Toward the construction of a technology platform for chemicals production from methanol: D-lactic acid production from methanol by an engineered yeast Pichia pastoris.

With the reduction in oil reserves and steady increases in the price of oil, alternative carbon sources like methanol are promising, but an efficient conversion process to fuels and other chemicals is still desired. In this study, we demonstrated for the first time the production of lactic acid from methanol using a lactate dehydrogenase copy number amplifying strategy in Pichia pastoris. We engineered methylotrophic yeast (Pichia pastoris) producing D-lactic acid by D-lactate dehydrogenase gene (d-LDH) integration into the non-transcribed spacer of the ribosomal DNA (rDNA) locus and post-transformational amplification. The resultant engineered strains GS115/S8/Z3 and GS115/S16/Z3 produced 3.48 and 3.26 g/L of D-lactic acid from methanol, respectively, in a 96-h test tube fermentation. To our knowledge, this is the first report about D-lactic acid production from methanol by an engineered P. pastoris strain. The technique of gene integration into the rDNA locus and post-transformational gene amplification could be useful for metabolic engineering in P. pastoris, and the chemical production from methanol by engineered P. pastoris represents a promising industrial technology.

A new human cadaver model for laparoscopic training using N-vinyl-2-pyrrolidone: a feasibility study.

The demand for cadavers for clinical skills training is increasing. However, conventional formalin-fixed bodies are often unsuitable for surgical training because the tissues become too hard. We recently developed a new formalin-free embalming method with N-vinyl-2-pyrrolidone (pyrrolidone) that has excellent fixative, disinfectant, and preservative properties, while still keeping tissues soft and pliable. In the study reported here we investigated the feasibility of laparoscopic manipulation using pyrrolidone-fixed cadavers. Donated cadavers were embalmed either with pyrrolidone (n = 7) or with formalin-containing fixative (n = 3). A laparoscopic 12-mm trocar was inserted into the umbilical region, and CO2 gas was insufflated. Intra-abdominal structures were observed with an endoscopic camera. In the pyrrolidone-embalmed cadavers, the abdomen remained soft and depressed. In addition, CO2 injection resulted in a marked expansion of the abdominal cavity, and it was possible to move the laparoscope freely in all directions. Clear endoscopic images of the abdominal viscera were obtained. The gallbladder and rectum were identified by grasping the surrounding organs with forceps. By contrast, in the formalin-fixed bodies, the abdominal wall was rigid, and it was difficult to move the laparoscope in the peritoneal space and observe structures in detail. The amount of CO2 and changes in abdominal diameter and circumference in response to CO2 injection were significantly larger in the pyrrolidone group. In conclusion, we successfully created a sufficient pneumoperitoneum state and obtained clear endoscopic images in the pyrrolidone-embalmed cadavers. Handling and dissection of the intra-abdominal structures with forceps closely replicated real-life surgery. These findings suggest the feasibility of laparoscopic training on cadavers embalmed with this pyrrolidone fixative.

Cough peak flow with different mechanically assisted coughing approaches under different conditions in patients with neuromuscular disorders.

PURPOSE: Mechanically assisted coughing (MAC) is an airway clearance method in which the thorax/abdomen is compressed in synchronization with mechanical insufflation-exsufflation (MI-E). MAC can be performed with manual assistance at the upper thorax (MAC-UT), lower thorax (MAC-LT), and upper thorax + abdomen (MAC-UT/A). This study aimed to determine the most effective approach under different conditions (air stacking or tracheostomy) in patients with neuromuscular disorders (NMDs). METHODS: The study included 34 patients with NMDs. The patients were categorized into air stacking group (n=15), no air stacking group (n=9), and tracheostomy/tracheostomy positive-pressure ventilation (TPPV) group (n=10). RESULTS: In each group, the cough peak flow (CPF) at 75% of the forced vital capacity (V̇75), V̇50, V̇25, and V̇10 were investigated during the approaches. In the air stacking group, the CPF was higher with MAC-UT, MAC-LT, and MAC-UT/A than with MI-E (p < 0.05). Additionally, V̇75 was higher with MAC-LT and MAC-UT/A than with MI-E (p < 0.05 and p < 0.01, respectively). In the no air stacking group, V̇75 was higher with MAC-UT/A than with MI-E (p < 0.05). In the tracheotomy/TPPV group, there were no significant differences. CONCLUSIONS: MAC approaches, especially MAC-LT and MAC-UT/A, are preferred in air stacking patients. However, in tracheostomy/TPPV patients, the CPF might not increase with MAC.

Approaches to Cough Peak Flow Measurement With Duchenne Muscular Dystrophy.

BACKGROUND: Manually-assisted coughing and mechanical insufflation-exsufflation (MI-E) are commonly used in patients with Duchenne muscular dystrophy (DMD). Few studies have compared cough peak flow (CPF) with manually-assisted coughing to other methods, such as MI-E + manually-assisted coughing. In addition, few studies have reported the reliability of the measured CPF values. This study aimed to compare CPF with different cough-assistance methods and to examine the reliability of CPF data. METHODS: The study included 12 subjects with DMD (mean age, 34 ± 8 y). CPF, CPF + manually-assisted coughing (assisted CPF), maximum insufflation capacity (MIC) + CPF (MIC-CPF), MIC + manually-assisted coughing (MIC+assisted CPF), MI-E (MI-E-CPF), and MI-E + assisted CPF were measured. A spirometer was used to compare CPF measurements obtained with each of the cough-assist techniques. The reliability of the measured CPF values was analyzed using Bland-Altman analysis. RESULTS: CPF was 59 ± 34 L/min, assisted CPF was 113 ± 32 L/min, MIC-CPF was 170 ± 30 L/min, MIC+assisted CPF was 224 ± 62 L/min, MI-E-CPF was 199 ± 40 L/min, and MI-E + assisted CPF was 240 ± 38 L/min. A fixed and proportional bias was found in the CPF measurements made with the peak flow meter and the spirometer. The average 95% CI in the difference between peak flow meter, MI-E, and CPF obtained using the spirometer were -7.45 to -1.95 and -1.45 to 4.95, respectively. Test for correlation was r = 0.54 (P < .001) for CPF (peak flow meter) and CPF (spirometer) and r = 0.17 (P = .17) in CPF (MI-E) and CPF (spirometer), respectively. CONCLUSION: MI-E + assisted CPF was the highest. The CPF measured with the peak flow meter suggested underestimation.

An efficient synthesis of 7α,12α-dihydroxy-4-cholesten-3-one and its biological precursor 7α-hydroxy-4-cholesten-3-one: Key intermediates in bile acid biosynthesis.

This paper describes a method for the chemical synthesis of 7α,12α-dihydroxy-4-cholesten-3-one (1a) and its biological precursor, 7α-hydroxy-4-cholesten-3-one (1b), both of which are key intermediates in the major pathway of bile acid biosynthesis from cholesterol. The principal reactions involved were (1) building of the cholesterol (iso-octane) side chain by 3-carbon elongation of the cholane (iso-pentane) one, (2) oxidation sequence to transform the 3α-hydroxy group of the steroidal A/B-ring to the desired 4-en-3-one system, and (3) appropriate protection strategy for hydroxy groups in the positions at C-7 and C-12 in the steroid nucleus. The absolute structure of 1a and 1b were confirmed by NMR and X-ray crystallography. The targeted compounds 1a and 1b, prepared in 11 steps from 2a and 2b respectively, should be useful for biochemical studies of bile acid biosynthesis or clinical studies of bile acid metabolism, as plasma levels of 1b (also termed C4) have been shown to correlate highly with the rate of bile acid biosynthesis in man.