Contribution of Salvaged Blood Red Blood Cell Transfusion During Living Donor Liver Transplantation.

BACKGROUND: Transfusion of allogeneic blood products can have adverse effects and profoundly influence postoperative liver transplantation outcomes, including graft survival. To minimize allogeneic red blood cell (RBC) transfusion, salvaged blood can be used during liver transplantation. The aim of this study was to determine the contribution of salvaged blood to allogeneic RBC transfusion in living donor liver transplantation (LDLT) recipients. METHODS: Data of 311 adult-to-adult LDLT recipients between January 2015 and April 2019 were analyzed. The primary outcome was a change in requirement for allogeneic RBCs due to the use of salvaged blood. Additionally, predictors of intraoperative allogeneic RBC transfusion were investigated. RESULTS: One hundred fifty-three (49.2%) recipients required allogeneic RBC transfusion. If recipients did not receive salvaged blood, 253 (81.4%) recipients would have needed allogeneic RBC transfusion. The total volume of salvaged blood transfused into recipients during surgery was 269,165 mL, which corresponded to 993 units of allogeneic RBCs and accounted for 76.1% of total RBC transfusion volume. Multivariate analysis showed that male recipients, model for end-stage liver disease score, preoperative hemoglobin level, and volume of salvaged blood used during surgery were independent predictors of the need for intraoperative allogenic RBC transfusion. CONCLUSIONS: Intraoperative use of salvaged blood significantly reduced allogeneic RBC transfusion in LDLT recipients. It can help transplant teams manage transfusion of allogeneic RBCs during liver transplantation.

Ultrasound-guided erector spinae plane block for postoperative analgesia in laparoscopic liver resection: A prospective, randomised controlled, patient and observer-blinded study.

BACKGROUND: Erector spinae plane block (ESPB) has been reported to manage postoperative pain effectively after various types of surgery. However, there has been a lack of study on the effect of ESPB after liver resection. OBJECTIVES: To investigate the analgesic effects of ESPB on pain control after laparoscopic liver resection compared with conventional pain management. DESIGN: Prospective, randomised controlled study. SETTING: A single tertiary care centre from February 2019 to February 2020. PATIENTS: A total of 70 patients scheduled to undergo laparoscopic liver resection. INTERVENTIONS: In the control group (n = 35), no procedure was performed. In the ESPB group (n = 35), ESPB was performed after induction of general anaesthesia. A total of 40 ml of ropivacaine 0.5% was injected at the T9 level bilaterally. After surgery, intravenous fentanyl patient-controlled analgesia was initiated. Fentanyl and hydromorphone were administered as rescue analgesics. MAIN OUTCOME MEASURES: The primary outcome was the cumulative postoperative opioid consumption at 24 h (morphine equivalent). The secondary outcomes were rescue opioid (fentanyl) dose in the postanaesthesia care unit (PACU) and pain severity at 1, 6, 12, 24, 48 and 72 h, assessed using a numerical rating scale (NRS) score. RESULTS: The median [IQR] postoperative opioid consumption during 24 hours following surgery was 48.2 [17.1] mg in the control group and 45.5 [35.8] mg in the ESPB group (median difference, 4.2 mg; 95% CI, -4.2 to 13.3 mg; P = 0.259). Conversely, rescue opioid in PACU was 5.3 [5.0] mg in the control group and 3.0 [1.5] mg in the ESPB group (median difference, 2.5 mg; 95% CI, 1.0 to 5.0 mg; P < 0.001). There was no significant difference in NRS scores point between the groups at any time. CONCLUSION: ESPB does not provide analgesic effect within 24 h after laparoscopic liver resection. TRIAL REGISTRATION: Clinical Trial Registry of Korea (https://cris.nih.go.kr.), identifier: KCT0003549).

Isolevuglandins and mitochondrial enzymes in the retina: mass spectrometry detection of post-translational modification of sterol-metabolizing CYP27A1.

We report the first peptide mapping and sequencing of an in vivo isolevuglandin-modified protein. Mitochondrial cytochrome P450 27A1 (CYP27A1) is a ubiquitous multifunctional sterol C27-hydroxylase that eliminates cholesterol and likely 7-ketocholesterol from the retina and many other tissues. We investigated the post-translational modification of this protein with isolevuglandins, arachidonate oxidation products. Treatment of purified recombinant CYP27A1 with authentic iso[4]levuglandin E(2) (iso[4]LGE(2)) in vitro diminished enzyme activity in a time- and phospholipid-dependent manner. A multiple reaction monitoring protocol was then developed to identify the sites and extent of iso[4]LGE(2) adduction. CYP27A1 exhibited only three Lys residues, Lys(134), Lys(358), and Lys(476), that readily interact with iso[4]LGE(2) in vitro. Such selective modification enabled the generation of an internal standard, (15)N-labeled CYP27A1 modified with iso[4]LGE(2), for the subsequent analysis of a human retinal sample. Two multiple reaction monitoring transitions arising from the peptide AVLK(358)(-C(20)H(26)O(3))ETLR in the retinal sample were observed that co-eluted with the corresponding two (15)N transitions from the supplemented standard. These data demonstrate that modified CYP27A1 is present in the retina. We suggest that such protein modification impairs sterol elimination and likely has other pathological sequelae. We also propose that the post-translational modifications identified in CYP27A1 exemplify a general mechanism whereby oxidative stress and inflammation deleteriously affect protein function, contributing, for example, to cholesterol-rich lesions associated with age-related macular degeneration and cardiovascular disease. The proteomic protocols developed in this study are generally applicable to characterization of lipid-derived oxidative protein modifications occurring in vivo, including proteins bound to membranes.

Sample prefractionation for mass spectrometry quantification of low-abundance membrane proteins.

Use of stable isotope-labeled full-length proteins as an internal standard prior to multiple reaction monitoring (MRM) analysis enables prefractionation of the target proteins and quantification of those low-abundance proteins, which cannot be reached without biological sample enrichment. In terms of membrane proteins, this benefit can be used if a sample processing workflow allows entire solubilization of membrane proteins. We have developed a universal workflow for sample processing and enrichment by optimizing washing and solubilization conditions and implementing sample fractionation by Whole Gel Eluter. The optimized protocol was applied to various membrane-bound cytochromes P450 (CYPs) and their electron transferring protein partners, cytochrome P450 reductase (CPR), ferredoxin reductase (FdR), and ferredoxin (Fdx), all important proteins for cholesterol elimination from different organs. Both, weakly associated (CPR and FdR) and tightly associated (CYP7B1, CYP11A1, CYP27A1, and CYP46A1) membrane proteins were quantified. Measurements were performed on three human tissues (temporal lobe of the brain, retina, and retinal pigment epithelium) obtained from multiple donors. The biological implications of our quantitative measurements are also discussed.

Features of the retinal environment which affect the activities and product profile of cholesterol-metabolizing cytochromes P450 CYP27A1 and CYP11A1.

The retina is the sensory organ in the back of the eye which absorbs and converts light to electrochemical impulses transferred to the brain. Herein, we studied how retinal environment affects enzyme-mediated cholesterol removal. We focused on two mitochondrial cytochrome P450 enzymes, CYPs 27A1 and 11A1, which catalyze the first steps in metabolism of cholesterol in the retina and other tissues. Phospholipids (PL) from mitochondria of bovine neural retina, retinal pigment epithelium, liver and adrenal cortex were isolated and compared for the effect on kinetic properties of purified recombinant CYPs in the reconstituted system in vitro. The four studied tissues were also evaluated for the mitochondrial PL and cholesterol content and levels of CYPs 27A1, 11A1 and their redox partners. The data obtained were used for modeling the retinal environment in the in vitro enzyme assays in which we detected the P450 metabolites, 22R-hydroxycholesterol and 5-cholestenoic acid, unexpectedly found by us in the retina in our previous studies. The effect of the by-product of the visual cycle pyridinium bis-retinoid A2E on kinetics of CYP27A1-mediated cholesterol metabolism was also investigated. The results provide insight into the retina's regulation of the enzyme-mediated cholesterol removal.

Conversion of 7-ketocholesterol to oxysterol metabolites by recombinant CYP27A1 and retinal pigment epithelial cells.

Of the different oxygenated cholesterol metabolites, 7-ketocholesterol (7KCh) is considered a noxious oxy-sterol implicated in the development of certain pathologies, including those found in the eye. Here we elucidated whether sterol 27-hydroxylase cytochrome P450 27A1 (CYP27A1) is involved in elimination of 7KCh from the posterior part of the eye: the neural retina and underlying retinal pigment epithelium (RPE). We first established that the affinities of purified recombinant CYP27A1 for 7KCh and its endogenous substrate cholesterol are similar, yet 7KCh is metabolized at a 4-fold higher rate than cholesterol in the reconstituted system in vitro. Lipid extracts from bovine neural retina and RPE were then analyzed by isotope dilution GC-MS for the presence of the 7KCh-derived oxysterols. Two metabolites, 3ß,27-dihydroxy-5-cholesten-7-one (7KCh-27OH) and 3ß-hydroxy-5-cholesten-7-one-26-oic acid (7KCh-27COOH), were detected in the RPE but not in the neural retina. 7KCh-27OH was also formed when RPE homogenates were supplemented with NADPH and the mitochondrial redox system. Quantifications in human RPE showed that CYP27A1 is indeed expressed in the RPE at 2-4-fold higher levels than in the neural retina. The data obtained represent evidence for the role of CYP27A1 in retinal metabolism of 7KCh and suggest that, in addition to cholesterol removal, the functions of this enzyme could also include elimination of toxic endogenous compounds.

Quantification of cholesterol-metabolizing P450s CYP27A1 and CYP46A1 in neural tissues reveals a lack of enzyme-product correlations in human retina but not human brain.

Cytochrome P450 enzymes (CYP or P450) 46A1 and 27A1 play important roles in cholesterol elimination from the brain and retina, respectively, yet they have not been quantified in human organs because of their low abundance and association with membrane. On the basis of our previous development of a multiple reaction monitoring (MRM) workflow for measurements of low-abundance membrane proteins, we quantified CYP46A1 and CYP27A1 in human brain and retina samples from four donors. These enzymes were quantified in the total membrane pellet, a fraction of the whole tissue homogenate, using ¹5N-labled recombinant P450s as internal standards. The average P450 concentrations/mg of total tissue protein were 345 fmol of CYP46A1 and 110 fmol of CYP27A1 in the temporal lobe, and 60 fmol of CYP46A1 and 490 fmol of CYP27A1 in the retina. The corresponding P450 metabolites were then measured in the same tissue samples and compared to the P450 enzyme concentrations. Investigation of the enzyme-product relationships and analysis of the P450 measurements based on different signature peptides revealed a possibility of retina-specific post-translational modification of CYP27A1. The data obtained provide important insights into the mechanisms of cholesterol elimination from different neural tissues.

Comparison of Bispectral Index and Patient State Index values according to recovery from moderate neuromuscular block under steady-state total intravenous anesthesia.

There were insufficient researches of the comparison between Bispectral Index (BIS) and Patient State Index (PSI) values during the recovery of moderate NMB. We investigated the response of these indices during neuromuscular blockade (NMB) reversal by sugammadex under steady-state total intravenous anesthesia (TIVA) using propofol/remifentanil. In this prospective, observational study, patients undergoing laparoscopic cholecystectomy were enrolled. At the end of surgery, after confirming that train-of-four (TOF) count as 1 or 2, we maintained a steady state (BIS value of 40-50). After administration of 2 mg kg-1 sugammadex, BIS, PSI, and electromyography (EMG) signal values were recorded at one-minute intervals for 10 min. The primary outcome was the difference between the changes in BIS and PSI from baseline to a TOF ratio (TOFR) of 90 after sugammadex administration in steady-state TIVA. A total of 48 patients completed this trial. There was no significant difference between the changes in BIS and PSI values from baseline to TOFR 90 (- 0.333 ± 4.955 vs. - 0.188 ± 4.616; 95% confidence interval [CI] - 2.095 to 1.803; p = 0.882). Both BIS-EMG and PSI-EMG values at baseline and TOFR 90 were not statistically different (95% CI - 0.550 to 1.092; p = 0.510, 95% CI - 1.569 to 0.527; p = 0.322, respectively). No patient experienced any complications. Changes in BIS and PSI values after NMB reversal during steady-state TIVA were not significantly different. Both BIS and PSI provide trustworthy values for monitoring anesthetic depth during NMB reversal under TIVA.Trial Registration: This study was registered in the Clinical Trial Registry of Korea ( https://cris.nih.go.kr : KCT 0003805).

Optimizing the conditions of a multiple reaction monitoring assay for membrane proteins: quantification of cytochrome P450 11A1 and adrenodoxin reductase in bovine adrenal cortex and retina.

Approximately 30% of naturally occurring proteins are predicted to be embedded in biological membranes. Nevertheless, this group of proteins is traditionally understudied due to limitations of the available analytical tools. To facilitate the analysis of membrane proteins, the analytical methods for their soluble counterparts must be optimized or modified. Multiple reaction monitoring (MRM) assays have proven successful for the absolute quantification of proteins and for profiling protein modifications in cell lysates and human plasma/serum but have found little application in the analysis of membrane proteins. We report on the optimization of sample preparation conditions for the quantification of two membrane proteins, cytochrome P450 11A1 (CYP11A1) and adrenodoxin reductase (AdR). These conditions can be used for the analysis of other membrane proteins. We have demonstrated that membrane proteins that are tightly associated with the membrane, such as CYP11A1, can be quantified in the total tissue membrane pellet obtained after high-speed centrifugation, whereas proteins that are weakly associated with the membrane, such as AdR, must be quantified in the whole tissue homogenate. We have compared quantifications of CYP11A1 using two different detergents, RapiGest SP and sodium cholate, and two different trypsins, sequencing grade modified trypsin and trypsin, type IX-S from porcine pancreas. The measured concentrations in these experiments were similar and encouraged the use of either combination of detergent/trypsin for quantification of other membrane proteins. Overall, the CYP11A1 and AdR quantified in this work ranged from 110 pmol to 10 fmol per milligram of tissue protein.

Deletion of xylR gene enhances expression of xylose isomerase in Streptomyces lividans TK24.

Glucose (xylose) isomerases from Streptomyces sp. have been used for the production of high fructose corn syrup for industrial purposes. An 11-kb DNA fragment containing the xyl gene cluster was isolated from Streptomyces lividans TK24 and its nucleotide sequences were analyzed. It was found that the xyl gene cluster contained a putative transcriptional repressor (xylR), xylulokinase (xylB), and xylose isomerase (xylA) genes. The transcriptional directions of the xylB and xylA genes were divergent, which is consistent to those found in other streptomycetes. A gene encoding XylR was located downstream of the xylB gene in the same direction, and its mutant strain produced xylose isomerase regardless of xylose in the media. The enzyme expression level in the mutant was 4.6 times higher than that in the parent strain under xylose-induced condition. Even in the absence of xylose, the mutant strain produce over 60% of enzyme compared with the xylose-induced condition. Gel mobility shift assay showed that XylR was able to bind to the putative xyl promoter, and its binding was inhibited by the addition of xylose in vitro. This result suggested that XylR acts as a repressor in the S. lividans xylose operon.

Analysis of kimchi microflora using denaturing gradient gel electrophoresis.

A polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique was used to determine the microfloral composition during the fermentation of kimchi, a traditional Korean fermented vegetable food. The kimchi was fermented at 10 degrees C or 20 degrees C for 30 or 20 days, respectively. DGGE of the partially amplified 16S rDNA was performed and the most intense bands sequenced. The application of this culture-independent molecular technique determined that the lactic acid bacteria Weissella confusa, Leuconostoc citreum, Lactobacillus sakei, and Lactobacillus curvatus were the main microorganisms responsible for kimchi fermentation.

Molecular cloning and expression of a thermostable xylose (glucose) isomerase gene, xylA, from Streptomyces chibaensis J-59.

In the present study, the xylA gene encoding a thermostable xylose (glucose) isomerase was cloned from Streptomyces chibaensis J-59. The open reading frame of xylA (1167 bp) encoded a protein of 388 amino acids with a calculated molecular mass of about 43 kDa. The XylA showed high sequence homology (92% identity) with that of S. olivochromogenes. The xylose (glucose) isomerase was expressed in Escherichia coli and purified. The purified recombinant XylA had an apparent molecular mass of 45 kDa, which corresponds to the molecular mass calculated from the deduced amino acid and that of the purified wild-type enzyme. The N-terminal sequences (14 amino acid residues) of the purified protein revealed that the sequences were identical to that deduced from the DNA sequence of the xylA gene. The optimum temperature of the purified enzyme was 85 degrees C and the enzyme exhibited a high level of heat stability.

The Methanothermobacter thermautotrophicus MCM helicase is active as a hexameric ring.

The minichromosome maintenance (MCM) complex is thought to function as the replicative helicase in archaea and eukarya. The structure of the single MCM protein homologue from the archaeon Methanothermobacter thermautotrophicus is not yet clear, and hexameric, heptameric, octameric, and dodecameric structures, open rings, and filamentous structures have been reported. Using a combination of biochemical and structural analysis, it is shown here that the M. thermautotrophicus MCM helicase is active as a hexamer.

Cloning, Purification, and Partial Characterization of the Halobacterium sp. NRC-1 Minichromosome Maintenance (MCM) Helicase.

The MCM gene from the archaeon Halobacterium, with and without its intein, was cloned into an Escherichia coli expression vector, overexpressed and the protein was purified and antibodies were generated. The antibodies were used to demonstrate that in vivo only the processed enzyme, without the intein, could be detected.