Effect of butorphanol,midazolam or ketamine on romifidine based sedation in horses during standing cheek tooth removal

Background

Standing surgery, especially dental procedures, are commonly performed in horses. This leads to an increasing demand for reliable sedation protocols. Therefore, it was the purpose of this study to investigate the influence of butorphanol, midazolam or ketamine on romifidine based sedation in horses during cheek tooth removal.

Methods

Forty horses presented for tooth extraction were divided in four groups using matched pair randomization. Group R was sedated with romifidine (bolus 0.03 mg/kg, followed by a constant rate infusion (CRI) 0.05 mg/kg/h) and group RB with romifidine (same dose) and butorphanol (0.02 mg/kg; CRI 0.04 mg/kg/h). Group RM received romifidine (same dose) and midazolam (0.02 mg/kg; CRI 0.06 mg/kg/h) whereas group RK was administered romifidine (same dose) and ketamine (0.5 mg/kg; CRI 1.2 mg/kg/h). If sedation was not adequate a top up bolus of romifidine (0.01 mg/kg) was administered. The quality of sedation and the conditions for tooth extraction, the level of ataxia, chewing, head and tongue movement were evaluated by using a scoring system. The investigator was blinded to the applied sedation protocol. Furthermore, serum cortisol concentrations before, during and after the procedure were analyzed to gain more information about the stress level of the horses.

Results

Horses in group RM showed significantly less chewing and tongue activity compared to horses sedated with romifidine alone or with butorphanol additionally, but also significantly higher levels of ataxia. The quality of sedation was significantly better if romifidine was administered in combination with ketamine compared to romifidine alone. Furthermore, horses of group RK needed less additional romifidine boli compared to all other groups. Blood cortisol concentrations during surgery in groups RB and RM remained unchanged. Horses of group R showed higher cortisol concentrations during sedation compared to horses of groups RB and RM.

Conclusion

Romifidine alone at an initial bolus dose of 0.03 mg/kg followed by a constant rate infusion of 0.05 mg/kg/h was insufficient to obtain an adequate level of sedation and led to increased stress levels, whereas the addition of butorphanol inhibited the stress response. The combination of romifidine with either midazolam or ketamine improved sedation quality and surgical conditions.

     

Effect of food intake on myocardial performance index

Background

Myocardial performance index (MPI) has been investigated in a variety of populations, but the effect of food intake has not been evaluated. We assessed whether myocardial performance index is affected by food intake in healthy subjects.

Methods

Twenty-three healthy subjects aged 25.6?±?4.5 years were investigated. MPI was measured before, 30 min after, and 110 min after a standardized meal.

Results

MPI decreased significantly (P?<?0.05) from fasting values 30 min after the meal, and had almost returned to baseline after 110 min. MPI decreased from 0.28?±?0.06 (fasting) to 0.20?±?0.07 30 min after eating. At 110 min after eating the index value was almost back to the baseline value 0.26?±?0.06. (P?=?0.15).

Conclusions

This study shows that myocardial performance index is affected by food intake in healthy subjects.

     

Development of a recombinant immunotoxin for the immunotherapy of autoreactive lymphocytes expressing MOG-specific BCRs

Objective

Myelin oligodendrocyte glycoprotein (MOG) is one of the major autoantigens in multiple sclerosis (MS), therefore selective depletion of autoreactive lymphocytes exposing MOG-specific B cell receptors (BCRs) would be beneficial in terms of MS treatment.

Results

Using E. coli we generated an efficient protocol for the purification of the recombinant immunotoxin DT-MOG composed of the extracellular Ig-like domain of MOG fused in frame with the catalytic and translocation subunits of diphtheria toxin (DT, Corynebacterium diphtheriae) under native conditions with a final yield of 1.5 mg per liter of culture medium. Recombinant DT-MOG was recognized in vitro by MOG-reactive antibodies and has catalytic activity comparable with wild-type DT.

Conclusion

Enhanced pharmacokinetics (mean residence time in the bloodstream of 61 min) and minimized diminished nonspecific toxicity (LD₅₀ = 1.76 mg/kg) of the DT-MOG makes it a potential candidate for the immunotherapy of MS.

     

Does centrifugation matter? Centrifugal force and spinning time alter the plasma metabolome

Background

Centrifugation is an indispensable procedure for plasma sample preparation, but applied conditions can vary between labs.

Aim

Determine whether routinely used plasma centrifugation protocols (1500×g 10 min; 3000×g 5 min) influence non-targeted metabolomic analyses.

Methods

Nuclear magnetic resonance spectroscopy (NMR) and High Resolution Mass Spectrometry (HRMS) data were evaluated with sparse partial least squares discriminant analyses and compared with cell count measurements.

Results

Besides significant differences in platelet count, we identified substantial alterations in NMR and HRMS data related to the different centrifugation protocols.

Conclusion

Already minor differences in plasma centrifugation can significantly influence metabolomic patterns and potentially bias metabolomics studies.

     

Cetirizine per os: exposure and antihistamine effect in the dog

Background

Cetirizine is an antihistamine used in dogs, but plasma concentrations in relation to effect after oral administration are not well studied. This study investigated cetirizine exposure and the plasma cetirizine concentration-antihistamine response relation in the dog following oral administration of cetirizine.

Results

Eight Beagle dogs were included in a cross-over study consisting of two treatments. In treatment one, cetirizine 2–4 mg/kg was administered per os once daily for 3 days. The other treatment served as a control. Wheal diameter induced by intra-dermal histamine injections served as response-biomarker. Cetirizine plasma concentration was quantified by UHPLC-MS/MS. Median (range) cetirizine plasma terminal half-life was 10 h (7.9–16.5). Cetirizine significantly inhibited wheal formation compared with the premedication baseline. Maximum inhibition of wheal formation after treatment with cetirizine per os was 100% compared with premedication wheal diameter. The median (range) IC₅₀-value for reduction in wheal area was 0.33 µg/mL (0.07–0.45). The median (range) value for the sigmoidicity factor was 1.8 (0.8–3.5). A behavioral study was also conducted and revealed no adverse effects, such as sedation.

Conclusion

The results indicate that a once-daily dosing regimen of 2–4 mg/kg cetirizine per os clearly provides a sufficient antihistamine effect. Based on this experimental protocol, cetirizine may be an option to treat histamine-mediated inflammation in the dog based on this experimental protocol but additional clinical studies are required.

     

Association between chronological depressive changes and physical symptoms in postoperative pancreatic cancer patients

Background

Pancreatic cancer (PC) has poorer prognosis and higher surgical invasiveness than many other cancers, with associated psychiatric symptoms including depression and anxiety. Perioperative depression has not been investigated in PC patients regarding surgical stress and relevant interventions.

Methods

We evaluated chronological depressive changes and subjective physical symptoms in surgically treated PC patients preoperatively and at 3 and 6 months postoperatively.Enrolled patients undergoing pancreatic tumor surgery completed questionnaires based on the Self-Rating Depression Scale (SDS) and Functional Assessment of Cancer Therapy for Patients with Hepatobiliary Cancer (FACT-Hep) preoperatively, and at 3 and 6 months postoperatively. Responses were analyzed with JMP® Pro using one-way and two-way ANOVA, Spearman’s rank correlation coefficient, and multiple regression analysis.

Results

Malignancy was diagnosed in 73 of 101 patients postoperatively; SDS score was significantly higher in these patients than in those with benign tumors at all timepoints: malignant/benign, 41.8/37.9 preoperatively (p?=?0.004); 43.5/37.8 3 months postoperatively (p?=?0.006); and 42.9/37.7 6 months postoperatively (p?=?0.020). SDS scores were significantly higher in patients <?65 years old with malignancy at 3 months than at 6 months postoperatively (44.6/42.5, p?=?0.046) and in patients with malignancy who underwent pancreaticoduodenectomy at 3 months postoperatively than preoperatively (43.4/41.1; p?=?0.028). SDS scores moderately correlated with 8 physical symptom-related FACT-Hep items 3 months postoperatively (p?<?0.05), showing low-to-moderate correlation with 16 physical symptom-related FACT-Hep items at 6 months postoperatively (p?<?0.05). Multiple regression analysis of FACT-Hep symptoms significantly correlated with SDS scores revealed the following significant variables: “lack of energy” (p?<?0.000) and “pain” (p?=?0.018) preoperatively (R²?=?0.43); “able to perform usual activities” (p?=?0.031) and “lack of energy” (p?<?0.000) at 3 months postoperatively (R²?=?0.51); and “stomach swelling or cramps” (p?=?0.034) and “bowel control” (p?=?0.049) at 6 months postoperatively (R²?=?0.52).

Conclusions

PC patients experience persistently high levels of depression preoperatively through 6 months postoperatively, with associated subjective symptoms including pain and gastrointestinal symptoms.

Trial registration

UMIN Clinical Trials Registry 000009592, Registered 20 December 2012.

     

Improved production of carotenoid-free welan gum in a genetic-engineered <Emphasis Type="Italic">Alcaligenes</Emphasis> sp. ATCC31555

Objective

To improve the production of welan gum and obtain a carotenoid-free strain while reducing the fermentation and post-treatment costs.

Results

The vitreoscilla globin (vgb) gene combined with the β-galactosidase (lacZ) promoter was inserted into the phytoene synthase (crtB) gene region of the chromosome in Alcaligenes sp. ATCC31555. When the recombinant strain was grown in a 5 l fermentor, welan gum was produced at 24 ± 0.4 g l^?1 compared to 21 g ± 0.4 g l^?1 in the wild type. Furthermore, the carotenoid-free welan gum produced using Alcaligenes sp. ATCC31555 VHb strain was less expensive with improved properties.

Conclusions

Alcaligenes sp. ATCC31555 VHb strain was a better neutral welan-producing strain with a higher production than the wild-type strain.

     

Corneal Collagen Cross-Linking for the Management of Mycotic Keratitis

Purpose

To evaluate the efficiency of corneal collagen cross-linking (CXL) in addition to topical voriconazole in cases with mycotic keratitis.

Design

Retrospective case series in a tertiary university hospital.

Participants

CXL was performed on 13 patients with mycotic keratitis who presented poor or no response to topical voriconazole treatment.

Methods

The clinical features, symptoms, treatment results and complications were recorded retrospectively. The corneal infection was graded according to the depth of infection into the stroma (from grade 1 to grade 3). The visual analogue scale was used to calculate the pain score before and 2 days after surgery.

Main Outcome Measures

Grade of the corneal infection.

Results

Mean age of 13 patients (6 female and 7 male) was 42.4 ± 17.7 years (20–74 years). Fungus was demonstrated in culture (eight patients) or cytological examination (five patients). Seven of the 13 patients (54%) were healed with topical voriconazole and CXL adjuvant treatment in 26 ± 10 days (15–40 days). The remaining six patients did not respond to CXL treatment; they initially presented with higher grade ulcers. Pre- and post-operative pain score values were 8 ± 0.8 and 3.5 ± 1, respectively (p < 0.05).

Conclusions

The current study suggests that adjunctive CXL treatment is effective in patients with small and superficial mycotic ulcers. These observations require further research by large randomized clinical trials.

     

Long-duration general anesthesia influences the intelligence of school age children

Background

General anesthesia has been linked to impaired brain development in immature animals and young children. In this study the influence of orthopedic surgery under general anesthesia on the intelligence of school age children has been evaluated.

Methods

A total of 209 subjects aged 6–12 years were recruited and allocated into 4 groups according to the duration of general anesthesia, including a control group (n?=?30), short (< 1 h, n?=?49), moderate- (1–3 h, n?=?51) and long-duration groups (> 3 h, n?=?79), respectively. The intelligence quotient (IQ) of the subjects was measured by the Raven’s Standard Progressive Matrices (RSPM) before and after orthopedic surgery under general anesthesia of various durations (vide supra).

Results

The IQ score decreased significantly in the long-duration group at 1 month post-operation compared with the pre-operation score (P?<?0.001), and IQ did not recover completely at 3 months postoperatively (P?<?0.05), but had recovered when measured at the 1-year follow-up. Moreover, this study showed that the development of children’s intelligence was affected by the exposure time to anesthetics at a younger age (OR?=?5.26, 95% CI:2.70–8.41, P?<?0.001), having a mother with a low education level (OR?=?2.71, 95% CI:1.24–6.14, P?=?0.014) and premature birth (OR?=?2.76, 95% CI:1.34–5.46, P?=?0.005).

Conclusions

More than 3 h general anesthesia influenced the IQ of school age children for up to 3 months after orthopedic surgery. Beside extended exposure time to anesthetics additional factors for post-operative IQ reduction were younger children age, mothers with low educational levels and premature birth.

Trial registration

Chinese Clinical Trial Registry with registration number ChiCTR-OOC-17013497 retrospectively registered on 11/23/2017.

     

Potential benefit of bosentan therapy in borderline or less severe pulmonary hypertension secondary to idiopathic pulmonary fibrosis—an interim analysis of results from a prospective,single-center,randomized, parallel-group study

Background

No drugs have been approved for the treatment of patients with pulmonary hypertension (PH) secondary to idiopathic pulmonary fibrosis (IPF), particularly those with idiopathic honeycomb lung. This study was conducted to investigate the long-term efficacy and safety of bosentan for PH based on changes in prognosis and respiratory failure.

Methods

IPF patients with borderline or less severe PH and completely organized honeycomb lung were randomized (1:1) to bosentan or no treatment for PH for 2 years and assessed at baseline and every 6 months for respiratory failure, activities of daily living (ADL), lung and heart functions by right cardiac catheterization, and other parameters. An interim analysis was performed, however, following detection of a significant survival benefit favoring bosentan therapy.

Results

Significant differences were noted for the bosentan-treated (n?=?12) vs. untreated (n?=?12) groups in hospital-free survival (603.44?±?50.074 days vs. 358.87?±?68.65 days; hazard ratio [HR], 0.19; P?=?0.017) and overall survival (671 days vs. 433.78?±?66.98 days; HR, 0.10; P?=?0.0082). Again, significant improvements were noted for the bosentan-treated group from baseline to month 6 or 12 in several indices in ADL, pulmonary circulation, and %DLCO. Without requiring O₂ inhalation, bosentan was associated with no increase but a trend toward a decrease in adverse events and an improvement in respiratory status.

Conclusions

Bosentan tended to improve prognosis and ADL without worsening respiratory failure in IPF patients with borderline or less severe PH and completely organized honeycomb lung alone.

Trial registration

This study was registered on December 18, 2010 with UMIN-CTR Clinical Trial as UMIN000004749 to investigate the long-term influence of bosentan on cardiac function, as well as its cardioprotective efficacy and safety, in patients with pulmonary hypertension secondary to concurrent COPD and IPF, respectively.

     

Long-term follow-up in adults after tetralogy of Fallot repair

Background

Tetralogy of Fallot (ToF) is the most common cyanotic congenital heart disease and the population of ToF repair survivors is growing rapidly. Adults with repaired ToF develop late complications. The aim of this study was to describe and analyze long-term follow-up of patients with repaired ToF.

Methods

This is a retrospective cohort study. Consecutive 83 patients with repaired ToF who did not undergo pulmonary valve replacement were included. Mean age of all patients was 30.5?±?10.7. There were 49 (59%) male. Patients were divided into two groups according to the time since the repair (<?25 years and?≥?25 years). The electrocardiographic (ECG), cardiopulmonary exercise testing (CPET), echocardiographic and cardiac magnetic resonance (CMR) data were reviewed retrospectively.

Results

In CPET values were not significantly different in the two groups. In CMR volumes of left and right ventricles were not significantly different in the two groups. There were no differences between the groups in ventricular ejection fraction, mass of ventricles, or pulmonary regurgitation fraction. Among all the patients, ejection fraction and left and right ventricle mass, indexed pulmonary regurgitation volume measured by CMR did not correlate with the time since repair. In ECG among all the patients, ejection fraction of the RV, measured in CMR, negatively correlated with QRS duration (r?=???0.43; p?<?0.001). There was a positive correlation between QRS duration and end diastolic volume of the RV (r?=?0.30; p?<?0.02), indexed end diastolic volume of the RV (r?=?0.29; p?=?0.04), RV mass (r?=?0.36; p?<?0.001) and left ventricle mass (r?=?0.26; p?=?0.04).

Conclusion

Long-term survival and clinical condition after surgical correction of ToF in infancy is generally good and the late functional status in ToF – operated patients could be excellent up to 25 years after the repair. QRS duration could be an utility and easy factor to assessment of right ventricular function.

Trial registration

The study protocol was approved by the local Ethics Committee. Each participant provided informed consent to participate in the study (license number 122.6120.88.2016 from 28.04.2016).

     

The usefulness of the McGrath MAC laryngoscope in comparison with Airwayscope and Macintosh laryngoscope during routine nasotracheal intubation: a randomaized controlled trial

Background

McGrath MAC video laryngoscope offers excellent laryngosopic views and increases the success rate of orotracheal intubation in some cases. The aim of this study was to determine the usefulness of McGrath MAC for routine nasotracheal intubation by comparing McGrath MAC with Airway scope and Macintosh laryngoscope.

Methods

A total of 60 adult patients with ASA physical status class 1 or 2, aged 20–70 years were enrolled in this study. Patients were scheduled for elective oral surgery under general anesthesia with nasotracheal intubation. Exclusion criteria included lack of consent and expected difficult airway. Patients were randomly allocated to three groups: McGrath MAC (n?=?20), Airway scope (n?=?20), and Macintosh laryngoscope (n?=?20). After induction, nasotracheal intubation was performed by six expert anesthesiologists with more than 6 years of experience.

Results

There were no significant differences in preoperative airway assessment among the three groups. Successful tracheal intubation time was 26.8?±?5.7 (mean?±?standard deviation) s for McGrath MAC, 36.4?±?11.0 s for Airway scope, and 36.5?±?8.9 s for Macintosh laryngoscope groups. The time for successful tracheal intubation for McGrath MAC group was significantly shorter than that for Airway scope and Macintosh laryngoscope (p?<?0.01). McGrath MAC significantly improved the Cormack Lehane grade for nasotracheal intubation compared with Macintosh laryngoscope (p?<?0.05).

Conclusion

McGrath MAC significantly facilitates routine nasotracheal intubation compared with Airwayscope and Macintosh laryngoscope by shortening the tracheal intubation time and improving the Cormack Lehane grade.

Trial registration

UMINCTR Registration number UMIN000023506. Registered 5 Aug 2016.

     

Apolipoprotein E polymorphism and the risk of aneurysmal subarachnoid hemorrhage in a South Indian population

Background

The rupture of a brain aneurysm causes bleeding in the subarachnoid space. This is known as aneurysmal subarachnoid hemorrhage (aSAH). We evaluated the association of apolipoprotein E (APOE) polymorphism and the risk of aSAH in a South Indian population.

Methods

The study was performed on 200 subjects with aSAH and 253 healthy control subjects. Blood samples (5 ml) were used to isolate DNA and genotyping was performed for rs7412 and rs429358 using a Taqman allelic discrimination assay. Statistical software R.3.0.11 was used to statistically analyze the data and a p value <?0.05 was considered as statistically significant.

Results

We found a significant association with the risk of aSAH in ε3/ ε4 genetic model (OR?=?1.91, 95% CI?=?1.16–3.14, p?=?0.01). However, in the other genetic models and allele frequency, there was no significant association with the risk of aSAH. In subtyping, we found a significant association of ε2 allele frequency with posterior communicating artery (PCOM) aneurysm (OR?=?3.59, 95% CI?=?1.11–11.64, p?=?0.03).

Conclusion

Our results suggest that APOE polymorphism has an influence on the risk of aSAH in this South Indian population, specifically in the PCOM subtype.

     

Catesbeianin-1, a novel antimicrobial peptide isolated from the skin of <Emphasis Type="Italic">Lithobates catesbeianus</Emphasis> (American bullfrog)

Objectives

To identify and characterize a novel antimicrobial peptide, catesbeianin-1.

Results

Catesbeianin-1 is 25 amino acids long and is α-helical, cationic and amphipathic. It had antimicrobial activity against Gram-positive and Gram-negative bacteria. It was resistant against trypsin and pepsin. Catesbeianin-1 exhibited moderate hemolytic activity (approx 8%) at 100 μg/ml, and its HC₅₀ (50% hemolytic concentration) was 300 μg/ml. Its cytotoxicity was approx 10–20% at 100 μg/ml, and its CC₅₀ (50% cytotoxic concentration) was >100 μg/ml. The LD₅₀ of catesbeianin-1 in mice was 80 mg/kg. At 3.1 µg/ml, catesbeianin-1 significantly inhibited the growth of methicillin-resistant Staphylococcus aureus.

Conclusions

A new antimicrobial peptide from the skin of Lithobates catesbeianus (American bullfrog) may represent a template for the development of novel antimicrobial agents.

     

Effect of pre-warming on perioperative hypothermia during holmium laser enucleation of the prostate under spinal anesthesia: a prospective randomized controlled trial

Background

The purpose of this study is to assess whether the application of preoperative forced air warming set to high temperature (>?43?°C) for brief period can increase temperature on admission to the postanesthesia care unit (PACU) and prevent hypothermia or shivering during holmium laser enucleation of the prostate performed under spinal anesthesia.

Methods

Fifty patients were enrolled were assigned randomly to receive passive insulation (control group, n?=?25) or forced-air skin surface warming for 20?min before spinal anesthesia (pre-warming group, n?=?25). The primary outcome was temperature at PACU admission.

Results

The pre-warming group had a significantly higher temperature on admission to the PACU than the control group (35.9?°C [0.1] vs 35.6?°C [0.1], P?=?0.023; 95% confidence interval of mean difference, 0.1?°C–0.5?°C). The trend of decreasing core temperature intraoperatively was not different between groups (P?=?0.237), but intraoperative core temperature remained approximately 0.2?°C higher in the pre-warming group (P?=?0.005). The incidence of hypothermia on admission to the PACU was significantly lower in the pre-warming group (56% vs 88%, P?=?0.025). Shivering occurred in 14 patients in the control group, and 4 patients in the pre-warming group (P?=?0.007).

Conclusion

Brief pre-warming at 45?°C increased perioperative temperature and decreased the incidence of hypothermia and shivering. However, it was not sufficient to modify the decline of intraoperative core temperature or completely prevent hypothermia and shivering. Continuing pre-warming to immediately before induction of spinal anesthesia or combining pre-warming with intraoperative active warming may be necessary to produce clearer thermal benefits in this surgical population.

Trial registration

This trial was registered with Clinicaltrials.gov, NCT03184506, 5th June 2017.

     

Frailty has a stronger association with inflammation than age in older veterans

Background

Upregulation of pro-inflammatory cytokines has not only been associated with increased morbidity and mortality in older adults but also has been linked to frailty. In the current study we aimed to compare the relative relationship of age and frailty on inflammation and thrombosis in older veterans.

Results

We analyzed 117 subjects (age range 62–95 years; median 81) divided into 3 cohorts: non-frail, pre-frail and frail based on the Fried phenotype of frailty. Serum inflammatory markers were determined using commercially available ELISA kits. Frail and pre-frail (PF) subjects had higher levels than non-frail (NF) subjects of IL-6 (NF vs. PF: p?=?0.002; NF vs. F: p?<?0.001), TNFR1 (NF vs. F: p?=?0.012), TNFRII (NF vs. F: 0.002; NF vs. PF: p?=?0.005) and inflammatory index: = 0.333*log(IL-6)?+?0.666*log(sTNFR1) (NF vs. F: p?=?0.009; NF vs. PF: p?<?0.001). Frailty status explained a greater percent of variability in markers of inflammation than age: IL-6 (12 % vs. 0.3 %), TNFR1 (5 % vs. 4 %), TNFR2 (11 % vs. 6 %), inflammatory index (16 % vs. 8 %). Aging was significantly associated with higher fibrinogen (p?=?0.04) and D-dimer levels (p?=?0.01) but only among NF subjects.

Conclusion

In conclusion, these data suggest that among older veterans, frailty status has a stronger association with inflammation and the inflammatory index than age does. Larger studies, in more diverse populations are needed to confirm these findings.

     

Biodegradation of saline phenolic wastewater in a biological contact oxidation reactor with immobilized cells of <Emphasis Type="Italic">Oceanimonas</Emphasis> sp.

Objective

To develop a method to treat saline phenolic wastewater in a biological contact oxidation reactor (BCOR) with immobilized cells of a marine microorganism, Oceanimonas sp., isolated from seawater.

Results

Cells were immobilized on fibre carriers in the BCOR. Saline wastewater with phenol at 1.5 g/l and NaCl at 6 % (w/v) was treated. In continuous assays, 99 % removal of phenol was achieved and a kinetic model for the phenol degradation is presented based on Monod’s equation.

Conclusion

The BOCR system using immobilized cells of Oceanimonas efficiently treats saline phenolic wastewaters without having decrease the salinity of the wastewater.

     

Reengineering substrate specificity of <Emphasis Type="Italic">E. coli</Emphasis> glutamate dehydrogenase using a position-based prediction method

Objective

To re-engineer the active site of proteins for non-natural substrates using a position-based prediction method (PBPM).

Results

The approach has been applied to re-engineer the E. coli glutamate dehydrogenase to alter its substrate from glutamate to homoserine for a de novo 1,3-propanediol biosynthetic pathway. After identification of key residues that determine the substrate specificity, residue K92 was selected as a candidate site for mutation. Among the three mutations (K92V, K92C, and K92M) suggested by PBPM, the specific activity of the best mutant (K92 V) was increased from 171 ± 35 to 1328 ± 71 μU mg^?1.

Conclusion

The PBPM approach has a high efficiency for re-engineering the substrate specificity of natural enzymes for new substrates.