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Post-esophagectomy chylothorax is a therapeutic dilemma. Understanding anatomy and leakage site is essential, although imaging techniques for thoracic lymphatics are challenging and not always available. We describe a patient with chylothorax post-esophagectomy from a left-sided thoracic duct traversing to the right chest. We applied intranodal Lipiodol® lymphangiography, computed tomographic lymphangiography, and performed fluorescence-guided thoracic duct ligation at the level of the aortic arch. These techniques are not only useful in a postoperative setting but may be used as preventive strategies to reduce morbidity and mortality from chylothorax after any thoracic surgical procedure where the thoracic duct is at risk.

Objectives: To report a case of a stoma reversal in a patient with an acquired, extreme microcolon after a long-standing transversostomy and to give a review of the current literature. Methods: Case report and literature review by performing a PubMed database search, using the keywords Santulli enterostomy, anal atresia, posterior sagittal anorectoplasty and acquired microcolon. Results: An 18-year-old patient with a previous history of anal, rectal and sigmoid atresia, was admitted to our hospital with an acquired microcolon due to a long-standing transversostomy. The patient had a posterior sagittal anorectoplasty at the age of one year, but by reason of an enormous dilatation of the ascending colon and the associated discrepancy of the caliber of the proximal and distal colon, the colostomy was maintained. The patient was lost in follow-up during several years. Now, 16 years later, the patient requested closure of the colostomy. Since barium enemas still showed a dilated colon ascendens and a microcolon descendens, a staged approach was chosen. First, a right hemicolectomy was performed and a Santulli enterostomy was created by constructing an ileocolostomy just proximal of an end ileostomy. Progressively, more transanal bowel movements were seen and barium enemas showed a progressive expansion of the remaining colon and rectum. Test closing of the enterostomy using an inflated bladder catheter did not cause signs of obstruction. During the second stage, 17 months later, at the age of 20 years, the stoma was closed. Now, the patient has two to three solid stools a day, with a complete fecal continence. Discussion: The used technique is well known in pediatric surgery. We successfully implemented it in the treatment of a microcolon in an adult. The advantage is that we can feed the distal colon to achieve expansion, while the enterostomy functions as a venting system, hereby preventing obstructive complaints when the caliber of the colon is still narrow. In conclusion, long-standing colostomas with a concurrent microcolon can be closed but require a step-by-step approach.

Introduction: The incidence of incisional hernias (IHs) after open repair of an abdominal aortic aneurysm (AAA) is high. Several randomized controlled trials have reported favorable results with the use of prophylactic mesh to prevent IHs, without increasing complications. In this analysis, we report on the results of the 60-month follow-up of the PRIMAAT trial. Methods: In a prospective, multicenter, open-label, randomized design, patients were randomized between prophylactic retrorectus mesh reinforcement (mesh group), and primary closure of their midline laparotomy after open AAA repair (no-mesh group). This article reports on the results of clinical follow-up after 60 months. If performed, ultrasonography or computed tomography were used for the diagnosis of IHs. Results: Of the 120 randomized patients, 114 were included in the intention-to-treat analysis. Thirty-three patients in the no-mesh group (33/58-56.9%) and 34 patients in the mesh group (34/56-60.7%) were evaluated after 5 years. In each treatment arm, 10 patients died between the 24-month and 60-month follow-up. The cumulative incidence of IHs in the no-mesh group was 32.9% after 24 months and 49.2% after 60 months. No IHs were diagnosed in the mesh group. In the no-mesh group, 21.7% (5/23) underwent reoperation within 5 years due to an IH. Conclusions: Prophylactic retrorectus mesh reinforcement after midline laparotomy for the treatment of AAAs safely and effectively decreases the rate of IHs. The cumulative incidence of IHs after open AAA repair, when no mesh is used, continues to increase during the first 5 years after surgery, which leads to a substantial rate of hernia repairs.

Purpose Therapy of juvenile neurogenic flatfoot (JNF) with subtalar arthroereisis (SA) is currently under critical clinical investigation. In this retrospective matched pair analysis, the radiological outcome after arthroereisis in paediatric patients with infantile cerebral palsy and JNF was compared with children with juvenile flatfeet (JF) without neurological diseases. Methods From October 2007 to April 2018 80 patients with 149 flatfeet underwent surgery with SA. Inclusion criteria were: 1) JNF or JF with age at surgery ≤ 13 years; 2) treatment with SA; 3) presence of three sets of biplane radiographs (preoperative, postoperative and follow-up (FU)). The radiographs were analyzed for: 1) navicular-cuboidal-index (NCI); 2) talocalcaneal angle anteroposterior; 3) talocalcaneal angle lateral; 4) calcaneal-pitch (CP); and 5) talometatarsal-index (TMTI). Following this, 25 patients with 38 flatfeet could be included. Results The mean age at SA of the JNF group was 9.2 years (JF group: 9.3 years) and the mean time of FU was 35.2 months (JF group: 39.4 months). In comparison with preoperatively, a significant decrease of the NCI was seen in both groups (p = ≤ 0.05 and p = ≤ 0.001) in the FU radiographs. The analysis of CP and TMTI in the JF group also resulted in a significant improvement (p = ≤ 0.001 and p = ≤ 0.05). Overall, the comparison between the JNF and JF group showed no significant differences in regard to the analyzed postoperative parameters. Conclusion Based on this data, treatment of flatfeet by SA in patient with neurological disorders shows an improvement of radiological parameters comparable with neurologically unimpaired patients and might be considered as additional treatment option. Level of evidence IV

The majority of patients with type 2 diabetes (T2DM) achieve remission after bariatric surgery. Several models are available to preoperatively predict T2DM remission. This study compares the performance of these models in a Western population one year after surgery and explores their predictive value in comparison to a model specifically designed for our study population.Prediction models were retrieved using a literature search. Patients were retrospectively selected from a database of the Antwerp University Hospital. Performance of the models was assessed by determining the area under the receiver operating characteristic curve (AUROC), the accuracy, and the goodness of fit, and by comparing them to a custom-made logistic model.The probability of T2DM remission was calculated using 11 predictive scoring models and 8 regression models in a cohort of 250 patients. Complete T2DM remission occurred in 64.0% of patients. The IMS score (AUROC = 0.912; accuracy = 83.6%), DiaBetter score (0.907; 82.0%), and Ad-DiaRem score (0.903; 82.8%) best predicted T2DM remission and closely approached the performance of the custom-constructed model (0.917; 84.0%). The model by Ioffe et al. (0.630; 69.2%), Umemura et al. (0.692; 71.4%), and the ABCD score (0.757; 72.8%) were the least accurate.Most T2DM remission models reliably predicted one-year T2DM remission, with limited inter-model differences. The accuracy of most models approached that of the custom-constructed model, indicating a high predictive capability and performance in our patient cohort. To date, most models are only validated to estimate T2DM remission one year after surgery and they do not predict long-term remission.

Objectives The subtalar screw arthroereisis (SSA) is a treatment option for painful pediatric flexible flatfeet (PFF). Hence, the optimal time point for a SSA is discussed controversially. Therefore the present study evaluates the influence of the patient's age at surgery on the radiological outcome to provide further evidence in this matter. Methods From 08/2007 to 12/2015 50 patients with 95 PFF were included. Inclusion criteria were: 1) Patients with PFF under or equal 15 years of age, 2) treatment with SSA and 3) presence of pre-op, post-op and follow up (FU) routine biplane radiographs. A subdivision was made into group A: 5–8 years, group B: 9–12 years and group C: 13–15 years. The radiographs were analyzed for: 1) calcaneal-pitch (CP), 2) lateral talocalcaneal angle (lat. TCA), 3) a.p. talocalcaneal angle (a.p. TCA, kite angle) and 4) navicular-cuboidal-index (NCI) and meary angle. Results Our study showed the best deformity correction when surgery was conducted between 9 and 12 years of age (group B), with significant improvement in all measured parameters without secondary deterioration during FU. In group A, the SSA show inferior results with poorer long-term success with only an improvement in the a.p. TCA. Group C showed mixed results. While CP and NCI improved, the lat. TCA deteriorated in FU. In conclusion, the ideal age for surgical intervention by SSA is between 9 and 12 years. Surgery before the age of 8 years did not show long-term success and delayed treatment at the age of 13–15 was only partially successful with deterioration during FU period. Level of evidence IV.

Background:The incidence of incisional hernias after abdominal aortic aneurysm repair is high. Prophylactic mesh-augmented reinforcement during laparotomy closure has been proposed in patients at high risk of incisional hernia.Methods:A multicenter randomized trial was conducted on patients undergoing elective abdominal aortic aneurysm repair through a midline laparotomy (Clinical.Trials.gov: NCT00757133). In the study group, retromuscular mesh-augmented reinforcement was performed with a large-pore polypropylene mesh (Ultrapro, width 7.5cm). The primary endpoint was the incidence of incisional hernias at 2-year follow-up.Results:Between February 2009 and January 2013, 120 patients were recruited at 8 Belgian centers. Patients' characteristics at baseline were similar between groups. Operative and postoperative characteristics showed no difference in morbidity or mortality. The cumulative incidence of incisional hernias at 2-year follow-up after conventional closure was 28% (95% confidence interval [CI], 17%-41%) versus 0% (95% CI, 0%-6%) after mesh-augmented reinforcement (P < 0.0001; Fisher exact test). The estimated freedom of incisional hernia curves (Kaplan-Meier estimate) were significantly different across study arms ((2) = 19.5, P < 0.0001; Mantel-Cox test). No adverse effect related to mesh-augmented reinforcement was observed, apart from an increased mean time to close the abdominal wall for mesh-augmented reinforcement compared with the control group: 46 minutes (SD, 18.6) versus 30 minutes (SD, 18.5), respectively (P < 0.001; Mann-Whitney U test).Conclusions:Prophylactic retromuscular mesh-augmented reinforcement of a midline laparotomy in patients with abdominal aortic aneurysm is safe and effectively prevents the development of incisional hernia during 2 years, with an additional mean operative time of 16 minutes.

Der Fall einer 43-jahrigen Patientin mit bewegungsabhangigen rechtsseitigen Schulterschmerzen, ohne Trauma und im Magnetresonanztomogramm (MRT) nachgewiesenem 3 cm grosen Ganglion unmittelbar dorsal des knochernen Glenoids mit Verbindung zum dorsalen apikalen Labrum wird vorgestellt. Nach arthroskopischer Glattung der halftig aufgefaserten langen Bizepssehne, des leicht aufgefaserten apikalen Labrums im Bizepssehnenankerberich und nach partieller Synovektomie erfolgte die offene chirurgische Zystenresektion uber einen longitudinalen dorsalen Zugang. Nach komplikationslosem postoperativem Verlauf blieb die Patientin uber den gesamten Nachuntersuchungszeitraum von 12 Monaten mit freier Schulterfunktion beschwerdefrei. Die Symptomatik, die intraoperativen arthroskopischen Befunde und Therapieverfahren einer spinoglenoidalen Zyste werden nach Literaturrecherche beschrieben und mit dem beschriebenen Fallbericht verglichen und diskutiert.

An 8-month-old female infant presented with a 2-day history of vomiting, diarrhoea and fever. Physical examination revealed a painful right abdomen. Laboratory tests showed a C-reactive protein of 140 mg/L (normal

Secondary limb lengthening after intercalary bone resection in pediatric patients is still challenging. After the resection, a free fibula autograft can be used to reconstruct the osseous gap. However, in particular in young pediatric patients, insufficient growth of the epiphyseal plate after transplantation may lead to a significant limb-length discrepancy (LLD). In this case, the autograft was used for limb lengthening. We report on the lengthening of a humerus regenerate after fibula autograft transplantation into a humeral defect in a pediatric/young adolescent patient. Because of LLD, she underwent callus distraction of the humerus regenerate after transplantation of a fibula autograft using a unilateral external fixator device. An 18-year-old female patient with status postintercalary proliferating chondroma resection (at the age of 7 years) required treatment for correction of a 13 cm humerus shortening. She reported no pain, had no functional limitations, and had a full range of motion of the shoulder and elbow joint. She complained about her short humerus and overall body scheme. A unilateral external fixator device for callus distraction was applied to the transplanted humerus regenerate after free fibula autograft transplantation. Bone lengthening was performed by distraction (2×0.5 mm/day). After 62 days and a lengthening of 6 cm in total, bone distraction was stopped mainly to avoid any complication. At this time, there was no limitation in the range of motion. No nerve palsy or other problems occurred at any time of the distraction. In pediatric patients, the transplantation of a fibula autograft is a well-established option to bridge an osseous gap after intercalary bone resection. This case report shows that even the humerus regenerate after fibula autograft transplantation has excellent potential for callotasis and bone remodeling and therefore bone lengthening in patients with LLD is an option even after transplantation of an autograft. This method provides a new therapeutic option for patients with LLD after fibula transplantation.

OA gold With disease progression, avascular necrosis (AVN) of the femoral head may lead to a collapse of the articular surface. The exact pathophysiology of AVN remains unclear, although several conditions are known that can result in spontaneous cell death, leading to a reduction of trabecular bone and the development of AVN. Hip AVN treatment is stage-dependent in which two main stages of the disease can be distinguished: pre-collapse (ARCO 0-II) and post-collapse stage (ARCO III-IV, crescent sign). In the pre-collapse phase, core decompression (CD), with or without the addition of bone marrow (e.g. bone marrow aspirate concentrate, BMAC) or bone graft, is a common treatment alternative. In the postcollapse phase, THA (total hip arthroplasty) must be performed in most of the patients. In addition to surgical treatment, the intravenous application of Iloprost has been shown to have a curative potential and analgesic effect. From October 2009 to October 2014, 49 patients with AVN (stages I-III) were treated with core decompression at our institution. All patients were divided into group A (CD + BMAC) and group B (CD alone). Of these patients, 20 were included in a matched pair analysis. The patients were matched to age, gender, ARCO-stage, Kerboul combined necrotic angle, the cause of AVN, and whether Iloprost-therapy was performed. The Merle d’Aubigné Score and the Kerboul combined necrotic angle in a-p and lateral radiographs were evaluated pre- and postoperatively. The primary endpoint was a total hip arthroplasty. In group A, two patients needed THA while in group B four patients were treated with THA. In group A, the Merle d’Aubigné Score improved from 13.5 (pre-operatively) to 15.3 (postoperatively). In group B there was no difference between the pre- (14.3) and postoperative (14.1) assessment. The mean of the Kerboul angle showed no difference in both groups compared pre- to postoperatively (group A: pre-op 212°, postop 220°, group B: pre-op 213, postop 222°). Regarding radiographic evaluation, the interobserver variability revealed a moderate agreement between two raters regarding the pre- (ICC 0.594) and postoperative analysis (ICC 0.604).This study demonstrates that CD in combination with the application of autologous bone marrow aspirate concentrate into the femoral head seems to be a safe and efficient treatment alternative in the early stages of AVN of the femoral head when compared to CD alone.

Two similar enzymes with different biosynthetic function in one species have evolved to catalyze two distinct reactions. X-ray structures of both enzymes help reveal their most important differences. The Rauvolfia alkaloid biosynthetic network harbors two O-glucosidases: raucaffricine glucosidase (RG), which hydrolyses raucaffricine to an intermediate downstream in the ajmaline pathway, and strictosidine glucosidase (SG), which operates upstream. RG converts strictosidine, the substrate of SG, but SG does not accept raucaffricine. Now elucidation of crystal structures of RG, inactive RG-E186Q mutant, and its complexes with ligands dihydro-raucaffricine and secologanin reveals that it is the "wider gate" of RG that allows strictosidine to enter the catalytic site, whereas the "slot-like" entrance of SG prohibits access by raucaffricine. Trp392 in RG and Trp388 in SG control the gate shape and acceptance of substrates. Ser390 directs the conformation of Trp392. 3D structures, supported by site-directed mutations and kinetic data of RG and SG, provide a structural and catalytic explanation of substrate specificity and deeper insights into O-glucosidase chemistry.

The paper by Haug et al. presents an overview of copulas and their use for dependence modeling in multivariate risk analysis. Various aspects of rank-based inference are covered, from pointwise and interval estimation to goodness-of-fit testing. The exposition is clearly not comprehensive and many details are glossed over but this is offset by the inclusion of newmaterial, most notably empirical likelihood methods. Readers interested in further details can refer, e.g., to Genest and Favre (2007), where additional graphical tools and inferential procedures are illustrated on a small ‘‘learning data set’’. A distinctive feature of the paper is its emphasis on extreme-value and tail copulas. These concepts are clearly relevant for risk management, as failure to account for dependence between extreme events can have disastrous financial, economic or environmental consequences; see, e.g., Chavez-Demoulin and Embrechts (2010). There is a pressing need for inference techniques tailored to extreme dependence models and the area is currently undergoing rapid development. To attempt a survey at this early stage was clearly a challenge, and the authors lived up to it. Relevant additional contributions of recent vintage include new goodness-of-fit tests for extreme-value copulas by Kojadinovic and Yan (2010) and Quessy (in press), and alternative estimators of the Pickands dependence function due to Bucher, Dette, and Volgushev (2010), Guillotte and Perron (2008), and Guillotte, Perron, and Segers (in press). In this discussion, wewould like to complement thework of Haug et al. by highlighting some of the difficulties associated with the occurrence of ties in rank-based inference for copulamodels. As illustrated in Section 2, this phenomenon is present in the Danish fire insurance data set and cannot be ignored. Unfortunately, the validity of rank-based procedures is often compromised when the margins are discontinuous (Genest & Neslehova, 2007). The Danish data are used in Sections 3 and 4 to illustrate the impact of ties on testing for extremeness and making inference for extreme-value copulas, respectively. While the problem is easily recognized, solutions are hard to come by. Some of the methodological challenges at hand are sketched in Section 5, and concluding remarks are made in Section 6.

A dual vector (pQE-70-STR1-SG) containing coding regions of strictosidine synthase (STR1, EC 4.3.3.2) and strictosidine glucosidase (SG, EC 3.2.1.105) from the Indian medicinal plant Rauvolfia serpentina was constructed. Functional expression of the vector in Escherichia coli cells (M15 strain) was proven by isolation of prepurified enzyme extracts, which show both STR1 and SG activities. Incubation of the enzyme in the presence of tryptamine and secologanin delivered the indole alkaloid cathenamine, demonstrating functional co-expression of both STR1- and SG-cDNAs. Cathenamine reduction by sodium borohydride leading to tetrahydroalstonine revealed the chemo-enzymatic indole alkaloid synthesis.

Strictosidine synthase (STR1) catalyzes the stereoselective formation of 3alpha(S)-strictosidine from tryptamine and secologanin. Strictosidine is the key intermediate in the biosynthesis of 2,000 plant monoterpenoid indole alkaloids, and it is a key precursor of enzyme-mediated synthesis of alkaloids. An improved expression system is described which leads to optimized His(6)-STR1 synthesis in Escherichia coli. Optimal production of STR1 was achieved by determining the impact of co-expression of chaperones pG-Tf2 and pG-LJE8. The amount and activity of STR1 was doubled in the presence of chaperone pG-Tf2 alone. His(6)-STR1 immobilized on Ni-NTA can be used for enzymatic synthesis of strictosidines on a preparative scale. With the newly co-expressed His(6)-STR1, novel 3alpha(S)-12-azastrictosidine was obtained by enzymatic catalysis of 7-azatryptamine and secologanin. The results obtained are of significant importance for application to chemo-enzymatic approaches leading to diversification of alkaloids with novel improved structures.

Perakine reductase (PR) catalyzes an NADPH-dependent step in a side-branch of the 10-step biosynthetic pathway of the alkaloid ajmaline. The enzyme was cloned by a "reverse-genetic" approach from cell suspension cultures of the plant Rauvolfia serpentina (Apocynaceae) and functionally expressed in Escherichia coli as the N-terminal His(6)-tagged protein. PR displays a broad substrate acceptance, converting 16 out of 28 tested compounds with reducible carbonyl function which belong to three substrate groups: benzaldehyde, cinnamic aldehyde derivatives and monoterpenoid indole alkaloids. The enzyme has an extraordinary selectivity in the group of alkaloids. Sequence alignments define PR as a new member of the aldo-keto reductase (AKR) super family, exhibiting the conserved catalytic tetrad Asp52, Tyr57, Lys84, His126. Site-directed mutagenesis of each of these functional residues to an alanine residue results in97.8% loss of enzyme activity, in compounds of each substrate group. PR represents the first example of the large AKR-family which is involved in the biosynthesis of plant monoterpenoid indole alkaloids. In addition to a new esterase, PR significantly extends the Rauvolfia alkaloid network to the novel group of peraksine alkaloids.

The biosynthetic pathway leading to the monoterpenoid indole alkaloid ajmaline in Rauvolfia serpentiin serpentina is one of the most studied in the field of natural product biosynthesis. Ajmaline has a complex structure which is based on a six-membered ring system harbouring nine chiral carbon atoms. There are about fifteen enzymes involved, including some involving the side reactions of the ajmaline biosynthetic pathway. All enzymes exhibit pronounced substrate specificity. In the recent years isolation and sequencing of their cDNAs has allowed a detailed sequence analysis and comparison with functionally related and occasionally un-related enzymes. Site-directed mutations of several of the ajmaline-synthesizing enzymes have been performed and their catalytic residues have been identified. Success with over-expression of the enzymes was an important step for their crystallization and structural analysis by X-ray crystallography. Crystals with sufficient resolution were obtained from the major enzymes of the pathway. Strictosidine synthase has a 3D-structure with a six-bladed β-propeller fold the first time such a fold found in the plant kingdom. Its ligand complexes with tryptamine and secologanin, as well as structure-based sequence alignment, indicate a possible evolutionary relationship to several primary sequence-unrelated structures with this fold. The structure of strictosidine glucosidase was determined and its structure has as a (β/α)8 barrel fold. Vinorine synthase provides the first 3D structure of a member of BAHD enzyme super-family. Raucaffricine glucosidase involved in a side-route of ajmaline biosynthesis has been crystallized. The ajmaline biosynthetic pathway is an outstanding example where many enzymes 3D-structure have been known and where there is a real potential for protein engineering to yield new alkaloid.

Perakine reductase (PR) is a novel member of the aldo-keto reductase enzyme superfamily from higher plants. PR from the plant Rauvolfia serpentina is involved in the biosynthesis of monoterpenoid indole alkaloids by performing NADPH-dependent reduction of perakine, yielding raucaffrinoline. However, PR can also reduce cinnamic aldehyde and some of its derivatives. After heterologous expression of a triple mutant of PR in Escherichia coli, crystals of the purified and methylated enzyme were obtained by the hanging-drop vapour-diffusion technique at 293 K with 100 mM sodium citrate pH 5.6 and 27% PEG 4000 as precipitant. Crystals belong to space group C222(1) and diffract to 2.0 A, with unit-cell parameters a = 58.9, b = 93.0, c = 143.4 A.

Acetylajmalan esterase (AAE) plays an essential role in the late stage of ajmaline biosynthesis. Based on the partial peptide sequences of AAE isolated and purified from Rauvolfia cell suspensions, a full-length AAE cDNA clone was isolated. The amino acid sequence of AAE has the highest level of identity of 40% to putative lipases known from the Arabidopsis thaliana genome project. Based on the primary structure AAE is a new member of the GDSL lipase superfamily. The expression in Escherichia coli failed although a wide range of conditions were tested. With a novel virus-based plant expression system, it was possible to express AAE functionally in leaves of Nicotiana benthamiana Domin. An extraordinarily high enzyme activity was detected in the Nicotiana tissue, which exceeded that in Rauvolfia serpentina (L.) Benth. ex Kurz cell suspension cultures about 20-fold. This expression allowed molecular analysis of AAE for the first time and increased the number of functionally expressed alkaloid genes from Rauvolfia now to eight, and the number of ajmaline pathway-specific cDNAs to a total of six.

Microsomal preparations from cell suspension cultures of the Indian plant Rauvolfia serpentina catalyze the hydroxylation of deoxysarpagine under formation of sarpagine. The newly discovered enzyme is dependent on NADPH and oxygen. It can be inhibited by typical cytochrome P450 inhibitors such as cytochrome c, ketoconazole, metyrapone, tetcyclacis and carbon monoxide. The CO-effect is reversible with light (450 nm). The data indicate that deoxysarpagine hydroxylase is a novel cytochrome P450-dependent monooxygenase. A pH optimum of 8.0 and a temperature optimum of 35 degrees C were determined. K(m) values were 25 microM for NADPH and 7.4 microM for deoxysarpagine. Deoxysarpagine hydroxylase activity was stable in presence of 20% sucrose at -25 degrees C for3 months. The analysis of presence of the hydroxylase in nine cell cultures of seven different families indicates a very limited taxonomic distribution of this enzyme.

X-ray measurements at room temperature (295 K) deliver high quality data sets with unprecedented speed (2 min), as shown for crystallized raucaffricine-O-β-D-glucosidase (RG), its mutant RG-Glu186Gln and several ligand complexes of the enzyme which participates in alkaloid biosynthesis in the plant Rauvolfia. The data obtained are compared with data sets measured under typical cryo conditions (100K). Under both conditions, density maps are highly comparable and favor the described protocol for room temperature measurements, potentially paving the way for future crystallographic studies capturing biosynthetic pathway intermediates.

The multi-step enzyme catalysed biosyntheses of monoterpenoid indole and isoquinoline alkaloids are described. Special emphasis is placed on those pathways leading to alkaloids of pharmacological and medicinal significance which have been fully elucidated at the enzyme level. The successful identification and cloning of cDNAs of single enzymes and their application provides great opportunities to develop novel strategies for both in vitro and in vivo alkaloid production in whole plants or tissue cultures, as well as in microbial systems such as Escherichia coli and yeast. Enzyme crystallisation, 3D analyses and site-directed mutation allowed rational engineering of enzyme substrate acceptance, which in turn can be used for reprogramming in vivo alkaloid biosynthesis and for the design of biomimetic alkaloid syntheses. These strategies broaden structural diversity and allow the creation of large libraries of unnatural alkaloid with expected optimised or novel biological activities. The chemo-enzymatic syntheses of the above-mentioned alkaloid groups and their precursors (in addition to selected examples of other alkaloid families) provides an overview of how enzyme reactions are integrated into the development of total chemical syntheses.

This chapter constitutes a survey on copula-based measures of multivariate association - i.e. association in a d-dimensional random vector \(X = (X_1 , \ldots ,X_d )\) where \(d \ge 2\). Some of the measures discussed are multivariate extensions of wellknown bivariate measures such as Spearman’s rho, Kendall’s tau, Blomqvist’s beta or Gini’s gamma. Others rely on information theory or are based on L p-distances of copulas. Various measures of multivariate tail dependence are derived by extending the coefficient of bivariate tail dependence. Nonparametric estimation of these measures based on the empirical copula is further addressed.

The multi-step enzyme catalysed biosyntheses of monoterpenoid indole and isoquinoline alkaloids are described. Special emphasis is placed on those pathways leading to alkaloids of pharmacological and medicinal significance which have been fully elucidated at the enzyme level. The successful identification and cloning of cDNAs of single enzymes and their application provides great opportunities to develop novel strategies for both in vitro and in vivo alkaloid production in whole plants or tissue cultures, as well as in microbial systems such as Escherichia coli and yeast. Enzyme crystallisation, 3D analyses and site-directed mutation allowed rational engineering of enzyme substrate acceptance, which in turn can be used for re-programming in vivo alkaloid biosynthesis and for the design of biomimetic alkaloid syntheses. These strategies broaden structural diversity and allow the creation of large libraries of unnatural alkaloid with expected optimised or novel biological activities. The chemo-enzymatic syntheses of the above-mentioned alkaloid groups and their precursors (in addition to selected examples of other alkaloid families) provides an overview of how enzyme reactions are integrated into the development of total chemical syntheses.

Strictosidine β-d-glucosidase (SG) follows strictosidine synthase (STR1) in the production of the reactive intermediate required for the formation of the large family of monoterpenoid indole alkaloids in plants. This family is composed of ∼2000 structurally diverse compounds. SG plays an important role in the plant cell by activating the glucoside strictosidine and allowing it to enter the multiple indole alkaloid pathways. Here, we report detailed three-dimensional information describing both native SG and the complex of its inactive mutant Glu207Gln with the substrate strictosidine, thus providing a structural characterization of substrate binding and identifying the amino acids that occupy the active site surface of the enzyme. Structural analysis and site-directed mutagenesis experiments demonstrate the essential role of Glu-207, Glu-416, His-161, and Trp-388 in catalysis. Comparison of the catalytic pocket of SG with that of other plant glucosidases demonstrates the structural importance of Trp-388. Compared with all other glucosidases of plant, bacterial, and archaeal origin, SG's residue Trp-388 is present in a unique structural conformation that is specific to the SG enzyme. In addition to STR1 and vinorine synthase, SG represents the third structural example of enzymes participating in the biosynthetic pathway of the Rauvolfia alkaloid ajmaline. The data presented here will contribute to deciphering the structure and reaction mechanism of other higher plant glucosidases.

SummaryThe highly substrate-specific strictosidine synthase (EC 4.3.3.2) catalyzes the biological Pictet-Spengler condensation between tryptamine and secologanin, leading to the synthesis of about 2000 monoterpenoid indole alkaloids in higher plants. The crystal structure of Rauvolfia serpentina strictosidine synthase (STR1) in complex with strictosidine has been elucidated here, allowing the rational site-directed mutation of the active center of STR1 and resulting in modulation of its substrate acceptance. Here, we report on the rational redesign of STR1 by generation of a Val208Ala mutant, further describing the influence on substrate acceptance and the enzyme-catalyzed synthesis of 10-methyl- and 10-methoxystrictosidines. Based on the addition of strictosidine to a crude strictosidine glucosidase preparation from Catharanthus cells, a combined chemoenzymatic approach to generating large alkaloid libraries for future pharmacological screenings is presented.

Raucaffricine glucosidase (RG) is an enzyme that is specifically involved in the biosynthesis of indole alkaloids from the plant Rauvolfia serpentina. After heterologous expression in Escherichia coli cells, crystals of RG were obtained by the hanging-drop vapour-diffusion technique at 293 K with 0.3 M ammonium sulfate, 0.1 M sodium acetate pH 4.6 buffer and 11% PEG 4000 as precipitant. Crystals belong to space group I222 and diffract to 2.30 A, with unit-cell parameters a = 102.8, b = 127.3, c = 215.8 A.

Fusobacterium nucleatum is one of the most frequently cultured bacteria from subgingival plaque. It can be isolated from active periodontal lesions as well as from healthy sites. Currently, F. nucleatum has been divided into five subspecies. An early hypothesis suggested an association between certain subspecies and periodontal disease or health. On the other hand, a broad heterogeneity between F. nucleatum isolates has been suggested, so that the subspeciation scheme and therefore the proposed association may not be valid. The purpose of the present study was to analyze if clonal differences exist between human F. nucleatum isolates from either periodontally healthy or diseased individuals. 23 F. nucleatum isolates from periodontally healthy and 17 isolates from periodontally diseased individuals were analysed by ribotyping. A broad genetic heterogeneity was present, although some of the isolates from periodontally healthy individuals were genotypically identical. However, no clonal differences between isolates from periodontally healthy or diseased individuals was detected.

Poor metabolic control of diabetes mellitus has often been associated with the severity of periodontal disease. This case report describes the diagnosis, therapeutic concept, treatment steps, and long-term monitoring of a 22-year-old female with generalized aggressive periodontitis who had a history of type 1 brittle diabetes mellitus. The main objective was to treat the patient with periodontal surgery despite the difficult baseline situation to avoid tooth loss that would have been accompanied by a need for prosthodontic treatment. The mean of glycosylated hemoglobin [figure: see text] measurement (HbA1c) was used to indicate the long-term control of diabetes mellitus. This report proves the efficiency of periodontal therapy in the prevention of future periodontal breakdown and improvement of the HbA1c level in a systemically compromised patient.

Hand instruments lead to application of excessive forces, resulting in extensive root substance removal. Ultrasonic debridement primarily removes accretions from root surfaces. However, there is a lack of information on procedural aspects of ultrasonic debridement. The purpose of the present study was to assess the forces applied by different therapists during ultrasonic root surface debridement of bicuspids in periodontal patients in vivo.Ten dentists and 10 dental hygienists each debrided 1 aspect of a bicuspid with moderate chronic periodontitis using an ultrasonic curet. The forces applied were recorded using a piezo-electric receiver built into the handle of the ultrasonic curet, an electronic transducer, and an analogous writer. The force recordings were converted from millivolts into Newtons (N). Mean positive and negative forces were calculated for each therapist, and t tests were used to assess differences in these forces between dentists and dental hygienists.The results showed that the mean positive force applied by dentists was 1.00 N (range 0.53 to 1.34 N). The corresponding mean value for dental hygienists was 0.77 N (range 0.20 to 1.22 N). The mean root debridement time was 110.2 seconds (range 38 to 165 seconds) for dentists and 90.8 seconds (range 38 to 244 seconds) for dental hygienists. The mean percentage of total debridement time used for positive force application by dentists was 76.1%, and for dental hygienists 63.9%. Negative forces impacting on the periodontal soft tissues were also recorded. These negative forces reached a mean of 0.16 N for dentists and 0.31 N for dental hygienists.From these observations, it may be concluded that application of ultrasonic debridement therapy is highly subjective, and that dentists and dental hygienists may not be adequately trained to use these instruments optimally.

A multivariate measure of association is proposed, which extends the bivariate copula-based measure Phi-Square introduced by Hoeffding [22]. We discuss its analytical properties and calculate its explicit value for some copulas of simple form; a simulation procedure to approximate its value is provided otherwise. A nonparametric estimator for multivariate Phi-Square is derived and its asymptotic behavior is established based on the weak convergence of the empirical copula process both in the case of independent observations and dependent observations from strictly stationary strong mixing sequences. The asymptotic variance of the estimator can be estimated by means of nonparametric bootstrap methods. For illustration, the theoretical results are applied to financial asset return data.

In diesem Aufsatz wird ein stochastisches Optimierungsverfahren vorgestellt, das die Losung beliebiger Optimierungsprobleme ohne Kenntnis des Gradienten der Gutefunktion erlaubt. Das Verfahren ist eine Weiterentwicklung der mehrgliedrigen Evolutionsstrategie dahingehend, das die implizit in der Objektmenge enthaltene Gradienteninformation ausgenutzt wird. Anhand von Testrechnungen wird nachgewiesen, das die Konvergenzsicherheit sehr hoch ist.