Fungal Metabolism of Sorbic Acid

The metabolism of sorbic acid (S A) by 11 species of Mucor and 14 other fungi in addition to Mucor sp. A–73 were studied. All species of Mucor metabolized SA to trans-4-hexenol (4–HEL) without exception. The metabolic pathway was investigated precisely by using the resting cells of Mucor sp. A–73. The reduction of SA to 4–HEL by the fungus demanded glucose. The reduction of SA to sorbic alcohol (SAL) was catalyzed by the inducible enzymic system and that of SAL to 4–HEL by the constitutive enzyme. In the phosphate buffer, a part of 4–HEL produced from SA was further transformed into mono-trans-4-hexenyl phosphate by Mucor sp. A–73.

The reductive action of the fungus upon several trans-2-alkenoic acids was also examined.     

New laparoscopic procedure for left-sided pancreatic cancer—artery-first approach laparoscopic RAMPS using 3D technique

Background

For left-sided pancreatic ductal adenocarcinoma (PDAC), radical antegrade modular pancreatosplenectomy (RAMPS) is a reasonable surgical approach for tumor-free margin resection and systemic lymph node clearance. In pancreaticoduodenectomy for PDAC, the superior mesenteric artery (SMA)-first approach (or the “artery-first approach”) has become the standard procedure. With improvements in laparoscopic instruments and techniques, some surgeons attempted to apply laparoscopic RAMPS (L-RAMPS) for carefully selected patients with left-sided PDAC. However, owing to several technical difficulties in this procedure, its application remains uncommon. Moreover, the artery-first approach in L-RAMPS has not been reported. Here, we developed the artery-first approach L-RAMPS for left-sided PDAC and have presented the same in this report.

Case presentation

Between June 2014 and July 2015, 16 patients with left-sided PDAC were referred to our division for pancreatic resection. The following technique was used for performing L-RAMPS on 3 of the 16 patients (19%). Six trocars were placed. After opening the omental bursa, only the middle segment of the pancreas was initially separated from both the left renal vein and the SMA. We termed this procedure as the “artery-first approach using a dome-shaped dorsomedial dissection (3D) technique.” This 3D technique enabled the interruption of the entire arterial supply to the specimen while preserving the venous drainage through the splenic vein for preventing venous congestion. The technique also contributed to the early detection of no tumor infiltration into the SMA and the early determination of posterior dissection plane. After pancreatic neck transection, the splenic artery and vein were divided. Finally, the pancreatic tail and spleen were dissected in a right-to-left direction. All operations were completed without any intraoperative complications. The median blood loss and retrieved lymph node count were 75 mL and 37, respectively, which were superior to those reported by other previous studies on L-RAMPS. All resection margins were free of carcinoma. No severe postoperative complications were observed.

Conclusions

The artery-first approach L-RAMPS using 3D technique is safe and feasible to perform. The significance of our proposed procedure is minimal blood loss and precise lymphadenectomy. Therefore, this novel technique may become the preferred treatment for left-sided PDAC in selected cases.

     

Optimized prime editing efficiently generates heritable mutations in maize

Low efficiency is the main obstacle to using prime editing in maize(Zea mays). Recently, prime-editing efficiency was greatly improved in mammalian cells and rice(Oryza sativa) plants by engineering primeediting guide RNAs(pegRNAs), optimizing the prime editor(PE) protein, and manipulating cellular determinants of prime editing. In this study, we tested PEs optimized via these three strategies in maize. We demonstrated that the ePE5max system, composed of PEmax, epegRNAs(pegRNA-evopreQ. 1), nick...     

Double column-switching high-performance liquid chromatographic method for the determination of TAK-603 and its metabolites in human serum

A double column-switching high-performance liquid chromatographic (HPLC) method for the determination of concentrations for TAK-603 (T) and its metabolites, T-72258 (M-I) and T-72294 (M-III), in human serum was developed. The analytes were extracted with ethyl acetate from human serum samples treated with triethylamine and injected into the HPLC system. Separation of the analytes was performed on the HPLC system with double column-switching technique. The mobile phases A and B for the first column and the mobile phase C for the second column used were a mixture of methanol–10 mM aqueous ammonium acetate solution (1:1, v/v), methanol and a mixture of methanol–10 mM aqueous ammonium acetate solution (11:9, v/v), respectively. The eluate was monitored with a UV detector at a wavelength of 253 nm. The work-up procedure was reproducible and more than 90% of the analytes could be recovered from human serum. The lower limits of quantitation were all 1 ng/ml for the analytes when 0.5 ml of human serum was used. Standard curves were linear with a correlation coefficient (R) of more than 0.999 in the range of 1–500 ng/ml for T, M-I and M-III in human serum. The intra- and inter-day precision of the method for the various analytes were below 4.8%. The accuracy was good with the deviations between spiked and calculated concentrations of the analytes being within 11.0%. The method was successfully applied to analyze serum samples after an oral administration of T to healthy male volunteers.     

Association of nicotinic acetylcholine receptors with central respiratory control in isolated brainstem-spinal cord preparation of neonatal rats

Nicotine exposure is a risk factor in several breathing disorders Nicotinic acetylcholine receptors (nAChRs) exist in the ventrolateral medulla, an important site for respiratory control. We examined the effects of nicotinic acetylcholine neurotransmission on central respiratory control by addition of a nAChR agonist or one of various antagonists into superfusion medium in the isolated brainstem-spinal cord from neonatal rats. Ventral C4 neuronal activity was monitored as central respiratory output, and activities of respiratory neurons in the ventrolateral medulla were recorded in whole-cell configuration. RJR-2403 (0.1-10 mM), alpha4beta2 nAChR agonist induced dose-dependent increases in respiratory frequency. Non-selective nAChR antagonist mecamylamine (0.1-100 mM), alpha4beta2 antagonist dihydro-beta-erythroidine (0.1-100 mM), alpha7 antagonist methyllycaconitine (0.1-100 mM), and a-bungarotoxin (0.01-10 mM) all induced dose-dependent reductions in C4 respiratory rate. We next examined effects of 20 mM dihydro-beta-erythroidine and 20mM methyllycaconitine on respiratory neurons. Dihydro-beta-erythroidine induces hyperpolarization and decreases intraburst firing frequency of inspiratory and preinspiratory neurons. In contrast, methyllycaconitine has no effect on the membrane potential of inspiratory neurons, but does decrease their intraburst firing frequency while inducing hyperpolarization and decreasing intraburst firing frequency in preinspiratory neurons. These findings indicate that alpha4beta2 nAChR is involved in both inspiratory and preinspiratory neurons, whereas alpha7 nAChR functions only in preinspiratory neurons to modulate C4 respiratory rate.     

Live piglets derived from in vitro-produced zygotes vitrified at the pronuclear stage

We report the successful cryopreservation of in vitro-produced porcine zygotes. Follicular oocytes from prepubertal gilts were matured (IVM), fertilized (IVF), and cultured (IVC) in vitro. At 10 or 23 h after IVF, the oocytes were centrifuged to visualize pronuclei. Zygotes with two or three pronuclei were used for solid surface vitrification (SSV). Survival of vitrified-warmed zygotes was determined by their morphology. To assess their developmental competence, vitrified (SSV), cryoprotectant-treated (CPA), and untreated (control) zygotes were subjected to IVC for 6 days. Survival and developmental competence did not differ between control and CPA zygotes. The proportion of live zygotes after SSV and warming (93.4%) was similar to that in the controls (100%). Cleavage and blastocyst formation rates of SSV zygotes after vitrification (71.7% and 15.8%, respectively) were significantly lower than those of controls (86.3% and 24.5%, respectively; ANOVA P<0.05). Blastocyst cell numbers of SSV and control embryos were similar (41.2+/-3.4 and 41.6+/-3.3, respectively). There was no difference in developmental ability between zygotes cryopreserved at an early (10 h after IVF) or late (23 h after IVF) pronuclear stage. Storage in liquid nitrogen had no effect on the in vitro developmental competence of vitrified zygotes beyond the reduction induced by the vitrification itself. When the embryo culture medium was supplemented with 1 muM glutathione, the rate of development of cryopreserved zygotes to the blastocyst stage did not differ significantly from that of control glutathione-treated zygotes (18.6% and 22.1%, respectively). To test their ability to develop to term, vitrified zygotes were transferred to five recipients, resulting in three pregnancies and the production of a total of 17 piglets. These data demonstrate that IVM-IVF porcine zygotes can be cryopreserved at the pronuclear stage effectively without micromanipulation-derived delipation, preserving their full developmental competence to term.     

Treatment with proteasome inhibitor MG132 during cloning improves survival and pronuclear number of reconstructed rat embryos

In several mammalian species including rats, successfully cloned animals have been generated using somatic cell nuclear transfer (SCNT). However, in the case of rats, additional treatment with MG132, a proteasome inhibitor, before enucleation of oocytes seems to be required for successful cloning because ovulated rat oocytes are spontaneously activated, and hence, their suppression is the key to successful cloning. A previous study on rats demonstrated that matured oocytes potentially possess lower cytostatic factor (CSF) activity compared to mouse oocytes, resulting in a low incidence of premature chromosome condensation in the reconstructed embryos after SCNT. It is known that mice having more than two pronuclei are generally observed in nuclear-transferred oocytes after induction of premature chromosome condensation, which implies successful reprogramming. This leads us to the hypothesis that MG132 treatment affects not only the inhibition of spontaneous activation but also the reprogramming and developmental ability of reconstructed rat embryos. If so, prolonged MG132 treatment during and/or after SCNT may further improve the survivability. However, the effect of MG132 treatment on reconstructed embryos after SCNT has been very limited in rats and other species. We show here that prolonged MG132 treatment during and after SCNT improves survival and the number of pronuclei in reconstructed rat embryos after activation. These reconstructed embryos treated before, during, and after SCNT showed significantly higher p34(cdc2) kinase activity involving CSF activity compared to that of the control embryos. On the other hand, p34(cdc2) kinase activity was not recovered in nuclear-transferred oocytes without MG132, which suggested that the enucleation had detrimental effects on the development of reconstructed oocytes. Taken together, MG132 treatment during SCNT increases survival and pronuclear numbers in reconstructed rat embryos via maintenance of high CSF activity. The data suggest that MG132 treatment is indispensable for at least rat SCNT.