Effects of glucagon-like peptide-1 receptor agonists on spermatogenesis-related gene expression in mouse testis and testis-derived cell lines.

Glucagon-like peptide-1 (GLP-1) is an incretin released into the gastrointestinal tract after food ingestion, and stimulates insulin secretion from the beta cells of the pancreatic islets. Incretins have recently been reported to have extrapancreatic actions, and they are anticipated to have potential efficacy for conditions such as male infertility as well as diabetes. However, the effects of incretins on male reproductive function remain unclear. In this study, GLP-1 receptor expression and the effects of GLP-1 on spermatogenesis-associated genes were investigated using mouse testes and testis-derived cultured cell lines. Glp1r mRNA and GLP-1 protein were expressed in mouse testes at levels comparable to or greater than those in positive control adipose tissue, and the liver and intestine, and also in a Sertoli cell line (TM4) and a Leydig cell line (MA-10) as well as the GC-1 spg and GC-2 spd (ts) germ cell lines. TM4 cells treated with the GLP-1 receptor agonist exenatide showed transiently and significantly upregulated Kitl, Pdgfa, and Glp1r mRNA expression. Furthermore, at 1 hr post-exenatide administration to male mice, Kitl and Glp1r mRNA expression levels were significantly increased, and Pdgfa mRNA expression level also showed a tendency toward increase. TM4 cells were treated with various cell-activating agents, and bucladesine elicited significantly increased Glp1r mRNA expression. We suggest that GLP-1 provides acute stimulation of Sertoli cells in the mouse testis and has a stimulatory effect on the expression of spermatogenesis-related genes.

Arresting calcium-regulated sperm metabolic dynamics enables prolonged fertility in poultry liquid semen storage.

The preservation of liquid semen is pivotal for both industrial livestock production and genetic management/conservation of species with sperm that are not highly cryo-tolerant. Nevertheless, with regard to poultry semen, even brief in vitro storage periods can lead to a notable decline in fertility, despite the in vivo capacity to maintain fertility for several weeks when within the hen's sperm storage tubules. For fertility in sperm, intracellular calcium ions ([Ca2+]i) play a key role in signaling towards modifying energy metabolism. While reducing [Ca2+]i has been found to enhance the preservation of sperm fertility in some mammals, the connection between semen fertility and calcium availability in avian sperm has received limited attention. In this study, we demonstrate that the use of extracellular and intracellular calcium chelators in liquid semen extenders, specifically EGTA and EGTA-AM, has distinct effects on prolonging the fertility of chicken sperm. These results were validated through in vivo fertility tests. Mechanistically, the effects observed were linked to coordination of mitochondrial metabolism and ATP catabolism. Despite both calcium chelators inducing hypoxia, they differentially regulated mitochondrial respiration and ATP accumulation. This regulation was closely linked to a bimodal control of dynein ATPase activity; a direct initial activation with reduction in [Ca2+]i, and subsequent suppression by cytoplasmic acidification caused by lactic acid. These findings not only contribute to advancing poultry liquid semen preservation techniques, but also elucidates biologically relevant mechanisms that may underlie storage within the female reproductive tract in birds.

Segmental differentiation of the murine epididymis: identification of segment-specific, GM1-enriched vesicles and regulation by luminal fluid factors†.

The murine epididymis has 10 distinct segments that provide the opportunity to identify compartmentalized cell physiological mechanisms underlying sperm maturation. However, despite the essential role of the epididymis in reproduction, remarkably little is known about segment-specific functions of this organ. Here, we investigate the dramatic segmental localization of the ganglioside GM1, a glycosphingolipid already known to play key roles in sperm capacitation and acrosome exocytosis. Frozen tissue sections of epididymides from adult mice were treated with the binding subunit of cholera toxin conjugated to AlexaFluor 488 to label GM1. We report that GM1-enriched vesicles were found exclusively in principal and clear cells of segment 2. These vesicles were also restricted to the lumen of segment 2 and did not appear to flow with the sperm into segment 3, within the limits of detection by confocal microscopy. Interestingly, this segment-specific presence was altered in several azoospermic mouse models and in wild-type mice after efferent duct ligation. These findings indicate that a lumicrine factor, itself dependent on spermatogenesis, controls this segmental differentiation. The RNA sequencing results confirmed global de-differentiation of the proximal epididymal segments in response to efferent duct ligation. Additionally, GM1 localization on the surface of the sperm head increased as sperm transit through segment 2 and have contact with the GM1-enriched vesicles. This is the first report of segment-specific vesicles and their role in enriching sperm with GM1, a glycosphingolipid known to be critical for sperm function, providing key insights into the segment-specific physiology and function of the epididymis.

Novel Cytochrome P450 2C94 Functionally Metabolizes Diclofenac and Omeprazole in Dogs.

Cytochromes P450 (P450s or CYPs) are important drug-metabolizing enzymes. Because dogs are frequently used in drug metabolism studies, knowledge of dog CYP2C enzymes is essential because in humans these enzymes are abundant and play major roles in liver and intestine. The present study identified and characterized novel dog CYP2C94 along with previously identified dog CYP2C21 and CYP2C41. Dog CYP2C21, CYP2C41, and CYP2C94 cDNAs, respectively, contained open reading frames of 490, 489, and 496 amino acids and shared high-sequence identities (70%, 75%, and 58%) with human CYP2Cs. Dog CYP2C94 mRNA was preferentially expressed in liver, just as dog CYP2C21 and CYP2C41 mRNAs were. In dog liver, CYP2C21 mRNA was the most abundant, followed by CYP2C94 and CYP2C41 mRNAs. Moreover, the hepatic expressions of all three dog CYP2C mRNAs varied in four individual dogs, two of which did not express CYP2C41 mRNA. The three dog CYP2C genes had similar gene structures, and CYP2C94, although located on the same chromosome, was in a genomic region far from the gene cluster containing CYP2C21 and CYP2C41 Metabolic assays with recombinant proteins showed that dog CYP2C94, along with CYP2C21 and CYP2C41, efficiently catalyzed oxidations of diclofenac, warfarin, and/or omeprazole, indicating that dog CYP2C94 is a functional enzyme. Novel dog CYP2C94 is expressed abundantly in liver and encodes a functional enzyme that metabolizes human CYP2C substrates; it is, therefore, likely responsible for drug clearances in dogs. SIGNIFICANCE STATEMENT: Novel dog cytochrome P450 2C94 (CYP2C94) was identified and characterized along with dog CYP2C21 and CYP2C41. Dog CYP2C94, isolated from liver, had 58% sequence identity and a close phylogenetic relationship with its human homologs and was expressed in liver at the mRNA level. Dog CYP2C94 (and CYP2C21 and CYP2C41) catalyzed oxidations of diclofenac and omeprazole, human CYP2C9 and CYP2C19 substrates, respectively, but CYP2C41 also hydroxylated warfarin. CYP2C94 is therefore a functional drug-metabolizing enzyme likely responsible for drug clearances in dogs.

A case of squamous cell carcinoma arising from a suprapubic cystostomy tract in a patient with spinal bifida: Immunohistochemical analysis and literature review.

Introduction: Squamous cell carcinoma arising from a suprapubic cystostomy tract is a rare complication of an indwelling catheter and is caused by long-term inflammation and mechanical irritation. Prognosis is relatively poor. Biomarkers in the cancer pathway have not been investigated. Case presentation: A 61-year-old woman with a 34-year history of suprapubic catheter placement presented with a rapidly growing elevated lesion around the cystostomy site. Tumor biopsy confirmed squamous cell carcinoma. Local excision with partial cystectomy was performed. Multiple metastases were identified 5 months later. The patient died 14 months after the initial treatment. Immunohistochemical analysis of the resected specimen revealed alterations in vascular endothelial growth factor, epidermal growth factor receptor, cyclooxygenase-2, and Ki-67. Conclusion: We encountered a case of squamous cell carcinoma arising from a suprapubic cystostomy tract. Immunohistochemical analysis revealed activation of multiple carcinogenic pathways in cancer cells, including those for angiogenesis, signal transduction by epidermal growth factor receptor, inflammation, and cell proliferation.

Blending to Make Nonhealable Polymers Healable: Nanophase Separation Observed by CP/MAS 13 C NMR Analysis.

Can commodity polymers are made to be healable just by blending with self-healable polymers? Here we report the first study on the fundamental aspect of this practically challenging issue. Poly(ether thiourea) (PTUEG3 ; Tg =27 °C) reported in 2018 is extraordinary in that it is mechanically robust but can self-heal even at 12 °C. In contrast, poly(octamethylene thiourea) (PTUC8 ; Tg =50 °C), an analogue of PTUEG3 , cannot heal below 92 °C. We found that their polymer blend self-healed in a temperature range above 32 °C even when its PTUEG3 content was only 20 mol %. Unlike PTUEG3 alone, this polymer blend, upon exposure to high humidity, barely plasticized, keeping its excellent mechanical properties due to the non-hygroscopic nature of the PTUC8 component. CP/MAS 13 C NMR analysis revealed that the polymer blend was nanophase-separated, which possibly accounts for why such a small amount of PTUEG3 provided the polymer blend with humidity-tolerant self-healable properties.

Polymyxin B neutralizes endotoxin and improves the quality of chicken semen during liquid storage.

Despite its importance in gamete utilization for livestock production, poultry semen cryopreservation in a liquid state, is limited in the poultry industry due to a significant decline in sperm viability and functionality during liquid storage. Lipopolysaccharide (LPS) is released from gram-negative bacteria and impairs sperm function in mammals. Using exogeneous LPS, we show this endotoxin compromises sperm viability and function, including motility and penetrability to the inner peri-vitelline layer (IPVL) during liquid storage at 4 °C. This outcome was supported by LPS quantification showing an extreme increase in the first 24 h of storage. Polymyxin B (PMB) is an LPS neutralizer previously shown to improve fertility in boar semen, thus we explored the effect of PMB on chicken semen quality during liquid storage. Sperm viability and penetrability tests showed that PMB completely abolishes the deleterious effect by LPS. However, co-addition of PMB with penicillin G (PenG), an antibiotic against gram positive bacteria, reduces IPVL-penetrability while improving sperm viability post-storage. Furthermore, artificial insemination trials showed that PMB addition improves semen fertility at the post liquid storage. Our results show that chicken semen quality during liquid storage is impaired by bacterial LPS, but improved by PMB addition due to cancelled endotoxic effects, which offers a new approach for prolonged fertility of poultry semen storage in a liquid state.

A comprehensive analysis of six forms of cytochrome P450 2C (CYP2C) in pigs.

Pigs are an important species used in drug metabolism studies; however, the cytochromes P450 (P450s or CYPs) have not been fully investigated in pigs.In this study, pig CYP2C32, CYP2C33, CYP2C34, CYP2C36, CYP2C42, and CYP2C49 cDNAs were isolated and found to contain open reading frames of 490 or 494 amino acids that shared 64-82% sequence identity with human CYP2C8/9/18/19.Pig CYP2C genes formed a gene cluster in a genomic region that corresponded to that of the human CYP2C cluster; an additional gene cluster was formed by pig CYP2C33a and CYP2C33b distant from the first cluster but located in the same chromosome.Among the tissues analysed, these pig CYP2C mRNAs were preferentially expressed in liver, small intestine, and/or kidney; pig CYP2C49, CYP2C32, CYP2C34, and CYP2C33 mRNAs were the most abundant CYP2C mRNAs in liver, jejunum, ileum, and kidney, respectively.Metabolic assays showed that pig CYP2C proteins (heterologously expressed in Escherichia coli) metabolised typical human CYP2C substrates diclofenac, warfarin, and/or omeprazole.The results suggest that these pig CYP2Cs are functional enzymes able to metabolise human CYP2C substrates in liver and small intestine, just as human CYP2Cs do.

Effects of Oxygen Gas Injection on the Subsequent Development of Chick Embryos in a Shell-Less Culture System.

The effects of oxygen gas injection starting on day 17 of incubation (D17) in a chick shell-less culture system (cSLC) on the subsequent embryo development were examined on day 19 of incubation (D19). On D19 of cSLC, the plasma phosphorus and total cholesterol concentrations of the embryos were significantly higher (P<0.05), while the plasma calcium concentrations were significantly lower (P<0.05) than those in the intact control (IC) group. However, no significant differences in embryo viability and other major blood component levels were observed among the experimental groups (P>0.05). The percutaneous oxygen saturation was lower in D17-cSLC embryos before oxygen gas supplementation than in the IC (P<0.05) embryos. Severe renal tubular degeneration of the metanephros was observed in D19-cSLC embryos despite oxygen gas injection starting from D17. These results indicate that D19-cSLC embryos are hypoxia even after injecting oxygen gas starting on D17.

Molecular and Functional Characterization of N-Acetyltransferases in Common Marmosets and Pigs.

Arylamine N-acetyltransferases (NATs) are drug-metabolizing enzymes that are essential for the metabolism of endogenous substrates and xenobiotics. The molecular characteristics of NATs have been extensively investigated in humans but remain to be investigated in common marmosets and pigs, animal species that are often used in drug metabolism studies. In this study, marmoset NAT1 and pig NAT1 cDNAs were isolated from liver samples and were characterized by molecular analyses and drug-metabolism assays. These NAT genes were intronless and formed gene clusters with one other NAT gene in the genome, just as human NAT genes do. Marmoset NAT1 and pig NAT1 amino acid sequences showed high sequence identities (94% and 85%, respectively) to human NAT1. Phylogenetic analysis indicated that marmoset NAT1 and pig NAT1 were more closely clustered with human NATs than with rat or mouse NATs. Marmoset NAT1 and pig NAT1 mRNAs were expressed in all the tissue types analyzed, with the expression levels being highest in the small intestine. Metabolic assays using recombinant proteins found that marmoset NAT1 and pig NAT1 metabolized human NAT substrates p-aminobenzoic acid, 2-aminofluorene, sulfamethazine, and isoniazid. Marmoset NAT1 and pig NAT1 substantially acetylated p-aminobenzoic acid and 2-aminofluorene relevant human NAT1, but their activities were lower toward sulfamethazine and isoniazid than those of the relevant human NAT2. Therefore, marmoset and pig NATs are functional enzymes with molecular similarities to human NAT1, but their substrate specificities, while similar to human NAT1, differ somewhat from human NAT2. SIGNIFICANCE STATEMENT: Marmoset N-acetyltransferase NAT1 and pig NAT1 were identified and showed high sequence identities to human NAT1. These NAT mRNAs were expressed in various tissues. Marmoset and pig NAT1s acetylated typical human NAT substrates, although their substrate specificities differed somewhat from human NAT2. Marmoset NAT1 and pig NAT1 have similarities with human NAT1 in terms of molecular and enzymatic characteristics.

Cytochrome P450 2J Genes Are Expressed in Dogs, Cats, and Pigs, and Encode Functional Drug-Metabolizing Enzymes.

Cytochrome P450s (P450s) have been identified and analyzed in dogs and pigs, species that are often used in preclinical drug studies. Moreover, P450s are clinically important for drug therapy not only in humans, but also in species under veterinary care, including dogs and cats. In the present study, seven P450s homologous to human CYP2J2, namely, dog CYP2J2; cat CYP2J2; and pig CYP2J33, CYP2J35, CYP2J91, and CYP2J93, were newly identified and characterized, along with pig CYP2J34 previously identified. The cDNAs of these CYP2Js contain open reading frames of 502 amino acids, except for CYP2J35 (498 amino acids), and share high sequence identity (77%-80%) with human CYP2J2. Phylogenetic analysis revealed that dog and cat CYP2J2 were closely related, whereas pig CYP2Js formed a cluster. All seven CYP2J genes contain nine coding exons and are located in corresponding genomic regions, with the pig CYP2J genes forming a gene cluster. These CYP2J2 mRNAs were predominantly expressed in the small intestine with additional expression in the kidney and brain for dog CYP2J2 and pig CYP2J91 mRNAs, respectively. All seven CYP2Js metabolized human CYP2J2 substrates terfenadine, ebastine, and astemizole, indicating that they are functional enzymes. Dog CYP2J2 and pig CYP2J34 and CYP2J35 efficiently catalyzed ebastine primary hydroxylation and secondary carebastine formation at low substrate concentrations, just as human CYP2J2 does. Velocity-versus-substate plots exhibited sigmoidal relationships for dog CYP2J2, cat CYP2J2, and pig CYP2J33, indicating allosteric interactions. These results suggest that dog, cat, and pig CYP2Js have similar functional characteristics to human CYP2J2, with slight differences in ebastine and astemizole oxidations. SIGNIFICANCE STATEMENT: Dog CYP2J2; cat CYP2J2; and pig CYP2J33, CYP2J34, CYP2J35, CYP2J91, and CYP2J93, homologous to human CYP2J2, were identified and characterized by sequence, phylogenetic, and genomic structure analyses. Intestinal expression patterns of CYP2J mRNAs were characteristic in dogs, cats, and pigs. Dog, cat, and pig CYP2Js likely play roles as drug-metabolizing enzymes in the small intestine, similar to human CYP2J2.

Effects of Calcium Lactate on the Development of Chicken Embryos in a Shell-less Culture System up to Day Seventeen of Incubation.

This study examined the effects of calcium lactate on the development of chicken embryos in a shell-less culture system (cSLCS) up to the seventeenth day of incubation. In the presence of calcium lactate, a significant reduction in embryo viability was observed during the first week of incubation in cSLCS. On day 17 of embryo development, no significant difference was observed in the blood plasma calcium concentration or tibia bone density between cSLCS and intact control embryos, whereas the tibia length was significantly shorter in cSLCS embryos than in the intact control. These results suggest that calcium lactate supplementation in cSLCS supports bone formation in developing chicken embryos, but has adverse effects on the viability of embryos, particularly during the first week of embryo development.

Membrane-Mediated Regulation of Sperm Fertilization Potential in Poultry.

Fertilization requires successful completion of molecular events taking place at different spatiotemporal scales. Transcriptionally and translationally inactive sperm need to rely on pre-assembled pathways modulated by extracellular signals that traverse the plasma membranes. However, species differences in how sperm respond to them delay the progress toward a comprehensive understanding of how activation of the signaling cascades is coordinated in poultry sperm. In chickens, recent studies have found that membrane rafts are present on the sperm surface and play important roles in regulating multistage fertilization. In this review, we focus on three steps in which membrane alteration plays a key role. The first is post-testicular maturation, in which bird sperm acquire fertilization functions through biochemical changes. The second part of this review concerns membrane regulation of sperm-egg binding and the acrosome reaction. Finally, we extend our discussion to the translation of membrane raft theory into a technical principle for the commercial production and genetic preservation of poultry.

Androgen receptor-neuroendocrine double-negative tumor with squamous differentiation arising from treatment-refractory metastatic castration-resistant prostate cancer.

INTRODUCTION: Treatment-refractory metastatic castration-resistant prostate cancer is a heterogeneous disease classified into androgen receptor-high prostate cancer, androgen receptor-low prostate cancer, amphicrine prostate cancer co-expressing androgen receptor and neuroendocrine genes, double-negative prostate cancer lacking androgen receptor and neuroendocrine gene expression, and small cell or neuroendocrine prostate cancer without androgen receptor activity. Double-negative tumors can convert to the squamous phenotype. CASE PRESENTATION: A 62-year-old man was newly diagnosed with prostate cancer (serum prostate-specific antigen 2613 ng/mL, Gleason score 4 + 5 = 9, cT3aN1M1b) that progressed to castration resistance 4 months after starting abiraterone with androgen deprivation therapy. After enzalutamide and docetaxel failed, a right ilium metastasis newly emerged. Needle biopsy confirmed a metastatic tumor with squamous differentiation that was CK5/6-positive and chromogranin A-, synaptophysin-, and androgen receptor-negative. CONCLUSION: We encountered a case of double-negative prostate cancer with squamous differentiation identified by needle biopsy of a right ilium metastasis after abiraterone, enzalutamide, and docetaxel failure.

Functional difference of ATP-generating pathways in rooster sperm (Gallus gallus domesticus).

Adenosine triphosphate (ATP) production via glycolysis and oxidative phosphorylation is essential for the maintenance of flagellar motility in sperm; however, the primary energy production pathways supporting fertilization vary among species. Inconsistency in thought exists regarding which pathways maintain ATP production and sperm motility in poultry. Glycolysis and mitochondrial oxidation contribute to flagellar motion in chicken sperm, but the relative dependence on these pathways for motility and penetrability into the inner perivitelline layer remains unclear. In the present study, there was use of various inhibitors and energy substrates to evaluate the relative contribution of anaerobic glycolysis and mitochondrial oxidation to chicken sperm flagellar motility, ATP production, and penetrating capacity through the perivitelline layer. Although both pathways contributed to these processes to varying extent, glucose was the primary substrate for sperm penetration into the inner perivitelline layer in chickens. Furthermore, results from metabolic stress analyses indicated that there was less perivitelline penetrability in response to pyruvate that was not due to changes in reactive oxygen species or intracellular pH. Overall, results from the present study indicate glycolysis and mitochondrial oxidation pathways have distinct functions in the flagellar motility and penetrability of the perivitelline membrane by rooster sperm. There, therefore, are new insights as a result of findings in the present study into the energy production system of sperm through which there is utilization of extracellular metabolic substrates for maintaining sperm fertilization capacity.

Mechanically Robust, Self-Healable Polymers Usable under High Humidity: Humidity-Tolerant Noncovalent Cross-Linking Strategy.

Although mechanically robust polymer materials had not been thought to self-heal, we recently found that poly(ether thiourea) PTUEG3, which is a glassy polymer with high mechanical strength, self-heals even at ambient temperatures. This finding updated the above preconception. Nevertheless, it should also be noted that PTUEG3, under high humidity, absorbs water and is plasticized to lose its mechanical strength. Humidity-induced plasticization is a general problem for polymers with polar groups. Herein, we report that PTUEG3, if designed by copolymerization to contain only 10 mol % of a dicyclohexylmethane (Cy2M) thiourea unit (TUCy2M), serves as a humidity-tolerant, mechanically robust polymer material that can self-heal at ambient temperatures. This copolymer contained, in its ether thiourea (TUEG3)-rich domain, a humidity-tolerant, noncovalently cross-linked 3D network with mechanical robustness formed by stacking of the Cy2M group. The present work provides a promising design strategy for mechanically robust, self-healable polymers usable under high humidity.

Src family kinases-mediated negative regulation of sperm acrosome reaction in chickens (Gallus gallus domesticus).

The acrosome reaction (AR) is a strictly-regulated, synchronous exocytosis that is required for sperm to penetrate ova. This all-or-nothing process occurs only once in the sperm lifecycle through a sequence of signaling pathways. Spontaneous, premature AR therefore compromises fertilization potential. Although protein kinase A (PKA) pathways play a central role in AR across species, the signaling network used for AR induction is poorly understood in birds. Mechanistic studies of mammalian sperm AR demonstrate that PKA activity is downstreamly regulated by Src family kinases (SFKs). Using SFK inhibitors, our study shows that in chicken sperm, SFKs play a role in the regulation of PKA activity and spontaneous AR without affecting motility. Furthermore, we examined the nature of SFK phosphorylation using PKA and protein tyrosine phosphatase inhibitors, which demonstrated that unlike in mammals, SFK phosphorylation in birds does not occur downstream of PKA and is primarily regulated by calcium-dependent tyrosine phosphatase activity. Functional characterization of SFKs in chicken sperm showed that SFK activation modulates the membrane potential and plays a role in inhibiting spontaneous AR. Employing biochemical isolation, we also found that membrane rafts are involved in the regulation of SFK phosphorylation. This study demonstrates a unique mechanism for regulating AR induction inherent to avian sperm that ensure fertilization potential despite prolonged storage.

Localisation and function of glucose transporter GLUT1 in chicken (Gallus gallus domesticus) spermatozoa: relationship between ATP production pathways and flagellar motility.

Glucose plays an important role in sperm flagellar motility and fertility via glycolysis and oxidative phosphorylation, although the primary mechanisms for ATP generation vary between species. The glucose transporter 1 (GLUT1) is a high-affinity isoform and a major glucose transporter in mammalian spermatozoa. However, in avian spermatozoa, the glucose metabolic pathways are poorly characterised. This study demonstrates that GLUT1 plays a major role in glucose-mediated motility of chicken spermatozoa. Using specific antibodies and ligand, we found that GLUT1 was specifically localised to the midpiece. Sperm motility analysis showed that glucose supported sperm movement during incubation for 0-80min. However, this was abolished by the addition of a GLUT1 inhibitor, concomitant with a substantial decrease in glucose uptake and ATP production, followed by elevated mitochondrial activity in response to glucose addition. More potent inhibition of ATP production and mitochondrial activity was observed in response to treatment with uncouplers of oxidative phosphorylation. Because mitochondrial inhibition only reduced a subset of sperm movements, we investigated the localisation of the glycolytic pathway and showed glyceraldehyde-3-phosphate dehydrogenase and hexokinase I at the midpiece and principal piece of the flagellum. The results of this study provide new insights into the mechanisms involved in ATP production pathways in avian spermatozoa.

Formation of α-synuclein aggregates in aqueous ethylammonium nitrate solutions.

The effect of adding ethylammonium nitrate (EAN), which is an ionic liquid (IL), on the aggregate formation of α-synuclein (α-Syn) in aqueous solution has been investigated. FTIR and Raman spectroscopy were used to investigate changes in the secondary structure of α-Syn and in the states of water molecules and EAN. The results presented here show that the addition of EAN to α-Syn causes the formation of an intermolecular ß-sheet structure in the following manner: native disordered state → polyproline II (PPII)-helix → intermolecular ß-sheet (α-Syn amyloid-like aggregates: α-SynA). Although cations and anions of EAN play roles in masking the charged side chains and PPII-helix-forming ability involved in the formation of α-SynA, water molecules are not directly related to its formation. We conclude that EAN-induced α-Syn amyloid-like aggregates form at hydrophobic associations in the middle of the molecules after masking the charged side chains at the N- and C-terminals of α-Syn.

[OUTCOMES OF CHRONIC UNILATERAL HEMATURIA TREATED USING DIGITAL FLEXIBLE URETEROSCOPE].

(Objectives) We examined the treatment outcomes in cases of chronic unilateral hematuria treated using flexible ureteroscope for observation and hemostasis. (Methods) The study included 14 patients (7 men and 7 women) with a median age of 56.5 years who underwent ureteroscopy using a digital flexible ureteroscope for chronic unilateral hematuria between March 2014 and August 2019. All the patients presented with macroscopic hematuria as a clinical symptom, but in one patient, the hematuria was accompanied by anemia and required a blood transfusion. In addition, bleeding occurred on the left side in 8 patients and on the right side in 3 patients; however, for the remaining 3 patients, the affected side could not be identified. Fourteen patients were examined on the basis of the ureteroscopic findings, number of bleeding sites, hemostatic intervention, treatment effect, and presence or absence of recurrences. (Results) The ureteroscopic findings showed a hemangioma in 7 patients and minute venous rupture in 3, but the remaining 4 patients showed no clear findings. The site of the findings was in the superior calyces in 8 cases, middle calyces in 4 cases, inferior calyces in 4 cases, and renal pelvic wall in 1 case. In addition, the findings were located at multiple sites in 6 cases, including all renal calyces in 2 cases. Ten patients with findings underwent hemostatic interventions (electrocoagulation and laser treatment). The median postoperative follow-up period was 32.4 months (range, 6.4-65.4 months). In all the cases, the hematuria disappeared after treatment. One of the 2 patients with findings in all renal calyces showed recurrence of macroscopic hematuria at 1 year and 6 months, which disappeared after conservative treatment. (Conclusions) In this study, observation using digital flexible ureteroscope was useful in the treatment of chronic unilateral hematuria, and the hemostatic interventions performed on the bleeding sites in the renal pelvis were effective.