according to body mass index and albumin level. All patients received the conversion from CII to transdermal fentanyl using a 2-step taper of CII over 6 hours. Comparisons of efficacy, toxicity, and serum fentanyl concentrations between study groups were analyzed at baseline, 3, 6, 9, 12, 15, 18, and 24 hours after initiation of the conversion. RESULTS: The dose-adjusted serum fentanyl concentrations for all patients were significantly decreased at 15 to 24 hours after conversion compared with baseline, although pain intensity and the number of rescue events remained stable during the conversion. The dose-adjusted serum fentanyl concentrations at 9 to 24 hours were significantly reduced in the low albumin group compared with the normal albumin group (P<0.05). CONCLUSIONS: Our study demonstrated that the dose-adjusted serum fentanyl concentrations remained relatively stable, and pain intensity and the number of rescue events remained stable during conversion. Hypoalbuminemia was strongly associated with poor absorption of transdermally administered fentanyl.