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BACKGROUND: Acetabular retroversion is a form of hip dysplasia that may cause femoroacetabular impingement syndrome (FAIS), leading to pain and restricted hip range of motion. An exercise intervention aiming at altering pelvis tilt and related functional biomechanics may be a useful first-line intervention for patients who are not eligible for surgical repositioning.RESEARCH QUESTION: Does squat and gait biomechanics change following an 8-week targeted exercise program in individuals with symptomatic acetabular retroversion and FAIS?METHODS: This prospective intervention study used participants as their own controls. Examinations were conducted at three time-points: T1 baseline; T2 following an 8-week control period; T3 after 8 weeks' intervention. At each time-point, three-dimensional motion analysis of a deep squat and level gait was performed, and pain intensity was recorded using a numerical rating scale (NRS 0-10). The intervention consisted of a home-exercise program to improve core stability and pelvic movement. Differences in waveforms between time-points across pelvis and lower-limb biomechanics were evaluated using statistical parametric mapping. Delta (Δ, differences between T1-T2 and T2-T3) was used to evaluate changes in spatiotemporal gait parameters and pain.RESULTS: Nineteen patients (18 females), mean age 22.6 (SD 4.5) years, BMI (kg/m2) 23.0 (SD 4.1), were included. Changes (Δ T1-T2 vs. Δ T2-T3) in squat biomechanics were observed as: (i) decreased anterior pelvic tilt, (ii) deeper vertical pelvis position, and (iii) increased knee flexion angle. Contrary, no significant changes in gait biomechanics, Δ walking speed, Δ step length, or NRS for pain were found.SIGNIFICANCE: Following a targeted exercise intervention, participants were able to squat deeper, potentially allowing better hip function. The deepened squat position was accompanied by increased knee flexion and reduced anterior pelvic tilt. Gait biomechanics and patient-reported pain remained unchanged post-intervention. These findings are important for future design of exercise interventions targeting pelvic tilt in symptomatic individuals.

Aim: Physical activity is important for health but injuries might affect the level of activity. The aim of this study was to evaluate change in physical activity after fracture and to determine pre...

Excessive anterior pelvic tilt is suspected of causing femoroacetabular impingement, low back pain, and sacroiliac joint pain. Non-surgical treatment may decrease symptoms and is seen as an alternative to invasive and complicated surgery. However, the effect of non-surgical modalities in adults is unclear. The aim of this review was to investigate patient- and observer-reported outcomes of non-surgical intervention in reducing clinical symptoms and/or potential anterior pelvic tilt in symptomatic and non-symptomatic adults with excessive anterior pelvic tilt, and to evaluate the certainty of evidence. MEDLINE, EMBASE, Web of Science and Cochrane (CENTRAL) databases were searched up to March 2019 for eligible studies. Two reviewers assessed risk of bias independently, using the Cochrane Risk of Bias tool for randomized trials and the ROBINS-I tool for non-randomized studies. Data were synthesized qualitatively. The GRADE approach was used to assess the overall certainty of evidence. Of 2013 citations, two randomized controlled trials (RCTs) (n = 72) and two non-RCTs (n = 23) were included. One RCT reported a small reduction (< 2°) in anterior pelvic tilt in non-symptomatic men. The two non-RCTs reported a statistically significant reduction in anterior pelvic tilt, pain, and disability in symptomatic populations. The present review was based on heterogeneous study populations, interventions, and very low quality of evidence. No overall evidence for the effect of non-surgical treatment in reducing excessive anterior pelvic tilt and potentially related symptoms was found. High-quality studies targeting non-surgical treatment as an evidence-based alternative to surgical interventions for conditions related to excessive anterior pelvic tilt are warranted.Cite this article: EFORT Open Rev 2020;5:37-45. DOI: 10.1302/2058-5241.5.190017

Acetabular retroversion is associated with femoroacetabular impingement syndrome (FAIS). Anterior pelvic tilt enhances risk of FAIS.To investigate feasibility and change in patient-reported symptoms of a home-based exercise intervention in patients with acetabular retroversion and excessive anterior pelvic tilt, in comparison with a prior control period.Prospective intervention study.Patients (18-40 years) not eligible for surgery, with radiographic signs of acetabular retroversion and excessive anterior pelvic tilt were recruited. An 8-week control period was followed by an 8-week training period. The home-based intervention consisted of education and exercises for core stability, muscle strengthening and stretching for reducing anterior pelvic tilt. Feasibility assessments were dropout (≤10%), adherence (≥75% of sessions), exercise-related pain, and adverse events. Primary outcome was change in the Copenhagen Hip and Groin Outcome Score (HAGOS) pain-subscale. Pelvic tilt was measured by EOS scanning.Forty-two patients (93% female, mean age 22.2 ± 4.2 years) were included. Dropout rate was 7% and satisfactory adherence was demonstrated by 85%. Exercise-related pain and adverse events were acceptable. Between-period mean change score for HAGOS-PAIN was 5.2 points (95% CI: -0.3-10.6) and -1.6° (95% CI: -3.9-0.7) of anterior pelvic tilt. Patients responding positively (≥10 points) (n = 10, 26%), had pre-exercise moderate pain (HAGOS-PAIN 47.5-70 points).Current exercise intervention was feasible. However, we found no clinically relevant changes in self-reported hip-related pain or anterior pelvic tilt. Post hoc responder analysis revealed that patients with moderate pain at baseline might benefit from this exercise.

Introduction The prevalence of acetabular retroversion is sparsely investigated. It may be associated with increased anterior pelvic tilt. The purpose of this study was to investigate whether patients with symptomatic and radiographically verified acetabular retroversion demonstrated increased anterior pelvic tilt compared to a control group, and furthermore to evaluate the prevalence of acetabular retroversion in the general population. Methods Anteroposterior pelvic radiographs in standing position of 111 patients with acetabular retroversion prior to anteverting periacetabular osteotomy (PAO) and 132 matched controls from the general Danish population were assessed. Pelvic tilt was assessed by the sacrococcygeal joint–symphysis distance and pelvic-tilt-ratio. Acetabular retroversion was defined as positive cross-over sign and posterior wall sign. Prior to assessments, interrater reliability analysis was performed. Measurements were agreed by two independent assessors. A nonparametric regression model was used to test between-group differences in median pelvic tilt. The prevalence was calculated as the ratio of subjects and hips with acetabular retroversion, respectively. Results The patient group had significantly larger median anterior pelvic tilt of 14.3 mm in sacrococcygeal joint–symphysis distance and −0.08 in pelvic-tilt-ratio, compared to controls. The prevalence of subjects in the general population with either unilateral or bilateral acetabular retroversion was 24% and 18% for all hips. Conclusion Our data demonstrated that patients with symptomatic acetabular retroversion have increased anterior pelvic tilt compared to the general population. Radiographic sign of acetabular retroversion was highly prevalent in the general population. Implication for practice Increased anterior pelvic tilt should be considered when diagnosing and treating patients with hip pain, as symptoms may be related to the functional position of the pelvis and not necessarily solely come from the radiographic verified acetabular retroversion.

Background: The limb occlusion pressure (LOP) is determined to calculate the relative LOP. The different levels of relative LOP (percentage of LOP), influence treatment effect, and perceived discomfort during low-load blood flow restriction (BFR) strength training. Hence, it is of outmost importance to determine the LOP when using BFR in clinical practice. Purpose: The objectives of this study were to investigate the concurrent validity and intra-rater (test-retest) intra-day reliability of an inexpensive, portable, easy-to-use handheld (HH) oximeter compared to a high-resolution Doppler ultrasound scanner to detect LOP in the lower extremity. Study design: Cross-sectional validity and reliability study Methods: 50 healthy participants (mean age 25.8 years) were simultaneously assessed by two raters blinded from each other. A 20-cm wide thigh cuff with an attached sphygmomanometer was inflated until the raters independently registered LOP with the HH oximeter and the Doppler ultrasound scanner. The test session was performed twice with a five-minute time interval. Results: Strong linear relationship was observed between the HH oximeter and Doppler ultrasound (r=0.84-0.86, p

BackgroundPatients with symptomatic acetabular retroversion is reported having reduced functional ability and quality of life but little is known about the effect of non-surgical interventions.Purpose/Aim of StudyTo investigate feasibility and change in patient-reported symptoms of a home-based exercise intervention in patients with acetabular retroversion and excessive anterior pelvic tilt, in comparison with a prior control period.Materials and MethodsPatients with symptomatic acetabular retroversion and excessive anterior pelvic tilt were included. Following an 8-week control period, patients were instructed to follow an 8-week targeted (3 times/week) progressive home-based exercise intervention. Feasibility assessment included; dropout, acceptable adherence (≥75% of sessions), exercise-related pain, and adverse events. Primary outcome was change in the Copenhagen Hip and Groin Outcome Score (HAGOS) pain subscale. Secondary outcomes included change in remaining HAGOS subscales, EQ-5D-3L questionnaire, and pelvic tilt measured by EOS® scanning.Findings / ResultsForty-two patients (39 women) (median [interquartile range (IQR)], 20.5 [19 - 25 years]) were included. Three patients were lost to follow-up (one regretting participating during the control period, one during the intervention period and one patient was lost at follow-up). Adherence to exercise sessions was 85%. Exercise-related pain and adverse events were acceptable. Between-period mean change score for the HAGOS-PAIN subscale was 5.2 points (95% confidence interval [CI]:[-0.3 – 10.6] and -1.6 degree [-3.9 – 0.7]) of anterior pelvic tilt. Additionally, patients who responded positively (≥ minimal clinically important difference) to the exercise intervention (n = 10, 26%), all had a pre-exercise HAGOS-PAIN score between 47.5 to 70 points.ConclusionsCurrent exercise intervention was feasible. However, no clinical relevant changes in self-reported hip-related pain, function, quality of life, nor anterior pelvic tilt were found. Post-hoc responder analysis revealed that patients with moderate pain at baseline might benefit from current exercise.

INTRODUCTION: Acetabular retroversion (AR) is a variant of hip dysplasia,1 enhancing the risk of pincer-morphology femoroacetabular impingement syndrome (FAIS).2 FAIS is associated with pain,3 reduced level of function,4 decreased health-related quality of life,5 and early development of hip osteoarthritis (OA).6, 7 Symptoms may be exacerbated by anterior pelvic tilt (APT)8 as it functionally increases anterosuperior femoral head cover.1, 9, 10 Currently, the standard treatment for symptomatic AR is an invasive anteverting periacetabular osteotomy.11 Exercise aiming at reducing APT may be a less-invasive alternative treatment strategy. However, the impact of a targeted exercise intervention in patients with symptomatic AR, and excessive APT is unknown. RESEARCH QUESTION: What is the change in patient-reported symptoms and pelvic tilt in patients with AR and excessive APT of an 8-week targeted progressive home-based exercise intervention compared with a prior control period?METHODS: The sample size was n=36 patients (age 18-40 years) not eligible for surgery, with radiographic signs of AR on an anterior/posterior radiograph in standing position. Patients were recruited from Odense University Hospital outpatient clinic (Denmark) from November 2018 to December 2019. Exclusion criteria were; pelvic-tilt-ratio greater than 0.512 (the height of the obturator foramen divided by the height of the lesser pelvis) indicating posterior pelvic tilt, radiographic sign of hip OA, related surgery, and a body mass index (BMI) above 35. The intervention consisted of education, activity modification, stretching for posterior pelvic tilt mobility, strengthening muscles for tilting the pelvis posteriorly, improving body core stability, and pelvic movement control three times per week. The primary outcome was change in the Copenhagen Hip and Groin Outcome Score (HAGOS) pain subscale. Secondary outcomes included change in pelvic tilt in standing position measured by EOS® scanning, as the angle between a horizontal line and a line connecting the upper border of the symphysis with the sacral promontory.13RESULTS: Forty-two patients (39 women) (median [interquartile range], 20.5 [19 – 25 years] were included. Three patients were lost to follow-up. Between-period mean change scores were 5.2 points (95% confidence interval [CI]: [-0.3 – 10.6]), for the HAGOS-PAIN subscale and -1.6 degrees [-3.9 – 0.7] of anterior pelvic tilt. DISCUSSION: No statistical or clinically relevant changes in self-reported hip-related pain and APT were found. However, post-hoc responder analysis revealed that patients with moderate pain at baseline benefited from exercise. Patients had an excessive APT of 74° ±8 (mean ±SD) at baseline, which is 14° more than the defined neutral pelvic tilt.13 The current study is limited by a short and unsupervised training period, and measuring static pelvic tilt only. Future results from three-dimensional gait analysis will provide answers to whether patients' ability to dynamically tilt their pelvis posteriorly has changed.REFERENCES: 1. Reynolds D, Lucas J, Klaue K. Retroversion of the acetabulum. A cause of hip pain. J Bone Joint Surg Br. 1999;81(2):281-8.2. Siebenrock KA, Schoeniger R, Ganz R. Anterior femoro-acetabular impingement due to acetabular retroversion. Treatment with periacetabular osteotomy. J Bone Joint Surg Am. 2003;85(2):278-86.3. Langlais F, Lambotte JC, Lannou R, Gedouin JE, Belot N, Thomazeau H, et al. Hip pain from impingement and dysplasia in patients aged 20-50 years. Workup and role for reconstruction. Joint Bone Spine. 2006;73(6):614-23.4. Rylander JH, Shu B, Andriacchi TP, Safran MR. Preoperative and postoperative sagittal plane hip kinematics in patients with femoroacetabular impingement during level walking. Am J Sports Med. 2011;39 Suppl:36S-42S.5. Clohisy JC, Baca G, Beaule PE, Kim YJ, Larson CM, Millis MB, et al. Descriptive epidemiology of femoroacetabular impingement: a North American cohort of patients undergoing surgery. Am J Sports Med. 2013;41(6):1348-56.6. Ganz R, Parvizi J, Beck M, Leunig M, Notzli H, Siebenrock KA. Femoroacetabular impingement: a cause for osteoarthritis of the hip. Clin Orthop Relat Res. 2003(417):112-20.7. Giori NJ, Trousdale RT. Acetabular retroversion is associated with osteoarthritis of the hip. Clin Orthop Relat Res. 2003(417):263-9.8. Riviere C, Hardijzer A, Lazennec JY, Beaule P, Muirhead-Allwood S, Cobb J. Spine-hip relations add understandings to the pathophysiology of femoro-acetabular impingement: A systematic review. Orthop Traumatol Surg Res. 2017;103(4):549-57.9. Dandachli W, Ul Islam S, Richards R, Hall-Craggs M, Witt J. The influence of pelvic tilt on acetabular orientation and cover: a three-dimensional computerised tomography analysis. Hip Int. 2013;23(1):87-92.10. Ross JR, Nepple JJ, Philippon MJ, Kelly BT, Larson CM, Bedi A. Effect of Changes in Pelvic Tilt on Range of Motion to Impingement and Radiographic Parameters of Acetabular Morphologic Characteristics. Am J Sports Med. 2014.11. Siebenrock KA, Schaller C, Tannast M, Keel M, Buchler L. Anteverting periacetabular osteotomy for symptomatic acetabular retroversion: results at ten years. J Bone Joint Surg Am. 2014;96(21):1785-92.12. Schwarz T, Benditz A, Springorum HR, Matussek J, Heers G, Weber M, et al. Assessment of pelvic tilt in anteroposterior radiographs by means of tilt ratios. Arch Orthop Trauma Surg. 2018.13. Tannast M, Murphy SB, Langlotz F, Anderson SE, Siebenrock KA. Estimation of pelvic tilt on anteroposterior X-rays--a comparison of six parameters. Skeletal Radiol. 2006;35(3):149-55.

Summary Barley (Hordeum vulgare L.) possesses a large and highly repetitive genome of 5.1 Gb that has hindered the development of a complete sequence. In 2012, the International Barley Sequencing Consortium released a resource integrating whole‐genome shotgun sequences with a physical and genetic framework. However, because only 6278 bacterial artificial chromosome (BACs) in the physical map were sequenced, fine structure was limited. To gain access to the gene‐containing portion of the barley genome at high resolution, we identified and sequenced 15 622 BACs representing the minimal tiling path of 72 052 physical‐mapped gene‐bearing BACs. This generated ~1.7 Gb of genomic sequence containing an estimated 2/3 of all Morex barley genes. Exploration of these sequenced BACs revealed that although distal ends of chromosomes contain most of the gene‐enriched BACs and are characterized by high recombination rates, there are also gene‐dense regions with suppressed recombination. We made use of published map‐anchored sequence data from Aegilops tauschii to develop a synteny viewer between barley and the ancestor of the wheat D‐genome. Except for some notable inversions, there is a high level of collinearity between the two species. The software HarvEST:Barley provides facile access to BAC sequences and their annotations, along with the barley–Ae. tauschii synteny viewer. These BAC sequences constitute a resource to improve the efficiency of marker development, map‐based cloning, and comparative genomics in barley and related crops. Additional knowledge about regions of the barley genome that are gene‐dense but low recombination is particularly relevant., Significance Statement We provide new genome sequence resources for barley and related species, to improve the efficiency of marker development, map‐based cloning, comparative genomics studies, and to provide insights into genome architecture. Notably, some of the gene‐rich islands in the barley genome deviate markedly from assumption that gene density and high recombination frequency are correlated.

There are several reports on the association between pubalgia and intra-articular hip disorders. The purpose of this study was to evaluate the long-term outcome in athletes who underwent tenotomy due to long-standing groin pain. A secondary purpose was evaluating the frequency of femoro-acetabular impingement (FAI) and its impact on the long-term outcome.Thirty-two high-level male athletes treated with adductor tenotomy, rectus abdominis tenotomy or both were included. At a median follow-up time of 6 years after the tenotomy, the subjects underwent standardised clinical examination, plain radiographs, completed web-based health-related patient-reported outcomes, including iHOT12, HAGOS (six subscales), EQ-5D (two subscales), HSAS for physical activity level and a VAS for overall hip function. Furthermore, patient satisfaction and return to sports were documented.Twenty-four of the 32 (75 %) athletes were satisfied with the outcome of the tenotomy, and 22 of the athletes (69 %) were able to return to their pre-injury sport. Before the long-term follow-up, two of these satisfied athletes had undergone repeat surgery (one hip arthroscopy due to FAI and one repeat tenotomy). Of the 24 satisfied athletes, eight (33 %) had a positive hip impingement test at the follow-up. Of the remaining eight athletes not satisfied with the outcome, only one returned to their pre-injury sport and three had undergone hip arthroscopy prior to follow-up. Five had positive hip impingement tests which was significantly more frequently than in the satisfied group (p = 0.008). The group with a positive hip impingement test reported significantly more pain and symptoms, more hip problems during sports and physical activity, as well as lower hip-related quality of life according to the HAGOS scores (p0.05), at follow-up.Tenotomy for pubalgia yielded a satisfactory long-term outcome, with three of four athletes being able to return to their pre-injury sport. The athletes that did not return to their pre-injury sport had higher frequency of positive hip impingement test and inferior functional outcome compared with the athletes that did return to their pre-injury sport. It is therefore recommended that the hip should be carefully evaluated for hip impingement before tenotomy is considered as treatment for athletes with pubalgia.

Lipase-catalyzed acidolysis was examined for the production of structured phospholipids in a hexane system. In a practical operation of the reaction system, the formation of lyso-phospholipids from hydrolysis is often a serious problem, as demonstrated from previous studies. A clear elucidation of the issue and optimization of the system are essential for the practical applications in reality. The effects of enzyme dosage, reaction temperature, solvent amount, reaction time, and substrate ratio were optimized in terms of the acyl incorporation, which led to the products, and lyso-phospholipids formed by hydrolysis, which led to the low yields. The biocatalyst used was the commercial immobilized lipase Lipozyme TL IM and substrates used were phosphatidylcholine (PC) from soybean and caprylic acid. A response surface design was used to evaluate the influence of selected parameters and their relationships on the incorporation of caprylic acid and the corresponding recovery of PC. Incorporation of fatty acids increased with increasing enzyme dosage, reaction temperature, solvent amount, reaction time, and substrate ratio. Enzyme dosage had the most significant effect on the incorporation, followed by reaction time, reaction temperature, solvent amount, and substrate ratio. However the parameters had also a negative influence on the PC recovery. Solvent amount had the most negative effect on recovery, followed by enzyme dosage, temperature, and reaction time. Individually substrate ratio had no significant effect on the PC recovery. Interactions were observed between different parameters. On the basis of the models, the reaction was optimized for the maximum incorporation and maximum PC recovery. With all of the considerations, the optimal conditions are recommended as enzyme dosage 29%, reaction time 50 h, temperature 54 degrees C, substrate ratio 15 mol/mol caprylic acid/PC, and 5 mL of hexane per 3 g substrate. No additional water is necessary. Under these conditions, an incorporation of caprylic acid up to 46% and recovery of PC up to 60% can be obtained from the prediction. The prediction was confirmed from the verification experiments.

Oxidative stability of liposomes made of (Docosahexaenoic acid) DHA-containing phosphatidylcholine (PC) was examined during preparation and storage. After preparation of the liposomes, the concentration of primary (conjugated dienes) and secondary oxidation products (Thiobarbituric acid-reactive substances, TBARS) were significantly higher compared to the initial value. During cold storage, formation of conjugated dienes and TBARS remained more or less constant in large unilamellar vesicles (LUV), whereas in mulitilamellar vesicles (MLV) they were seen to increase over a period of 21 days. Evaporation of solvent traces from a lipid film should preferably be done under nitrogen as vacuum evaporation was found to increase oxidation of the phospholipid.

Ileal pouch anal anastomosis (IPAA) is the preferred method for restorative surgery in patients with ulcerative colitis who have to undergo proctocolectomy. The most common complication is pouchitis and several studies have pointed to the microbiota of the pouch as being a risk factor. The aim of this study was to follow the development of the bacterial microbiota in pouches during the first year.Terminal restriction fragment length polymorphism (T-RFLP) combined with cloning and sequencing was used to identify the most predominant bacteria on the different sampling occasions. A total of 274 clones were grouped by T-RFLP and clones from each group were selected for sequencing and identified by comparison with known sequences.Differences in T-RFLP profiles and clone libraries were found between the patients, and also in changes apparent in each patient at different time-points. The main bacterial groups in the pouches resembled those of the normal colonic microbiota, with a predominance of the clostridia clusters XIVa and IV, Bacteroides and Enterobacteriaceae. Exceptions were clones with sequences resembling those of the Clostridium perfringens group, in both patients and on all sampling occasions, and the dominance of clones resembling Turicibacter in one of the patients at the time of pouch construction.The pouch microbiota showed similarities to the normal colon microbiota except for the presence of clones with sequences resembling those of the C. perfringens group and Turicibacter. The bacterial composition differed between the two patients and the microbiota changed with time, suggesting that the composition is not stable during the first year.

Barley (Hordeum vulgareL.) possesses a large and highly repetitive genome of 5.1 Gb that has hindered the development of a complete sequence. In 2012, the International Barley Sequencing Consortium released a resource integrating whole-genome shotgun sequences with a physical and genetic framework. However, since only 6,278 BACs in the physical map were sequenced, detailed fine structure was limited. To gain access to the gene-containing portion of the barley genome at high resolution, we identified and sequenced 15,622 BACs representing the minimal tiling path of 72,052 physical mapped gene-bearing BACs. This generated about 1.7 Gb of genomic sequence containing 17,386 annotated barley genes. Exploration of the sequenced BACs revealed that although distal ends of chromosomes contain most of the gene-enriched BACs and are characterized by high rates of recombination, there are also gene-dense regions with suppressed recombination. Knowledge of these deviant regions is relevant to trait introgression, genome-wide association studies, genomic selection model development and map-based cloning strategies. Sequences and their gene and SNP annotations can be accessed and exported via http://harvest-web.org/hweb/utilmenu.wc or through the software HarvEST:Barley (download from harvest.ucr.edu). In the latter, we have implemented a synteny viewer between barley andAegilops tauschiito aid in comparative genome analysis.

This work provides different strategies for the enzymatic modification of the fatty acid composition in soybean phosphatidylcholine (PC) and the subsequent purification. Enzymatic transesterification reactions with caprylic acid as acyl donor were carried out in continuous enzyme bed reactors with a commercial immobilized lipase (Lipozyme RM IM) as catalyst. Operative stability of the immobilized lipase was examined under solvent and solvent-free conditions. The long reaction time required to have a high incorporation, combined with rapid deactivation of the enzyme, makes the solvent-free transesterification reaction unfavorable. Performing the reaction in the presence of solvent (hexane) makes it possible to have high incorporation into PC and deactivation of the lipase is less pronounced as compared to solvent-free operations. For solvent-free operation, it is suggested to recycle the reaction mixture through the packed bed reactor, as this would increase incorporation of the desired fatty acids, due to increased contact time between substrate and enzyme in the column. Removal of free fatty acids from the reaction mixture can be done by ultrafiltration; however, parameters need to be selected with care in order to have a feasible process. No changes are observed in the phospholipid (PL) distribution during ultrafiltration, and other techniques as column chromatography may be required if high purity of individual PL species is desired. LC/MS analysis of transesterified PC revealed the presence of 8:0/ 8:0-PC, showing that acyl migration takes place during the acidolysis reaction.

This study examined the phenolic composition and the antioxidant potencies of black currant juices that had been experimentally clarified with acidic proteases and pectinases to retain the phenolics and which had been subjected to rat intestinal mucosa extract incubation to mimic gut cell mediated biotransformation of phenolics. When compared at equimolar levels of 2.5 microM gallic acid equivalents, the black currant juice samples prolonged the induction time of human low-density lipoprotein oxidation in vitro by 2.6-3.6 times, and the order of antioxidant potency of differently clarified black currant juices was centrifuged juicegelatin silica sol clarified juiceenzymatically clarified juice approximately raw juice. No immediate relationship between the, almost similar, phenolic profiles of the juice samples and their relative antioxidant activities could be established. Incubation of juices with a rat small intestine cell extract for 19 h promoted significant decreases in the contents of the anthocyanin 3-O-beta-glucosides (cyanidin 3-O-beta-glucoside and delphinidin 3-O-beta-glucoside), but did not affect the anthocyanin 3-O-beta-rutinosides (cyanidin 3-O-beta-rutinoside and delphinidin 3-O-beta-rutinoside) of the black currant juice. Black currant juice samples subjected to such intestinal cell extract incubation had approximately 30% decreased antioxidant capacity. Incubation of juices with the rat small intestine cell extracts at neutral pH appeared to decrease the levels of delphinidin glucosides more than the levels of cyanidin glucosides. The results provide an explanation for the predominant detection of anthocyanin rutinosides, and not anthocyanin glucosides, in plasma and urine in in vivo studies and provide important clues to better understand the complex mechanisms affecting dietary phenols in the gut.

The possibilities of applying ultrafiltration for the purification of structured phospholipids (PLs) produced by lipase-catalyzed acidolysis in a hexane system were examined. Commercial polymeric membranes with different cut-offs (1000–20,000 Da) were screened for their abilities to separate free fatty acids (FFA) from structured PLs. Suitable membranes were selected in terms of high selectivity between FFA and PLs. Several membranes showed to be able to reject more than 90% of phosphatidylcholine (PC), however, based on the solubility parameters of the polymers many of the membranes would not be suitable for long term with the solvents in use. One membrane was more stable with the solvents compared with the other membranes screened; it was a polysulphone (PSf) membrane on polyester (PE) support (GR70PE). GR70PE showed similar retention of PC as that of few other membranes, but showed relatively higher retention of FFA, resulting in lower selectivity. Increased pressure increased the retention of both PC and FFA, however, the selectivity was improved. With a discontinuous diafiltration process (11 batches) using GR70PE, it was possible to change the molar ratio between PC and FFA from 1:48 to 1:1.6. The results of this study show that membrane separation may be a promising route for downstream processing of structured PLs.

By-product formation is a serious problem in the lipase-catalyzed acyl exchange of phospholipids (PL). By-products are formed due to parallel hydrolysis reactions and acyl migration in the reaction system. A clear elucidation of these side reactions is important for practical operation in order to minimize by-products during reaction. In the present study we examined the lipozyme RM IM-catalyzed acidolysis for the production of structured phospholipids between phosphatidylcholine (PC) and caprylic acid in the solvent-free system. A five-factor response surface design was used to evaluate the influence of major factors and their relationships on a number of responses reflecting the turnover of main reactions as well as side reactions. The five factors, including enzyme dosage, reaction time, reaction temperature, substrate ratio (mol/mol caprylic acid/PC) and water addition, were varied at three levels with two star points. All parameters besides water addition had an effect on the incorporation of caprylic acid into PC and lysophosphatidylcholine (LPC). Reaction time and enzyme dosage showed increased effect on incorporation into PC, while substrate ratio and reaction temperature showed opposite effect. The PC content decreased with increase of all parameters except for substrate ratio. Optimal conditions are recommended as enzyme dosage 40%, reaction temperature 55 °C, water addition 1%, reaction time 70 h, and substrate ratio 6 mol/mol caprylic acid/PC. Under these conditions an incorporation of 46% with PC accounting for 53% of the PL fraction can be obtained. Regiospecific analysis of the product revealed that the caprylic acid was mainly incorporated into the sn-1 position accounting for 80% of the fatty acids incorporated.

Thin-layer chromatography with a flame ionization detector (TLC-FID) was used for monitoring the production of structured phospholipids (ML type: L, long-chain fatty acids; M, medium-chain fatty acids) by enzyme-catalyzed acidolysis between soybean phosphatidylcholine (PC) and caprylic acid. It was found that the structured PC fractionated into two to three distinct bands on both plate thin-layer chromatography (TLC) and Chromarod TLC. These three bands represented PC of the LL type, ML type, and MM type, respectively. The TLC-FID method was applied in the present study to examine the influence of enzyme dosage, reaction temperature, solvent amount, reaction time, and substrate ratio (caprylic acid/PC, mol/mol) on formation of ML-type PC in a batch reactor with Thermomyces lanuginosa lipase as the catalyst. The formation of ML-type PC was dependent on all parameters examined except for the substrate ratio. The ML-type PC content increased with increasing enzyme dosage, reaction temperature, solvent amount, and reaction time. The substrate ratio had no significant effect on the formation of ML-type PC within the tested range (3-15 mol/mol). The formation of MM-type PC was observed in some experiments, indicating that acyl migration is taking place during reaction since the lipase is claimed to be 1,3-specific. The TLC-FID method offers a simple and cheap technique for elucidation of product and byproduct formation during enzyme-catalyzed reactions for production of phospholipids containing mixtures of long- and medium-chain fatty acids.

The possibilities of producing structured phospholipids between soybean phospholipids and caprylic acid by lipase-catalyzed acidolysis were examined in continuous packedbed enzyme reactors. Acidolysis reactions were performed in both a solvent system and a solvent-free system with the commercially immobilized lipase from Thermomyces lanuginosa (Lipozyme TL IM) as catalyst. In the packed bed reactors, different parameters for the lipase-catalyzed acidolysis were elucidated, such as solvent ratio (solvent system), temperature, substrate ratio, residence time, water content, and operation stability. The water content was observed to be very crucial for the acidolysis reaction in packed bed reactors. If no water was added to the substrate during reactions under the solvent-free system, very low incorporation corporation of caprylic acid was observed. In both solvent and solvent-free systems, acyl incorporation was favored by a high substrate ratio between acyl donor and phospholipids, a longer residence time, and a higher reaction temperature. Under certain conditions, the incorporation of around 30% caprylic acid can be obtained in continuous operation with hexane as the solvent.

We describe the initial characterization of the starch granule proteome of barley, containing the proteins firmly associated with the granule. Starch granule preparations from mature barley seeds were subjected to SDS washes and enzymatic shaving with thermolysin prior to protein extraction in boiling SDS buffer. Proteins were examined by two- and one-dimensional electrophoresis. A typical granule proteome contained around 150 proteins spots. A total of 74 spots were identified by mass fingerprinting and sequencing using mass spectrometry with integrated NCBI database searches. Four of the central enzymes in starch synthesis, i.e. granule bound starch synthase I (GBSSI), SSI, SSII and SBEIIb, together with hordeins B and D, serpin Z4, pyruvate orthophosphate dikinase (PPDK) and an enzyme involved in thiamine biosynthesis, were identified. Unexpectedly, of the identified spots on the two-dimensional gel, 49 were found to belong to GBSSI, representing variations in both relative molecular mass and isoelectric point. The GBSSI fragmentation, which was evident from day 10 after pollination, was consistent between preparations and cultivars and is inferred to result from in vivo proteolysis. Based on the enzymatic shaving, we conclude that the starch synthesis enzymes, including the GBSSI fragments, are integral components of the granule, whereas the other granule-bound proteins are attached to the surface.

The aim of this study was to evaluate the reliability of the impression replica technique with a four-unit zirconia fixed dental prosthesis (FDP). Marginal and internal fit were measured by repeatedly placing the FDP on an epoxy cast using light-body silicone material corresponding to cement. All measured marginal and internal fit points showed varying values. The greatest variations were seen at the most distal margin (33 μm) and at the distal abutment of the FDP (77 μm). The results showed that the technique gives moderate variations and is a useful method to evaluate marginal and internal fit.

A full-length cDNA clone defining the new myrosinase gene family MC in Brassica napus was isolated and sequenced. Southern hybridization showed that the MC family probably consists of 3 or 4 genes in B. napus. MC genes are expressed in the developing seed, but not in the vegetative tissues investigated. In situ hybridizations to developing seeds showed that the MC genes are expressed in the myrosin cells of the embryo axis and the cotyledons. Complexes with myrosinase and myrosinase-binding protein (MBP) were purified and characterized. Sequencing of peptides from myrosinases occurring in the complexes showed that the 70 kDa myrosinase is encoded by the MC genes, whereas the 65 kDa myrosinase is encoded by the MB genes. This is in contrast to the 75 kDa myrosinase which occurs in free form and is encoded by the MA genes. Deglycosylations of the myrosinase complexes and the free myrosinase showed that the molecular sizes of the myrosinases could be reduced significantly by this treatment, and that the size differences between the different myrosinases are mainly due to differences in glycosylation.

In mature seeds of Brassica napus three major and three minor myrosinase isoenzymes were identified earlier. These myrosinases are known to be encoded by at least two different families of myrosinase genes, denoted MA and MB. In the work described in this paper the presence of different myrosinase isoenzymes in embryos, seedlings, and vegetative mature tissues of B. napus was studied and related to the expression of myrosinase MA and MB genes in the same tissues to facilitate future functional studies of these enzymes. In developing seeds, myrosinases of 75, 73, 70, 68, 66, and 65 kD were present. During seedling development there was a turnover of the myrosinase pool such that in 5-d-old seedlings the 75-, 70-, 66-, and 65-kD myrosinases were present, with the 70- and 75-kD myrosinases predominating. In 21-d-old seedlings the same myrosinases were present, but the 66- and 65-kD myrosinase species were most abundant. At flowering the mature organs of the plant contained only a 72-kD myrosinase. MA genes were expressed only in developing seeds, whereas MB genes were most highly expressed in seeds, seedling cotyledons, young leaves, and to a lesser extent other organs of the mature plant. During embryogenesis of B. napus, myrosinase MA and MB gene transcripts started to accumulate approximately 20 d after pollination and reached their highest level approximately 15 d later. MB transcripts accumulated to about 3 times the amount of MA transcripts. In situ hybridization analysis of B. napus embryos showed that MA transcripts were present predominatly in myrosin cells in the axis, whereas MB genes were expressed in myrosin cells of the entire embryo. The embryo axiz contained 75-, 70-, and 65-kD myrosinases, whereas the cotyledons contained mainly 70- and 65-kD myrosinases. Amino acid sequencing revealed the 75-kD myrosinase to be encoded by the MA gene family. The high degree of cell and tissue specificity of the expression of myrosinase genes suggests that studies of their transcription should provide interesting information concerning a complex type of gene regulation.

The aim of the present study was to explore lung microvascular leakage of protein and water in a feline model of septic shock, using a double isotope technique with external gamma camera detection and gravimetric lung water measurements. The experiments were performed on artificially ventilated cats. One group of cats (n = 8) was given an infusion of live Escherichia coli bacteria, and another group (n = 5) served as a control group receiving saline. Plasma transferrin was radiolabeled in vivo with indium-113m-chloride, and erythrocytes were labeled with technetium-99m. The distribution of these isotopes in the lungs was continuously measured with a gamma camera. A normalized slope index (NSI) was calculated, indicative of the transferrin accumulation corrected for changes in local blood volume that reflect protein leakage. In the septic group there was a protein leakage after bacterial infusion, with a NSI of 39 x 10(-4) +/- 5 x 10(-4) min-1 (mean +/- SEM), and the PaO2 diminished from 21 +/- 1 to 9.5 +/- 1 kPa. In control cats a slight protein leakage with a NSI of 9 +/- 10(-4) +/- 2 x 10(-4) min-1 was detected, probably caused by the operative procedure, but PaO2 did not change. Wet-to-dry-weight ratios of postmortem lungs were not significantly different between the groups. It was concluded that an intravenous infusion of live E. coli bacteria induces a lung capillary protein leakage without increased lung water and a concomitantly disturbed gas exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

We have isolated and sequenced a cDNA clone for the glucosinolate-degrading enzyme myrosinase from Brassica napus (oilseed rape). Using a probe derived from the cDNA clone we have studied the expression of myrosinase genes in different tissues of B. napus. The myrosinase genes are most highly expressed in the young leaves, the cotyledons and the developing seed. Other parts of the plant only show a relatively low expression of myrosinase. In the developing seed the expression of myrosinase is first detected at 15 days after pollination and at the highest levels around 30 days after pollination. The myrosinase genes are turned on earlier, but also silenced earlier than the genes for the storage protein cruciferin.

SUMMARY The Bipolaris sorokiniana tolerant 1 (bst1) barley mutant is derived from fast neutron-irradiated seeds of wild-type Bowman(Rph3). The induced mutation was genetically localized to a position on chromosome 5HL distal to the centromere using amplified fragment length polymorphism markers. In addition, the defence responses and related gene expression in the bst1 mutant after fungal challenge were compared with those occurring in wild-type plants. Hydrogen peroxide generation, determined by 3,3-diaminobenzidine staining, revealed a clearly reduced level of bst1, compared with the wild-type, during the entire experimental time: 8-120 h post-inoculation (hpi). At 48 hpi, the wild-type samples displayed twice as much fungal mass and three times greater H(2)O(2) production than bst1. At the same time, staining of B. sorokiniana showed less fungal growth in the spontaneous lesions of bst1 compared with the wild-type. Monitoring of defence-related genes at 48 hpi demonstrated strong expression of PR-1a, PR-2, PR-5 and PR-10 in bst1. A gene coding for a unique oxidoreductase enzyme, designated as HCP1, was expressed at much higher levels in inoculated leaves of the bst1 mutant than in those of the wild-type plant. Taken together, the results suggest that the defence to B. sorokiniana largely relies on salicylic acid-responsive pathogenesis-related (PR) genes, as well as selected reactive oxygen species and unknown HCP1-associated factors.

Immunocytochemical studies on Brassica napus (rapeseed) tissues using a monoclonal antibody against myrosinase (thioglucoside glucohydrolase) showed that the enzyme was only present in a small number of cells. In the developing embryo, scattered myrosinase-containing cells were present in both cotyledons and axis. The enzyme accumulated in these cells during the later stages of seed development, approximately from day 20 until day 40 after pollination. Parallel staining with the immunocytochemical technique and a histochemical method identified these cells as myrosin cells. Myrosinase appeared to be located outside the myrosin grains, although the occasional association with the membrane of the grains also was noted. In leaves, petals, and siliques, scattered parenchyma cells were stained in the mesophyll as well as in the vascular tissue. In young leaves, guard cells also contained myrosinase. The enzyme was also present in xylem cells of the stem.

Continuous registration of postoperative wound infections reduces the rate of infection per se at the same time as enabling special risk groups to be identified. By improving or changing hygiene and antibiotic routines, the rate of infection in these especially exposed groups can be reduced. Major postoperative infectious complications prolong postoperative hospital stay by 17 days on average. Reduction of the rate of infections would save hundreds of treatment days.

Acyl modification of the sn-2 position in phospholipids (PLs) was conducted by acidolysis reaction using immobilized phospholipase A(2) (PLA(2)) as the catalyst. In the first stage we screened different carriers for their ability to immobilize PLA(2). Several carriers were able to fix the enzyme and maintain catalytic activity; however, the final choice of carrier for the continued work was a non-ionic weakly polar macroreticular resin. Response surface methodology was applied to evaluate the influence of substrate ratio, reaction temperature, and water addition during acidolysis reaction between caprylic acid and soybean phosphatidylcholine (PC). Reaction temperature and water addition had significant effect on acidolysis reaction, however no effect was observed for substrate ratio (mol caprylic acid/mol PC) in range tested. In general an inverse relationship between incorporation of caprylic acid and PC recovery was observed. Highest incorporation obtained during acidolysis reactions was 36%. Such incorporation could be obtained under reaction temperature, 45 degrees C; substrate ratio, 9mol/mol caprylic acid/PC; water addition of 2%; 30wt.% immobilized enzyme; and reaction time, 48h. The yield under these conditions was however only 29%. Lysophosphatidylcholine (LPC) was the major by-product formed during the reaction. Incorporation of acyl donor into LPC was very low (4%), which indicates that acyl migration is only a minor problem for PLA(2) catalyzed synthesis reaction. Conjugated linoleic acid and docosahexaenoic acid were also tested as acyl donors, and were able to be incorporated into PC with 30 and 20%, respectively.

Phospholipider har stor anvendelse i levnedsmiddel-, kosmetik-, og farmaceutiske produkter for blandt andet deres emulgerende egenskaber samt evne til at danne liposomer. Interessen for at ændre på phospholipidernes struktur er stigende. Strukturændringer resulterer i ændret funktionalitet. Ved udskiftning af fedtsyrerne med andre ikke naturligt forekommende er det muligt at forbedre fysiske og kemiske, ernæringsmæssige, farmaceutiske og medicinske egenskaber. Indtil nu har der været relativt få forsøg på at optimere de funktionelle egenskaber ved at ændre fedtsyresammensætningen. Målet med projektet var at udvikle processer baseret på enzymatisk interesterificering til produktion af phospholipider med specifik fedtsyre profil (strukturerede phospholipider), og opsætning af membrane separationssystemer til oprensning af strukturerede phospholipider efter reaktion. Produktionen af strukturerede phospholipider blev udført i både ”packed bed”- og batchreaktorer med eller uden tilstedeværelse af solvent (organisk opløsningsmiddel). Under den praktiske udførelse af reaktionerne kan dannelsen af biprodukter, grundet sideløbende hydrolyse og acyl migrering, være et alvorligt problem. En grundig belysning af disse problemer og optimering af diverse reaktionssystemer blev udført i dette projekt. Til produktion i større skala er det praktisk at udføre reaktionerne i ”packed bed” reaktorer, da det tillader en kontinuerlig proces. Kontinuerlig proces i ”packed bed” reaktor blev dog vist at være vanskelig med et solvent frit system. En lang reaktionstid kombineret med hurtig deaktivering af enzymet gør processen ufavorabel. Et solvent system virker tilsyneladende til at være et godt valg for reaktionen, da høj inkorporering og udbytte opnås. Til adskillelse af strukturerede phospholipider fra fedtsyrer blev der under dette arbejde udviklet en ”downstream” proces, som involver ultrafiltrering. I apolære solventer har phospholipider tendens til a danne ”reverse micelles”, som kan adskilles fra fedtsyrer og solvent ved anvendelse af passende membraner. Ydermere blev fysiske egenskaber af specifikke strukturerede phospholipider undersøgt i emulsioner og liposom formuleringer. Projektet blev udført af Anders F. Vikbjerg ved BioCentrum-DTU under ledelse af Lektor Xuebing Xu. Andre deltagere af projektet var Huiling Mu (BioCentrum-DTU), Gunnar E. Jonsson (Institut for Kemiteknik, DTU) og LiPlasome Pharma A/S. This project is mainly a study on the enzyme catalyzed production of phospholipids with modified fatty acid profile (structured phospholipids). Besides production of structured phospholipids, membrane purification of structured phospholipids, and properties of selected structured phospholipids in emulsions and liposome formulations were also studied. Replacements of existing fatty acids in natural soybean phospholipids with others not natural occurring, were done by acidolysis using different commercial microbial lipases and porcine pancreatic phospholipase A2 (PLA2). Lipases were used for modification of sn-1 positioned fatty acids of the phospholipids, whereas PLA2 was used for modification of the sn-2 positioned fatty acids. Reactions were performed in both packed-bed and batch reactors with or without the presence of organic solvents. Effects of different reaction parameters, on primary- and side reactions, were examined for various reaction systems. TLC-FID method was developed during this work to assist the evaluation of product and byproduct formations. The incorporation of desired fatty acids into phospholipid and recovery in batch reactors was affected by enzyme load, reaction time, reaction temperature, water content, substrate molar ratio and solvent amount. Influence of temperature and substrate ratio seemed to depend on the particular reaction system. In solvent systems using immobilized Thermomyces lanuginosa lipase incorporation of desired fatty acid increased with increased temperature (35-55°) and substrate molar ratio (3-15 mol/mol), whereas in solvent free system using immobilized Rhizomucor miehei lipase, incorporation decreased with increase of these parameters in similar range. During PLA2 catalyzed acidolysis reaction, substrate ratio showed no effect on incorporation or yield, and maximum incorporation was observed at 45 °C. Individually, water content showed no effect on the incorporation in solvent-free system during lipase-catalyzed reactions; however it had significant effect during reactions involving PLA2. With both types of enzyme, the recovery of diacylphospholipids decreased with increase of watercontent due to parallel hydrolysis. Presence of solvent improves mixing in the system, and makes subsequent removal of enzyme more convenient; however increasing amounts of solvent was shown to reduce recovery of phospholipid more strongly than it increased fatty acid incorporation during batch operation. During lipase-catalyzed acidolysis reaction between phosphatidylcholine (PC) and acyl donor, the formation of glycerophosphorylcholine (GPC), the presence of acyl donor in the intermediate lysophosphatidylcholine (LPC) and migration into the sn-2 position of PC were observed, which are consequences of acyl migration. GPC formation increased especially with increasing water content in the reaction system; whereas incorporation into LPC and migration into sn-2 position increased with reaction time. Acyl migration should be minimized in the reaction system in order to achieve a high product yield and purity. Production of structured phospholipids in packed bed reactors was affected by the same reaction parameters tested during batch operation. Continuous operation in packed bed reactor was very difficult with a solvent free system. A long reaction time combined with rapid deactivation of the enzyme makes the process unfavorable. Solvent system seems to provide good choice for acidolysis reaction, as high incorporation and yields are achieved. Recovery of diacylphospholipids is considerably higher when reactions are performed in packed bed reactors as compared to batch operation. For the separation of structured phospholipids from free fatty acids, a downstream process involving ultrafiltration was developed during this work. In non-polar solvent phospholipids tend to form reverse micelles, which can be separated from free fatty acids and solvent by using appropriate membranes. Different commercial membranes with different cut-off values were screened in dead end operation. Polysulphone membrane with polyester support showed some good qualities in terms of flux and selectivity. Multiple steps with dilution of retentate to minimize the viscosity and fouling were done to improve the separation. Membrane performance was shown to be very dependent on the initial feed concentration, concentration factor in each step and applied pressure. Two individual studies were made to examine the physical and chemical properties of specific structured phospholipids. In the first study, the ability of enzymatically synthesized structured phosphatidylcholine containing caprylic acid to form and stabilize oil-in-water emulsions, prepared with different triacylglycerols, was examined and compared with natural soybean PC and deoiled lecithin. In the other study, oxidative properties of structured phospholipid containing highly unsaturated docosahexaenoic acid were examined in liposome formulations. The two studies demonstrate the potential usages of structured phospholipids, which have properties differing from natural soybean phospholipids. Overall, the study provides detailed information for practical application of enzyme catalyzed acidolysis of structured phospholipids including down stream processing, and property evaluation of specific structured phospholipids.