Engineering the expression system for Komagataella phaffii (Pichia pastoris): an attempt to develop a methanol-free expression system.

The construction of a methanol-free expression system of Komagataella phaffii (Pichia pastoris) was attempted by engineering a strong methanol-inducible DAS1 promoter using Citrobacter braakii phytase production as a model case. Constitutive expression of KpTRM1, formerly PRM1-a positive transcription regulator for methanol-utilization (MUT) genes of K. phaffii,was demonstrated to produce phytase without addition of methanol, especially when a DAS1 promoter was used but not an AOX1 promoter. Another positive regulator, Mxr1p, did not have the same effect on the DAS1 promoter, while it was more effective than KpTrmp1 on the AOX1 promoter. Removing a potential upstream repression sequence (URS) and multiplying UAS1DAS1 in the DAS1 promoter significantly enhanced the yield of C. braakii phytase with methanol-feeding, which surpassed the native AOX1 promoter by 80%. However, multiplying UAS1DAS1 did not affect the yield of methanol-free expression by constitutive KpTrm1p. Another important region to enhance the effect of KpTrm1p under a methanol-free condition was identified in the DAS1 promoter, and was termed ESPDAS1. Nevertheless, methanol-free phytase production using an engineered DAS1 promoter outperformed phytase production with the GAP promoter by 25%. Difference in regulation by known transcription factors on the AOX1 promoter and the DAS1 promoter was also illustrated.

The effect of steep Trendelenburg positioning on intraocular pressure and visual function during robotic-assisted radical prostatectomy.

BACKGROUND: To evaluate intraocular pressure (IOP) changes in patients undergoing robotic-assisted radical prostatectomy and to evaluate complications from increased IOP. METHODS: Thirty-one eyes scheduled for robotic prostatectomy were included. Perioperative IOP measurements were performed as follows: prior to induction of anaesthesia while supine and awake (T1); immediately post-induction while supine (T2); every hour from 0 to 5 h while anaesthetised in a steep Trendelenburg position (T3-T8); prior to awakening while supine (T9); and 30 min after awakening while supine (T10). A complete ophthalmic examination including visual acuity and retinal nerve fibre layer thickness (RNFL) was performed at enrolment and 1 month postoperatively. RESULTS: Average IOP (mm Hg) for each time point was as follows: T1=18.0, T2=9.8, T3=18.9, T4=21.6, T5=22.5, T6=22.3, T7=24.2, T8=24.0, T9=15.7 and T10=17.9. The proportion of eyes with intraoperative IOP ≧30 mm Hg were as follows: T3=0%, T4=3.23%, T5=9.68%, T6=6.45%, T7=22.22%, and T8=25%. Maximum IOP was 36 mm Hg. Mean visual acuity (logarithm of the minimal angle of resolution) and RNFL showed no statistically significant difference before and after operation and no other ocular complications were found at final examination. CONCLUSIONS: While IOP increased in a time-dependent fashion in anesthaetised patients undergoing robotic-assisted radical prostatectomy in a steep Trendelenburg position, visual function showed no significant change postoperatively and no complications were seen. Steep Trendelenburg positioning during time-limited procedures appears to pose little or no risk from IOP increases in patients without pre-existing ocular disease.

Long-term inhibition of angiotensin prevents reduction of periarterial innervation of calcitonin gene-related peptide (CGRP)-containing nerves in spontaneously hypertensive rats.

The aim of this study was to investigate age-related changes in the density of calcitonin gene-related peptide (CGRP)-containing nerve fibers in spontaneously hypertensive rats (SHR) and the effects of long-term inhibition of the renin-angiotensin system on these changes. The density of immunocytochemically stained nerve fibers in the mesenteric artery was quantified by computer-assisted image processing. An age-related decrease in the density of CGRP-like immunoreactive (LI)-containing nerve fivers but not neuropeptide Y (NPY)-LI-containing sympathetic nerve fibers was found in the mesenteric artery of SHR but not Wistar Kyoto rats (WKY). The density of NPY-LI-containing sympathetic nerve fibers was significantly greater in SHR than in WKY. SHR were treated for 7 weeks with angiotensin converting enzyme inhibitor (0.005% temocapril), angiotensin II type-1 (AT1) receptor antagonist (0.025% losartan) or vasodilator (0.01% hydralazine) in their drinking water. Each drug treatment significantly lowered the systolic blood pressure measured by tail-cuff method. Long-term treatment of SHR with temocapril and losartan significantly increased the density of CGRP-LI-containing nerve fibers in mesenteric arteries. However, the density after hydralazine treatment was similar to the level in non-treated SHR. The density of NPY-LI-containing nerve fibers was not increased by any of the drug treatments. These results suggest that long-term inhibition of the renin-angiotensin system in SHR prevents remodeling of CGRPergic nerve fibers and prevents the reduction of CGRPergic nerve function.