Consolidated bioprocessing of raw starch with Saccharomyces cerevisiae strains expressing fungal alpha-amylase and glucoamylase combinations.

Cost-effective consolidated bioprocessing (CBP) of raw starch for biofuel production requires recombinant Saccharomyces cerevisiae strains expressing α-amylases and glucoamylases. Native Aureobasidium pullulans apuA, Aspergillus terreus ateA, Cryptococcus sp. S-2 cryA and Saccharomycopsis fibuligera sfiA genes encoding raw-starch α-amylases were cloned and expressed in the S. cerevisiae Y294 laboratory strain. Recombinant S. cerevisiae Y294[ApuA] and Y294[AteA] strains produced the highest extracellular α-amylase activities (2.17 U mL-1 and 2.98 U mL-1, respectively). Both the ApuA and AteA α-amylases displayed a preference for pH 4 to 5 and retained more than 75% activity after 5 days at 30°C. When ateA was co-expressed with the previously reported Aspergillus. tubingensis glucoamylase gene (glaA), the amylolytic S. cerevisiae Y294[AteA-GlaA] strain produced 45.77 g L-1 ethanol after 6 days. Ethanol production by this strain was improved with the addition of either 2.83 µL STARGEN 002 (54.54 g L-1 ethanol and 70.44% carbon conversion) or 20 µL commercial glucoamylase from Sigma-Aldrich (73.80 g L-1 ethanol and 90.19% carbon conversion). This is the first report of an engineered yeast strain that can replace up to 90% of the enzymes required for raw starch hydrolysis, and thus contributes to the realisation of a CBP yeast for starch-based biofuel production.

Quantitative metabolomics of a xylose-utilizing Saccharomyces cerevisiae strain expressing the Bacteroides thetaiotaomicron xylose isomerase on glucose and xylose.

The yeast Saccharomyces cerevisiae cannot utilize xylose, but the introduction of a xylose isomerase that functions well in yeast will help overcome the limitations of the fungal oxido-reductive pathway. In this study, a diploid S. cerevisiae S288c[2n YMX12] strain was constructed expressing the Bacteroides thetaiotaomicron xylA (XI) and the Scheffersomyces stipitis xyl3 (XK) and the changes in the metabolite pools monitored over time. Cultivation on xylose generally resulted in gradual changes in metabolite pool size over time, whereas more dramatic fluctuations were observed with cultivation on glucose due to the diauxic growth pattern. The low G6P and F1,6P levels observed with cultivation on xylose resulted in the incomplete activation of the Crabtree effect, whereas the high PEP levels is indicative of carbon starvation. The high UDP-D-glucose levels with cultivation on xylose indicated that the carbon was channeled toward biomass production. The adenylate and guanylate energy charges were tightly regulated by the cultures, while the catabolic and anabolic reduction charges fluctuated between metabolic states. This study helped elucidate the metabolite distribution that takes place under Crabtree-positive and Crabtree-negative conditions when cultivating S. cerevisiae on glucose and xylose, respectively.

Expression and evaluation of enzymes required for the hydrolysis of galactomannan.

The cost-effective production of bioethanol from lignocellulose requires the complete conversion of plant biomass, which contains up to 30 % mannan. To ensure utilisation of galactomannan during consolidated bioprocessing, heterologous production of mannan-degrading enzymes in fungal hosts was explored. The Aspergillus aculeatus endo-ß-mannanase (Man1) and Talaromyces emersonii α-galactosidase (Agal) genes were expressed in Saccharomyces cerevisiae Y294, and the Aspergillus niger ß-mannosidase (cMndA) and synthetic Cellvibrio mixtus ß-mannosidase (Man5A) genes in A. niger. Maximum enzyme activity for Man1 (374 nkat ml(-1), pH 5.47), Agal (135 nkat ml(-1), pH 2.37), cMndA (12 nkat ml(-1), pH 3.40) and Man5A (8 nkat ml(-1), pH 3.40) was observed between 60 and 70 °C. Co-expression of the Man1 and Agal genes in S. cerevisiae Y294[Agal-Man1] reduced the extracellular activity relative to individual expression of the respective genes. However, the combined action of crude Man1, Agal and Man5A enzyme preparations significantly decreased the viscosity of galactomannan in locust bean gum, confirming hydrolysis thereof. Furthermore, when complemented with exogenous Man5A, S. cerevisiae Y294[Agal-Man1] produced 56 % of the theoretical ethanol yield, corresponding to a 66 % carbohydrate conversion, on 5 g l(-1) mannose and 10 g l(-1) locust bean gum.

Fungal ß-glucosidase expression in Saccharomyces cerevisiae.

Recombinant Saccharomyces cerevisiae strains expressing ß-glucosidases from Thermoascus aurantiacus (Tabgl1) and Phanerochaete chrysosporium (PcbglB and Pccbgl1) were constructed and compared to S. cerevisiae Y294[SFI], previously identified as the best ß-glucosidase-producing strain. The PcbglB was also intracellularly expressed in combination with the lac12 lactose permease of Kluyveromyces lactis in S. cerevisiae Y294[PcbglB + Lac12]. The recombinant extracellular ß-glucosidases indicated maximum activity in the pH range 4-5 and temperature optima varying from 50 to 75 °C. The S. cerevisiae Y294[Pccbgl1] strain performed best under aerobic and anaerobic conditions, producing 2.6 times more ß-glucosidase activity than S. cerevisiae Y294[SFI] and an ethanol concentration of 4.8 g l(-1) after 24 h of cultivation on cellobiose as sole carbohydrate source. S. cerevisiae Y294[Tabgl1] was unable to grow on cellobiose (liquid medium), whereas S. cerevisiae Y294[PcbglB + Lac12] exhibited limited growth.

Production and characterisation of recombinant α-L-arabinofuranosidase for production of xylan hydrogels.

A recombinant strain of the protease-deficient, non-acidifying pH mutant Aspergillus niger D15 (A. niger D15 [abfB]) strain was developed to secrete α-L-arabinofuranosidase (AbfB) free of endo-1,4-ß-xylanases for selective hydrolysis of xylan into hydrogels. The A. niger D15 [abfB] strain expressed the α-L-arabinofuranosidase abfB gene under the transcriptional control of the glyceraldehyde-3-phosphate dehydrogenase promoter (gpd(P)) and glucoamylase terminator (glaA(T)) in fermentation cultures containing 10 % glucose. The yield, activity, purity, kinetics and ability of the recombinant AbfB to selectively hydrolyse xylans into hydrogels were assessed. The recombinant AbfB secreted in 125-mL shake flasks and 10-L bioreactor fermentation cultures had specific activities against ρ-nitrophenyl-α-arabinofuranoside of up to 4.4 and 2.7 U g⁻¹ (dry weight), respectively. In addition, the recombinant AbfB was present as a single protein species on silver-stained 10 % sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The recombinant AbfB had optimal activity at 40-55 °C and pH 3.0 to pH 5.0 and was stable at temperature and pH of up to 60 °C and pH 6.0, respectively. About 20 % of the available arabinose in the xylan was released by the recombinant AbfB from the hydrolysis of low viscosity wheat and oat spelt arabinoxylans and about 9 and 5 % from bagasse and bamboo arabinoglucuronoxylans, respectively, that led to the formation of the hydrogels. Therefore, the constructed A. niger D15 [abfB] strain presented a microbial system for the production of recombinant AbfB with the required purity for the modification of xylans into hydrogels.

Production of the Aspergillus aculeatus endo-1,4-beta-mannanase in A. niger.

The beta-mannanase gene (man1) from Aspergillus aculeatus MRC11624 (Izuka) was patented for application in the coffee industry. For production of the enzyme, the gene was originally cloned and expressed in Saccharomyces cerevisiae. However the level of production was found to be economically unfeasible. Here we report a 13-fold increase in enzyme production through the successful expression of beta-mannanase of Aspergillus aculeatus MRC11624 in Aspergillus niger under control of the A. niger glyceraldehyde-3-phosphate dehydrogenase promoter (gpd (P)) and the A. awamori glucoamylase terminator (glaA(T)). The effect of medium composition on mannanase production was evaluated, and it was found that the glucose concentration and the organic nitrogen source had an effect on both the volumetric enzyme activity and the specific enzyme activity. The highest mannanase activity levels of 16,596 nkat ml(-1) and 574 nkat mg(-1) dcw were obtained for A. niger D15[man1] when cultivated in a process-viable medium containing corn steep liquor as the organic nitrogen source and high glucose concentrations.

Constitutive expression of the Trichoderma reesei beta-1,4-xylanase gene (xyn2) and the beta-1,4-endoglucanase gene (egl) in Aspergillus niger in molasses and defined glucose media.

The xylanase II (xyn2)- and endoglucanase I (egl)-encoding regions of Trichoderma reesei QM6a were successfully expressed in Aspergillus niger D15 under the transcriptional control of the glyceraldehyde-6-phosphate dehydrogenase (gpd) promoter from A. niger and the glaA terminator of Aspergillus awamori. A stable xyn2 transformant produced beta-xylanase activity of 8,000 nkat/ml and 5,000 nkat/ml in shake-flask cultures containing defined or 20% (v/v) molasses medium, respectively. The recombinant Xyn2 enzyme expressed highest activity at pH 5-6 and 50-60 degrees C and retained more than 75% of its activity after 3 h of incubation at 50 degrees C. A stable egl transformant produced endo-P-1,4-glucanase activity of 2,300 nkat/ml in shake-flask cultures containing defined media and about half the activity in 20% molasses medium. Maximum endoglucanase activity was obtained at pH 5 and 60 degrees C. Both Xyn2 and EgI retained >80% activity after incubation at 50 degrees C for 3 h. The heterologous Xyn2 and EgI represent a significant portion of the total extracellular proteins produced.

Recruitment of house staff into anesthesiology: factors responsible for house staff selecting anesthesiology as a career and individual training program.

STUDY OBJECTIVE: To identify factors responsible in the selection of anesthesiology as a career by Mayo Clinic house staff (i.e., residents and clinical fellows); to evaluate their level of satisfaction with their choice of career and training program, and their perceptions of the future for anesthesiology trainees. DESIGN: Cross-sectional analysis using a questionnaire survey of 67 house staff enrolled in the anesthesiology training program during the 1995-1996 academic year. SETTING: Mayo Clinic, Rochester, MN. MEASUREMENTS AND MAIN RESULTS: Forty-eight (72%) of those surveyed responded to the questionnaire. Data were analyzed using the Chi-square and Mann-Whitney rank sum tests. A p-value less than or equal to 0.05 was considered statistically significant. The most frequently cited reasons for selecting anesthesiology as a career included the following: it is a "hands-on" specialty, it involves clinical application of physiology and pharmacology, and it provides immediate gratification in one's work. The most frequently cited reasons for selecting our training program were the diversity of training experience, prestige associated with Mayo Clinic, and employment opportunities following training. Forty-four (92%) felt downsizing of anesthesiology training programs was a national trend, 26 (54%) anticipated difficulty obtaining a job following training, and 16 (33%) felt they had future job security. Overall, 47 (98%) were happy with their career choice, and 40 (83%) would choose anesthesiology as a career if they were now graduating from medical school. All 1996 graduates found suitable employment without difficulty. CONCLUSIONS: Our data indicate that selection of a career in anesthesiology and training program are strongly associated with concerns regarding educational experiences and postgraduate employment opportunities.

Recruitment of House Staff into Anesthesiology: Factors Responsible for House Staff Selecting Anesthesiology as a Career and Individual Training Program.

Due to a variety of factors, there was a recent redistribution of graduating American medical students toward primary care specialties and a decline in the number of applicants seeking training positions in anesthesiology. In the present study, factors responsible for Mayo house staff (i.e., residents and clinical fellows) selecting anesthesiology as a career, and Mayo Clinic as their training program, were identified. We also evaluated their level of satisfaction with their choice of career and training program, and their perceptions of the future for anesthesiology trainees. A cross sectional analysis was conducted using a questionnaire to survey 67 house staff enrolled in the anesthesiology training program at Mayo Clinic, Rochester, MN during the 1995-1996 academic year. All responses were anonymous. Data were analyzed using the chi-square and Mann-Whitney rank sum tests. P ? 0.05 was considered statistically significant. Forty-eight (72%) of those surveyed responded to the questionnaire. The most frequently cited reasons for selecting anesthesiology as a career included it: is a "hands-on" specialty, involves clinical application of physiology and pharmacology, and provides immediate gratification in one's work. The most frequently cited reasons for selecting our training program were the diversity of training experience, prestige associated with Mayo Clinic, and employment opportunities following training. Forty-four (92%) felt downsizing of anesthesiology training programs was a national trend, 26 (54%) anticipated difficulty obtaining a job following training, and 16 (33%) felt they had future job security. Overall, 47 (98%) were happy with their career choice, and 40 (83%) would choose anesthesiology as a career if they were now graduating from medical school. Our data indicate that selection of a career in anesthesiology and training program are strongly associated with concerns regarding educational experiences and postgraduate employment opportunities. More than half anticipated difficulty obtaining postgraduate employment; however, this skepticism was overshadowed by two findings: the trainees reported a very high level of satisfaction in their choice of career and training program, and the vast majority reported they would seek a career in anesthesiology if they were now selecting a medical specialty. Additionally, all 1996 graduates found suitable employment without difficulty.

Preoperative antiplatelet therapy does not increase the risk of spinal hematoma associated with regional anesthesia.

One thousand orthopedic procedures in 924 patients given spinal or epidural anesthesia were prospectively studied to determine the risk of hemorrhagic complications associated with regional anesthesia. A history of excessive bruising or bleeding was elicited in 115 (12%) patients. Preoperative antiplatelet medications were taken by 386 (39%) patients. Aspirin was the most frequently reported antiplatelet drug and was taken by 193 patients. Subcutaneous heparin was administered to 22 patients before surgery on the operative day. One patient of 774 tested had a preoperative platelet count less than 100,000/mm3. In addition, 26 of 171 preoperative prothrombin times and 10 of 115 preoperative activated partial thromboplastin times were longer than normal. Only 31 preoperative bleeding times were performed; five were prolonged. There were no documented spinal hematomas (major hemorrhagic complications). Blood was noted during needle or catheter placement (minor hemorrhagic complication) in 223 (22%) patients, including 73 patients with frank blood in the needle or catheter. Preoperative antiplatelet therapy did not increase the incidence of minor hemorrhagic complications. However, female gender, increased age, a history of excessive bruising/bleeding, surgery to the hip, continuous catheter anesthetic technique, large needle gauge, multiple needle passes, and moderate or difficult needle placement were all significant risk factors. The lack of correlation between antiplatelet medications and bloody needle or catheter placement (producing clinically insignificant collections of blood in the spinal canal or epidural space) is strong evidence that preoperative antiplatelet therapy is not a significant risk factor for the development of neurologic dysfunction from spinal hematoma in patients who undergo spinal or epidural anesthesia while receiving these medications.

Blood flow to contracting human muscles: influence of increased sympathetic activity.

The purpose of this study was to examine the effects of the increased sympathetic activity elicited by the upright posture on blood flow to exercising human forearm muscles. Six subjects performed light and heavy rhythmic forearm exercise. Trials were conducted with the subjects supine and standing. Forearm blood flow (FBF, plethysmography) and skin blood flow (laser Doppler) were measured during brief pauses in the contractions. Arterial blood pressure and heart rate were also measured. During the first 6 min of light exercise, blood flow was similar in the supine and standing positions (approximately 15 ml.min-1.100 ml-1); from minutes 7 to 20 FBF was approximately 3-7 ml.min-1.100 ml-1 less in the standing position (P less than 0.05). When 5 min of heavy exercise immediately followed the light exercise, FBF was approximately 30-35 ml.min-1.100 ml-1 in the supine position. These values were approximately 8-12 ml.min-1.100 ml-1 greater than those observed in the upright position (P less than 0.05). When light exercise did not precede 8 min of heavy exercise, the blood flow at the end of minute 1 was similar in the supine and standing positions but was approximately 6-9 ml.min-1.100 ml-1 lower in the standing position during minutes 2-8. Heart rate was always approximately 10-20 beats higher in the upright position (P less than 0.05). Forearm skin blood flow and mean arterial pressure were similar in the two positions, indicating that the changes in FBF resulted from differences in the caliber of the resistance vessels in the forearm muscles.(ABSTRACT TRUNCATED AT 250 WORDS)

Status of the match in anesthesiology: 1988.

The majority of anesthesia program directors agreed 2 years ago to use the National Resident Matching Program (NRMP) as the mechanism for appointing senior medical students to second postgraduate year positions in anesthesiology. Eighty-seven applicants for the Mayo Clinic program were surveyed to measure the level of cooperation with the NRMP by programs and applicants. Low percentages of applicants reported pressures to sign contracts (22.4%) or make verbal commitments before February (32.8%). The frequency of these incidents was extremely low when considered in light of the number of applicant interviews reported by the applicants who responded. Nonparticipation in the match is a more important problem than infractions of NRMP rules.

Doxacurium chloride for neuromuscular blockade before tracheal intubation and surgery during nitrous oxide-oxygen-narcotic-enflurane anesthesia.

The neuromuscular effects of doxacurium (BW A938U) were studied in 36 patients, divided into four groups of 9 patients each, given doxacurium either 50 micrograms/kg (2 x ED95) 5 or 4 minutes or 80 micrograms/kg (3 x ED95) 4 or 3 minutes before tracheal intubation. Adequate neuromuscular relaxation permitted successful intubation at 5 minutes for doxacurium 50 micrograms/kg and at 4 minutes for 80 micrograms/kg. Time to 90% blockade was 5.4 +/- 1.5 minutes for doxacurium 50 micrograms/kg and 3.5 +/- 1.2 minutes for 80 micrograms/kg. Time to 25% spontaneous recovery was 84.7 +/- 54.3 minutes for doxacurium 50 micrograms/kg and 164.4 +/- 85.2 minutes for 80 micrograms/kg. Either neostigmine 45 micrograms/kg, neostigmine 60 micrograms/kg, or edrophonium 1000 micrograms/kg was given for reversal when T1 had spontaneously recovered to 25% of baseline level, T1 being the first response to repetitive train-of-four (TOF) stimuli (2 Hz for 2 seconds at 10-second intervals) expressed as percent of baseline level. The T4:T1 ratio is the amplitude of the fourth twitch relative to the first twitch in a TOF stimulus expressed as a ratio. T1 rapidly achieved 90% of baseline in 5-10 minutes after reversal of neuromuscular blockade. In contrast, the T4:T1 ratio lagged, recovering to a mean of 0.6 at 20 minutes when T1 was over 90% of baseline. Recovery patterns were not statistically significantly different (unpaired t-test) among the three reversal regimens. Therefore, the reversal data were pooled. No clinically significant hemodynamic effects occurred in any group.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidemiologic features of humeral fractures.

Five hundred sixty-four Rochester, Minnesota, residents had a total of 586 humeral fractures during the period from 1965 to 1974. Of these, 47% involved the proximal humerus, and 20% the humeral shaft, and 33% the distal humerus. The incidence of humeral fractures associated with severe trauma was relatively higher among children and young adults, and distal humeral fractures predominated. Among the elderly, however, proximal humeral fractures associated with moderate trauma were most common and were responsible for the excess humeral fracture incidence rates among women and the dramatic increase in rates with age for both sexes. Rochester incidence rates for all three humeral fracture sites were substantially greater than those previously reported from the United Kingdom, although the proximal humeral fracture incidence was very similar to that in Malmo, Sweden. The Rochester rates appear to be the best available for use in determining the costs and benefits of efforts to prevent these and other aging or osteoporosis-related fractures in the United States.