Erythrocytapheresis in Children and Young Adults with Hemoglobinopathies and Iron Overload in Need of Iron Chelation Therapy.

Limited data regarding erythrocytapheresis in children, adolescents, and young adults have been published. The aim of this study was to evaluate erythrocytapheresis, either as a standalone therapy or in combination with iron chelation therapy, in children and young adults with hemoglobinopathies in whom current iron chelation therapy is not sufficient in decreasing the iron overload during management. We retrospectively analysed erythrocytapheresis in 19 patients with hemoglobinopathies in need of iron chelation therapy diagnosed with sickle cell disease (SCD) or ß-thalassemia major. Patients were divided into (1) a case cohort who received erythrocytapheresis alone or in combination with iron chelation therapy and (2) a control cohort who received oral iron chelation therapy alone. Serum ferritin and haemoglobin levels were compared at five different time points over a one-year period. In the erythrocytapheresis cohort, there was a significant decrease in serum ferritin (p < 0.001). In the iron chelation therapy alone cohort, there was no significant decrease in serum ferritin over time (p = 0.156). Comparing the evolution of median serum ferritin between therapy with erythrocytapheresis and iron chelation therapy showed a statistically significant difference (p = 0.008). Patients with ß-thalassemia major receiving erythrocytapheresis showed a greater reduction in serum ferritin compared to patients without (p = 0.036). A difference could not be shown between the erythrocytapheresis and iron chelation single therapies (p = 0.100). This study showed an overall significant reduction in serum ferritin in patients with hemoglobinopathies treated with erythrocytapheresis in addition to iron chelation. A clinical, although not statistical, trend of higher haemoglobin levels was maintained. Erythrocytapheresis in paediatric patients with ß-thalassemia major was as effective in decreasing ferritin levels as in previously reported studies with SCD. Erythrocytapheresis is a promising therapy for treating and preventing transfusion-related iron overload.

Influence of a Calcium Phosphate Coating (BONIT®) on the Proliferation and Differentiation Potential of Human Mesenchymal Stroma Cells in the Early Phase of Bone Healing.

When implanting osteosynthetic materials or orthopedic implants, the surface condition plays a decisive role for mid- to long-term osseointegration. BONIT®, an electrochemically produced calcium phosphate (CaP) coating, has been used in the surface refinement of implants since 1995. More than 3.5 million coated implants have been successfully placed so far. BONIT® has thus been able to demonstrate clinical success. However, due to its surface properties and solubility, and the resulting difficulty in culturing cells, there are no in vitro studies investigating its influence at the molecular level, particularly on bone metabolism. In a first step, the cells from a total of ten donors were seeded separately on four different surfaces: 1. a pure corundum-blasted titanium surface (CELLTex®, CT), 2. CT with additional BONIT® coating (CT + B), 3. a hydroxyapatite-blasted titanium surface (DUOTex®, DT), 4. DT with additional BONIT® coating (DT + B). In a second step, the cells were grown for 48 h. The proliferation behavior and differentiation potential of hMSCs were investigated at three consecutive time points (12 h, 24 h and 48 h) by quantifying the mRNA expression of ten important differentiation markers using quantitative real-time polymerase chain reaction (qRT-PCR). We were able to show that BONIT® has an influence on the early proliferation and differentiation behavior of hMSCs in patients of all age groups. The additional BONIT® coating on CELLTex® or DUOTex® led to a defined mRNA expression pattern for the investigated factors: a tendency towards a higher expression rate with coating present could be found for bone morphogenetic protein 2 (BMP2), osteopontin (OPN), osteocalcin (OC), receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG). A similar or lower expression rate was detected for runt-related transcription factor 2 (RUNX2), alpha-1 type I collagen (COL1A1), alkaline phosphatase (AP), osteonectin (ON) and insulin-like growth factor I (IGF1). We have developed a new method that allows the cultivation of human mesenchymal stromal cells (hMSCs) on the soluble coating BONIT® for gene expression analysis. BONIT® has a significant influence on the proliferation and differentiation behavior of human mesenchymal stroma cells. This study describes a defined gene expression pattern of bone metabolism that may help to understand the influence of this CaP coating on the early phase of implant osseointegration.

Prevention of Incisional Hernias by Prophylactic Mesh-augmented Reinforcement of Midline Laparotomies for Abdominal Aortic Aneurysm Treatment: Five-year Follow-up of a Randomized Controlled Trial.

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.

Fluorescence-Guided Thoracic Duct Dissection in Robotic en Bloc Esophagectomy.

En bloc resection of the thoracic duct compartment enhances adequate lymph node removal and may improve oncologic outcomes in esophagectomy for malignant esophageal diseases. However, it also increases the risk of postoperative chylothorax, with a reported incidence of 5% to 20%. This report describes a technique that facilitates intraoperative identification of the thoracic duct, as well as proximal and distal ligation, during robot-assisted esophagectomy by lymphangiography-guided injection of indocyanine green in the right groin in a patient in the left lateral position. This approach can be swiftly applied at any time during any thoracoscopic procedure using the lateral position when visualization of the thoracic duct anatomy is needed.

Review, Performance Comparison, and Validation of Models Predicting Type 2 Diabetes Remission After Bariatric Surgery in a Western European Population.

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Outcome after subtalar screw arthroereisis in children with flexible flatfoot depends on time of treatment: Midterm results of 95 cases.

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.

Postoperative continuation of antidepressant therapy is associated with reduced short-term weight loss following Roux-en-Y gastric bypass surgery.

PURPOSE: Bariatric surgery candidates are frequently treated with antidepressants. Several of these drugs have been associated with weight gain and could potentially interfere with weight loss after bariatric surgery. This cohort study aimed to investigate the short-term effects of antidepressants on weight loss during the first 24 months after a Roux-en-Y gastric bypass. METHODS: Patients with a fully documented antidepressant treatment status for at least 12 months were retrospectively included. Weight loss was expressed as the percentage excess BMI loss (%EBMIL) or percentage total weight loss (%TWL). A mixed linear effects model was used to determine the impact of continued and discontinued treatment with antidepressants on weight loss. RESULTS: A total of 751 patients were included in this study. At 24 months, patients had lost 77.38 ± 30.10 %EBMIL (30.63 ± 13.12 %TWL). In patients treated with antidepressants (n = 125), the %EBMIL and %TWL was reduced with - 2.81% (p = 0.025) and - 1.36% (p = 0.002) respectively, and with - 5.52 %EBMIL (p < 0.001; - 1.05 %TWL, p = 0.012) after multivariate adjustment. Serotonin-norepinephrine reuptake inhibitors (- 12.47 %EBMIL, p < 0.001) and tricyclic antidepressants (- 11.01 %EBMIL, p = 0.042) were predominantly responsible for worse outcomes. Beyond 24 months, at 36 months (- 4.83%, p < 0.001) and 48 months (- 3.54%, p = 0.006), the %EBMIL was still reduced. No significant effects of antidepressants on metabolic outcomes after surgery were observed. CONCLUSIONS: Treatment with antidepressants was associated with reduced weight loss after gastric bypass surgery, but only if treatment was continued for at least 1 year postoperatively. Mainly tricyclic antidepressants and serotonin-norepinephrine reuptake inhibitors were responsible for this reduction in weight loss.

Cronkhite-Canada syndrome causing pouch outlet obstruction 5 years after roux-en-y gastric bypass.

OBJECTIVE: We present the case of a 57-year-old man with a history of Roux-en-Y gastric bypass (RYGB) and colonic polyps who presented with an upper gastrointestinal obstruction based on massive stomach polyposis in the pouch. METHODS: Two months prior to this acute admission, he had undergone resection of the gastric remnant due to massive refractory intraluminal bleeding from a polypoid mass. Ten years earlier, right colectomy was performed due to hypertrophic polyposis unsuitable for endoscopic polypectomy. Upper gastrointestinal endoscopy showed a polypoid mass in the pouch causing obstruction. Benign biopsies were obtained. A resection of the stomach pouch with esophagojejunostomy was performed. Macroscopic evaluation of the pouch lumen showed massive polyposis with a sharp demarcation near the Z-line and at the gastrojejunostomy. On clinical examination, the presence of atrophic nail changes, alopecia, and palmar hyperpigmentation was noticed. RESULTS: Postoperative course was uneventful and feeding was restarted successfully. Histological analysis revealed hyperplastic polypoid tissue, which resembled the polyps in the stomach remnant and colon. Together with the ectodermal changes, the diagnose of Cronkhite-Canada syndrome was established. CONCLUSION: Diffuse polyposis in Cronkhite-Canada syndrome is a rare cause for pouch obstruction after RYGB. Clinical examination should focus on dermatologic findings.

Lengthening of newly formed humerus after autologous fibula graft transplantation following intercalary tumor resection.

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.

5-Year survival after splenectomy in a patient with recurrent cutaneous melanoma: a case report.

OBJECTIVE: Splenic metastases of melanoma are uncommon and generally part of a disseminated disease with a poor prognosis. In case of solitary visceral metastasis, surgical resection often remains an effective treatment. This case describes a long-term survival after splenectomy for a recurrent melanoma. METHODS: We present a 55-year-old female with a solitary splenic metastasis. Her medical history revealed a melanoma of the foot in 2007 and an in-transit metastasis in 2011. A PET-CT revealed in 2012 the presence of a small aspecific lesion in the spleen. Close follow-up was chosen. Since PET-CT and MRI showed a progressive lesion, a laparoscopic splenectomy was performed. RESULTS: Histological report confirmed the diagnosis of a metastatic lesion. Sixty months after the first radiographic evidence of the splenic metastasis, follow-up revealed no signs of residual disease. CONCLUSIONS: Although current systemic approaches demonstrate to provide clinically important benefit, patients with oligometastatic disease should be evaluated for surgical metastasectomy. A laparoscopic approach for splenic metastasis is shown to be a safe treatment. This disease-free survival of 60 months in a patient with a history of an in-transit metastasis has not been published before. A splenectomy for a solitary metastasis could prolong the survival, even in a recurrent melanoma.

Screening for rare variants in the PNPLA3 gene in obese liver biopsy patients.

BACKGROUND: Previous research has clearly implicated the PNPLA3 gene in the etiology of nonalcoholic fatty liver disease as a polymorphism in the gene was found to be robustly associated to the disease. However, data on the involvement of rare PNPLA3 variants in the development of nonalcoholic fatty liver disease (NAFLD) is currently limited. Therefore, we performed an extensive mutation analysis study on a cohort of obese liver biopsy patients to determine PNPLA3 variation and its correlation with fatty liver disease. METHODS: We screened the entire coding region of the PNPLA3 gene in DNA samples of 393 obese liver biopsy patients with varying degrees of fatty liver disease. Mutation analysis was performed by high-resolution melting curve analysis in combination with direct sequencing. RESULTS: We identified several common polymorphisms as well as one rare synonymous variant (c.867G>A rs139896256), one rare intronic variant (c.979+13C>T) and 3 nonsynonymous coding variants (p.A76T, p.A104V and p.T200M) in the PNPLA3 gene. In silico analysis indicated that the p.A104V variant will probably have no functional effect, whereas for the p.A76T and p.T200M variant a possible pathogenic effect is suggested. CONCLUSION: Overall, we showed that novel variants in PNPLA3 are very rare in our liver biopsy cohort, thereby indicating that their impact on the etiology of NAFLD is probably limited. Nevertheless, for the three rare coding variants that were identified in patients with advanced liver disease, further functional characterization will be essential to verify their potential disease causality.

Prevention of Incisional Hernias by Prophylactic Mesh-augmented Reinforcement of Midline Laparotomies for Abdominal Aortic Aneurysm Treatment: A Randomized Controlled Trial.

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 7.5 cm). 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 (χ = 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.

Association study of PNPLA2 gene with histological parameters of NAFLD in an obese population.

INTRODUCTION: The prevalence of non-alcoholic fatty liver disease (NAFLD) and the closely associated metabolic syndrome is high and is related to risk factors such as obesity and type 2 diabetes. A genetic basis for NAFLD has been suggested, but only few causal genes have been identified. The most significant association reported to date is the robust association of the PNPLA3 I148M variant with susceptibility to NAFLD. We therefore hypothesized that the PNPLA2 gene might also be involved in NAFLD pathogenesis, because of its close sequence similarity with PNPLA3 and its possible involvement in ectopic fat accumulation. METHODS: In this study, we investigated the association of PNPLA2 polymorphisms with the development of non-alcoholic fatty liver disease in a prospectively recruited Belgian obese population comprising 633 individuals with varying degrees of fatty liver disease. We selected 3 PNPLA2 SNPs for genotyping, including 2 tagSNPs that cover most information on common genetic variation in the selected region. RESULTS: After performing linear regression analysis, we found that 2 of the analyzed PNPLA2 SNPs were associated with anthropometric and metabolic parameters. In our subcohort of patients that underwent liver biopsy (n=372/633 or 58.7%), we assessed the influence of the PNPLA2 variants on the severity of histologically determined liver damage, but we did not find convincing evidence for association. CONCLUSION: Although we found evidence for moderate association between PNPLA2 tagSNPs and anthropometric and metabolic parameters in our cohort, no evidence for association between polymorphisms in the PNPLA2 gene and the presence and severity of NAFLD was identified.

Bone Marrow Aspirate Concentrate in Combination With Intravenous Iloprost Increases Bone Healing in Patients With Avascular Necrosis of the Femoral Head: A Matched Pair Analysis.

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 post-collapse 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.

Ligand structures of synthetic deoxa-pyranosylamines with raucaffricine and strictosidine glucosidases provide structural insights into their binding and inhibitory behaviours.

Insight into the structure and inhibition mechanism of O-ß-d-glucosidases by deoxa-pyranosylamine type inhibitors is provided by X-ray analysis of complexes between raucaffricine and strictosidine glucosidases and N-(cyclohexylmethyl)-, N-(cyclohexyl)- and N-(bromobenzyl)-ß-d-gluco-1,5-deoxa-pyranosylamine. All inhibitors anchored exclusively in the catalytic active site by competition with appropriate enzyme substrates. Thus facilitated prospective elucidation of the binding networks with residues located at <3.9 Å distance will enable the development of potent inhibitors suitable for the production of valuable alkaloid glucosides, raucaffricine and strictosidine, by means of synthesis in Rauvolfia serpentina cell suspension cultures.

High speed X-ray analysis of plant enzymes at room temperature.

X-ray measurements at room temperature (295 K) deliver high quality data sets with unprecedented speed (<2 min), as shown for crystallized raucaffricine-O-ß-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.

Structures of alkaloid biosynthetic glucosidases decode substrate specificity.

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.