InhA, the enoyl-thioester reductase from Mycobacterium tuberculosis forms a covalent adduct during catalysis.

The enoyl-thioester reductase InhA catalyzes an essential step in fatty acid biosynthesis of Mycobacterium tuberculosis and is a key target of antituberculosis drugs to combat multidrug-resistant M. tuberculosis strains. This has prompted intense interest in the mechanism and intermediates of the InhA reaction. Here, using enzyme mutagenesis, NMR, stopped-flow spectroscopy, and LC-MS, we found that the NADH cofactor and the CoA thioester substrate form a covalent adduct during the InhA catalytic cycle. We used the isolated adduct as a molecular probe to directly access the second half-reaction of the catalytic cycle of InhA (i.e. the proton transfer), independently of the first half-reaction (i.e. the initial hydride transfer) and to assign functions to two conserved active-site residues, Tyr-158 and Thr-196. We found that Tyr-158 is required for the stereospecificity of protonation and that Thr-196 is partially involved in hydride transfer and protonation. The natural tendency of InhA to form a covalent C2-ene adduct calls for a careful reconsideration of the enzyme's reaction mechanism. It also provides the basis for the development of effective tools to study, manipulate, and inhibit the catalytic cycle of InhA and related enzymes of the short-chain dehydrogenase/reductase (SDR) superfamily. In summary, our work has uncovered the formation of a covalent adduct during the InhA catalytic cycle and identified critical residues required for catalysis, providing further insights into the InhA reaction mechanism important for the development of antituberculosis drugs.

A conserved threonine prevents self-intoxication of enoyl-thioester reductases.

Enzymes are highly specific biocatalysts, yet they can promote unwanted side reactions. Here we investigated the factors that direct catalysis in the enoyl-thioester reductase Etr1p. We show that a single conserved threonine is essential to suppress the formation of a side product that would otherwise act as a high-affinity inhibitor of the enzyme. Substitution of this threonine with isosteric valine increases side-product formation by more than six orders of magnitude, while decreasing turnover frequency by only one order of magnitude. Our results show that the promotion of wanted reactions and the suppression of unwanted side reactions operate independently at the active site of Etr1p, and that the active suppression of side reactions is highly conserved in the family of medium-chain dehydrogenases/reductases (MDRs). Our discovery emphasizes the fact that the active destabilization of competing transition states is an important factor during catalysis that has implications for the understanding and the de novo design of enzymes.

The use of ene adducts to study and engineer enoyl-thioester reductases.

An improved understanding of enzymes' catalytic proficiency and stereoselectivity would further enable applications in chemistry, biocatalysis and industrial biotechnology. We use a chemical probe to dissect individual catalytic steps of enoyl-thioester reductases (Etrs), validating an active site tyrosine as the cryptic proton donor and explaining how it had eluded definitive identification. This information enabled the rational redesign of Etr, yielding mutants that create products with inverted stereochemistry at wild type-like turnover frequency.

Patient Blood Management is Associated With a Substantial Reduction of Red Blood Cell Utilization and Safe for Patient's Outcome: A Prospective, Multicenter Cohort Study With a Noninferiority Design.

OBJECTIVE: To determine whether the implementation of patient blood management (PBM) is effective to decrease the use of red blood cell without impairment of patient's safety. BACKGROUND: The World Health Organization encouraged all member states to implement PBM programs employing multiple combined strategies to increase and preserve autologous erythrocyte volume to minimize red blood cell transfusions. Data regarding safety issues are not sufficiently available. METHODS: In this prospective, multicenter study, surgical inpatients from four German University Hospitals were analyzed before (pre-PBM) and after the implementation of PBM. PBM program included multiple measures (ie, preoperative optimization of hemoglobin levels, blood-sparing techniques, and standardization of transfusion practice). Primary aim was to show noninferiority of the PBM cohort with a margin of 0.5%. Secondary endpoints included red blood cell utilization. RESULTS: A total of 129,719 patients discharged between July 2012 and June 2015 with different inclusion periods for pre-PBM (54,513 patients) and PBM (75,206 patients) were analyzed. The primary endpoint was 6.53% in the pre-PBM versus 6.34% in the PBM cohort. The noninferiority aim was achieved (P < 0.001). Incidence of acute renal failure decreased in the PBM cohort (2.39% vs 1.67%; P < 0.001, regression model). The mean number of red blood cell transfused per patient was reduced from 1.21 ±â€Š0.05 to 1.00 ±â€Š0.05 (relative change by 17%, P < 0.001). CONCLUSIONS: The data presented show that implementation of PBM with a more conscious handling of transfusion practice can be achieved even in large hospitals without impairment of patient's safety. Further studies should elucidate which PBM measures are most clinically and cost effective. TRIAL REGISTRATION: PBM-Study ClinicalTrials.gov, NCT01820949.

Direct evidence for a covalent ene adduct intermediate in NAD(P)H-dependent enzymes.

The pyridine nucleotides NADH and NADPH (NAD(P)H) are ubiquitous redox coenzymes that are present in all living cells. Although about 16% of all characterized enzymes use pyridine nucleotides as hydride donors or acceptors during catalysis, a detailed understanding of how the hydride is transferred between NAD(P)H and the corresponding substrate is lacking for many enzymes. Here we present evidence for a new mechanism that operates during enzymatic hydride transfers using crotonyl-CoA carboxylase/reductase (Ccr) as a case study. We observed a covalent ene intermediate between NADPH and the substrate, crotonyl-CoA, using NMR, high-resolution MS and stopped-flow spectroscopy. Preparation of the ene intermediate further allowed direct access to the catalytic cycle of other NADPH-dependent enzymes-including those from type II fatty acid biosynthesis-in an unprecedented way, suggesting that formation of NAD(P)H ene intermediates is a more general principle in catalysis.

Innovative all-in-one exome sequencing strategy for diagnostic genetic testing in male infertility: Validation and 10-month experience.

BACKGROUND: Current guidelines indicate that patients with extreme oligozoospermia or azoospermia should be tested for chromosomal imbalances, azoospermia factor (AZF) deletions and/or CFTR variants. For other sperm abnormalities, no genetic diagnostics are recommended. OBJECTIVES: To determine whether exome sequencing (ES) with combined copy number variant (CNV) and single nucleotide variant (SNV) analysis is a reliable first-tier method to replace current methods (validation study), and to evaluate the diagnostic yield after 10 months of implementation (evaluation study). MATERIALS AND METHODS: In the validation study, ES was performed on DNA of patients already diagnosed with AZF deletions (n = 17), (non-)mosaic sex chromosomal aneuploidies or structural chromosomal anomalies (n = 37), CFTR variants (n = 26), or variants in known infertility genes (n = 4), and 90 controls. The data were analyzed using our standard diagnostic pipeline, with a bioinformatic filter for 130 male infertility genes. In the evaluation study, results of 292 clinical exomes were included. RESULTS: All previously reported variants in the validation cohort, including clinically relevant Y-chromosomal microdeletions, were correctly identified and reliably detected. In the evaluation study, we identified one or more clinically relevant genetic anomalies in 67 of 292 of all cases (22.9%): these included aberrations that could have been detected with current methods in 30 of 67 patients (10.2% of total), (possible) (mono)genetic causes in the male infertility gene panel in 28 of 67 patients (9.6%), and carriership of cystic fibrosis in nine of 67 patients (3.1%). CONCLUSION: ES is a reliable first-tier method to detect the most common genetic causes of male infertility and, as additional genetic causes can be detected, in our evaluation cohort the diagnostic yield almost doubled (10.2%-19.8%, excluding CF carriers). A genetic diagnosis provides answers on the cause of infertility and helps the professionals in the counseling for treatment, possible co-morbidities and risk for offspring and/or family members. Karyotyping will still remain necessary for detecting balanced translocations or low-grade chromosomal mosaicism.

Osteosarcomas of the mandible: multidisciplinary management of a rare tumor of the young adult a cooperative study of the GSF-GETO, Rare Cancer Network, GETTEC/REFCOR and SFCE.

BACKGROUND: Mandibular osteosarcomas (MOS) mostly affect young adults. Their treatment is extrapolated from that of extragnathic osteosarcomas. MATERIAL AND METHODS: A retrospective multicooperative group study was conducted to determine the impact of chemotherapy, adjuvant radiation therapy and surgery on outcomes and to identify prognostic factors. This ethical committee-approved study included a centralized review of histology slides and operative reports. RESULTS: Of 111 patients, 58.6% were male, median age 35 years (13%, ≤18 years). Histology was osteoblastic, chondroblastic, fibroblastic, conventional not otherwise specified and others in 39.6%, 30.6%, 8.1%, 12.6% and 8.0%, respectively. Pathological World Health Organisation grades were low, intermediate and high grade in 6.4%, 11.8% and 81.8%, respectively. Surgery was carried out for 94.5% of patients. Neoadjuvant chemotherapy (mixed protocols) was carried out in 93.1% of patients. Postoperative chemotherapy and radiotherapy were carried out in 54.7% and 23.8%, respectively. Median follow-up was 59.6 months (range). Five-year local control, metastasis-free, disease-free and overall survival rates were 64.6%, 68.9%, 53.2% and 69.2%, respectively. Survival was significantly associated with age, tumor size and surgery. Wide surgery with clear margins and free flap reconstruction was the strongest prognostic factor. Neoadjuvant chemotherapy improved disease-free and metastatic-free survival and increased clear margins rates from 50% to 68%. Intermediate grades behaved like high grades in terms of metastatic-free and disease-free survival. CONCLUSION: This homogeneous series is the largest to date and emphasizes the major impact of clear margins and multidisciplinary management. Neoadjuvant chemotherapy improves disease-free survival and should be recommended for both high and intermediate grade MOS.

[Botulinum toxin A: analgesic treatment for temporomandibular joint disorders]. / Toxine botulinique de type A : traitement antalgique des dysfonctions de l'appareil manducateur.

INTRODUCTION: Pain, along with clicking, sub-luxation, and blocking, is one of the main symptoms for patients presenting with temporomandibular joint disorders. We assessed the effectiveness of botulinum toxin A (BOTOX(®), Allergan) as analgesic treatment for temporomandibular joint disorders. PATIENTS AND METHOD: Twenty-six patients with chronic pain linked to temporomandibular joint disorders were prospectively assessed. Botulinum toxin A was injected in masseter and temporalis muscles. Follow-up parameters, at one and three months, were: measuring mouth opening and diduction, muscle tenderness and pain using a visual analogue scale. RESULTS: There was a significant (P<0.0001) decrease of pain, an increased mouth opening, and diduction at three months. Seventy percent of the patients felt less muscle tenderness. The patient felt his psychological state had improved and so did his family. DISCUSSION: Botulinum toxin A significantly decreases pain and improves movements of patients presenting with temporomandibular joint disorders. The effects are prolonged three months after the injection.

[Autologous tissue engineering by means of distraction osteogenesis: Biomechanical considerations]. / Ingénierie tissulaire autologue par distraction ostéogénique : considérations biomécaniques.

Tissue engineering consists in producing functional replacement tissue. Distraction osteogenesis is a tissue engineering technique that uses the mechanical environment of cells to induce tissue regeneration, without need for exogenous biochemical factors. A better understanding of the optimal mechanical conditions of distraction callus stretching may reduce the duration, discomfort, and even social impact of distraction protocols, and complications and failures. We present the current state of knowledge in this field by addressing the fundamentals of elongating bone tissue biomechanics, the influence of rhythm and rate of distraction, and that of vectors and stability. Finally, we present the innovations currently studied, which may modify our clinical protocol in the short term.

[Oro-maxillofacial bone tissue engineering combining biomaterials, stem cells, and gene therapy]. / Ingénierie du tissu osseux oro-maxillofacial par combinaison de biomatériaux, cellules souches, thérapie génique.

Improvements have been made in regenerative medicine, due to the development of tissue engineering and cellular therapy. Bone regeneration is an ambitious project, leading to many applications involving skull, maxillofacial, and orthopaedic surgery. Scaffolds, stem cells, and signals support bone tissue engineering. The scaffold physical and chemical properties promote cell invasion, guide their differentiation, and enable signal transmission. Scaffold may be inorganic or organic. Their conception was improved by the use of new techniques: self-assembled nanofibres, electrospinning, solution-phase separation, micropatterned hydrogels, bioprinting, and rapid prototyping. Cellular biology processes allow us to choose between embryonic stem cells or adult stem cells for regenerative medicine. Finally, communication between cells and their environment is essential; they use various signals to do so. The study of signals and their transmission led to the discovery and the use of Bone Morphogenetic Protein (BMP). The development of cellular therapy led to the emergence of a specific field: gene therapy. It relies on viral vectors, which include: retroviruses, adenoviruses and adeno-associated vectors (AAV). Non-viral vectors include plasmids and lipoplex. Some BMP genes have successfully been transfected. The ability to control transfected cells and the capacity to combine and transfect many genes involved in osseous healing will improve gene therapy.

[Improvement of maxillofacial bone distraction osteogenesis: future prospects]. / Potentialisation de la distraction ostéogénique maxillofaciale, voies d'avenir.

Distraction osteogenesis is a tissue engineering technique based on Ilizarov's study on long bones. McCarthy transposed it rapidly to facial bones. His results in cranial and maxillofacial surgery are good and reproducible. However, the current protocols are long and the devices used are bulky. Finding new devices and association with other tissue engineering techniques should improve distraction osteogenesis and the patient's comfort.

[Engineering a bone free flap for maxillofacial reconstruction: technical restrictions]. / Ingénierie d'un lambeau osseux vascularisé à destinée maxillofaciale : les limites techniques.

Vascularisation is a key for success in bone tissue engineering. Creating a functional vascular network is an important concern so as to ensure vitality in regenerated tissues. Many strategies were developed to achieve this goal. One of these is cellular growth technique by perfusion bioreactor chamber. These new technical requirements came along with improved media and chamber receptacles: bioreactors (chapter 2). Some bone tissue engineering processes already have clinical applications but for volumes limited by the lack of vascularisation. Resorbable or non-resorbable membranes are an example. They are used separately or in association with bone grafts and they protect the graft during the revascularization process. Potentiated osseous regeneration uses molecular or cellular adjuvants (BMPs and autologous stem cells) to improve osseous healing. Significant improvements were made: integration of specific sequences, which may guide and enhance cells differentiation in scaffold; nano- or micro-patterned cell containing scaffolds. Finally, some authors consider the patient body as an ideal bioreactor to induce vascularisation in large volumes of grafted tissues. "Endocultivation", i.e., cellular culture inside the human body was proven to be feasible and safe. The properties of regenerated bone in the long run remain to be assessed. The objective to reach remains the engineering of an "in vitro" osseous free flap without morbidity.

[Bone substitutes: Classification and concerns]. / Les biomatériaux de substitution osseuse : classification et intérêt.

Autograft is considered as the "gold standard" for bone reconstruction. It provides osteoinductive factors, osteogenic cells, and appropriate osteoconductive scaffold. Donor site morbidity is the main limitation of autograft. Donor disease transmission limits the use of allograft. Synthetic bone substitutes still lack osteoinductive or osteogenic properties. Composite bone substitutes combining synthetic scaffold and biochemical substances initiating proliferation and cell differentiation, and possibly osteogenesis. Bone substitutes and grafts intended for clinical use are listed.

Staged autogenous calvarial bone grafting and dental implants placement in the management of oligodontia: a retrospective study of 20 patients over a 12-year period.

Oligodontia demands multidisciplinary management due to its repercussions on dentofacial growth. To place implants to realize implant-borne fixed denture, preimplant surgery may be necessary if bone volumes are insufficient. Our aim was to assess bone increase following autogenic bone grafting and to discuss prosthetic options. Twenty patients followed for oligodontia, who underwent bone grafting, were treated from 2008 to 2019. Transversal and vertical bone levels were measured pre- and postoperatively to assess alveolar ridge augmentation. Mean horizontal grafting increase was 4.60 mm [standard deviation (SD) 0.79 mm], mean sinus lift increase was 9.95 mm (SD 2.35 mm). Mean implants placed per patient was 9, mean implants placed on grafted site was 5 per patient. Overall implant survival rate was 100%. All patients benefited from prosthetic procedures when it was planned to perform implant-borne fixed dentures. Within the framework of a complete treatment plan (involving paediatric dentistry, dentofacial orthopaedics, oral and maxillofacial surgery, and prosthodontics), autologous bone grafting combined or not with orthognathic surgery is fully adapted to patients with oligodontia. It allows reconstruction of favourable bone volumes for placement of implants to realize implant-borne fixed dentures, with high implant survival rates and great improvements to quality of life.

TMJ related short-term outcomes comparing two different osteosynthesis techniques for bilateral sagittal split osteotomy.

OBJECTIVES: Bilateral sagittal split osteotomy (BSSO) is a morpho-functional surgery and post-surgical osteosynthesis may influence temporomandibular joint (TMJ) health. Our objective was to evaluate temporomandibular disorders (TMD) and TMJ symptoms after orthognathic surgery according to the type of osteosynthesis used in a population of patients with dentofacial deformities. MATERIALS AND METHODS: One hundred and eighty-three consecutive patients undergoing orthodontic and maxillofacial surgery treatment for correction of their malocclusion were recruited for a two-year period at Lille University Hospital. All patients had at least a mandibular BSSO using Epker's technique. Each patient was examined before and one year after orthognathic surgery. We compared osteosynthesis by miniplates fixed with monocortical screws (n=42) and the hybrid fixation with bicortical retro-molar screws used with miniplates (n=141). TMJ health was assessed by monitoring TMD signs and symptoms according to the Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD) and scores obtained from the "Jaw Pain Function" questionnaire. RESULTS: There was no significant difference in pre-operative and one year post-operative RDC/TMD assessments (p≥0.91) or JPF score (p≥0.29) between the two types of osteosynthesis. CONCLUSION: There was no difference in TMJ health between the two techniques of osteosynthesis after BSSO. CLINICAL RELEVANCE: In our experience the hybrid technique fixation affords many advantages and does not influence postoperative TMD compared with osteosynthesis by miniplates.

[Palatal necrosis due to cocaine abuse]. / Nécrose palatine par consommation de cocaïne.

The easier access to cocaine allows chronic and intensive consumption. Nasally inhaled cocaine causes important midfacial lesions called: Cocaine Induced Midline Destructive Lesions. These lesions are due to several factors, the anesthetic, vasoconstrictive, locally prothrombotic properties of cocaine and its components combined with cytotoxic effects and traumatic nasal injuries related to consumption mode. Functional signs are: nasal regurgitation, rhinolalia, rhinorrhea, and midfacial pain. The morphological modifications of the nasal pyramid feature the destruction of bone and cartilage structures. Endo-buccal examination, anterior rhinoscopy, and TDM reveal palatine necrosis of variable extension. Nasal fossae superinfection is always present. Sinus superinfection is frequent. Management is multidisciplinary. Weaning must be achieved before surgery. It is necessary to rehabilitate speech and swallowing functions by the means of various local or free flaps.

Le Fort I osteotomy and calvarial bone grafting for dental implants.

INTRODUCTION: We present a reconstruction technique for atrophied maxilla which combines a Le Fort I osteotomy and calvarial bone grafting. This retrospective study was carried out to evaluate bone volume increase and the possibility to insert dental implants. PATIENTS AND METHODS: Bone volume increase was assessed in 56 patients using preoperative and 6-month postoperative computed axial tomography (CAT). The implant rate success and the type of prosthesis were evaluated. RESULTS: The average increase at the first molar level was 10.2mm vertically and 8.2mm transversally. No infection was reported. The implant success rate was 97.9% and a prosthetic rehabilitation was performed in all patients (55% with removable bar-supported prostheses, and 45% with fixed prostheses). DISCUSSION: The technique is adapted to atrophied maxilla reconstruction for dental implant rehabilitation. The implant success rate was similar to that of other bone augmentation techniques.

Temporomandibular disorders in head and neck cancers: Overview of specific mechanisms and management.

Diagnosis classification system of Temporomandibular disorders (TMD) is based on the biopsychosocial model of pain. The pathogenesis is poorly understood, leading to difficulties in treating these multifactorial conditions. The predisposing factors are pathophysiological, psychological or structural processes that alter the masticatory system and lead to an increase in the risk of development of TMD. The purpose of this integrative review was then to point out the specific mechanisms of TMD in the oral oncologic context to optimize the TMJ functional results in the management of patients with oral oncologic conditions. We explored in this paper the role of Axis II assessment of the biopsychosocial model of pain, the involvement of mechanical concepts such as dental occlusion, mandibular condyle positioning and related-structures reconstruction, and the stomatognathic changes induced by radiation.

A modified method for a customized harvest of fibula free flap in maxillofacial reconstruction.

Mandibular reconstruction using computer-aided design/computer-aided manufacturing cutting guides is currently a common procedure. However, inaccurate positioning of the cutting guide onto the fibular bone may result in osteosynthesis difficulties or imprecision in the reconstruction. A novel way to improve the stability of the cutting guides may be to add pillars in order for them to be suspended from the fibula, avoiding soft tissues interactions. We present the case of a 39-year-old male who needed mandibular reconstruction after a self-inflicted ballistic injury. We designed a customized cutting guide which included a set of 8 pillars allowing a suspension of the cutting guide 8 millimeters above the bone level. The pillars were perpendicular to one another, and allowed the operator to screw the cutting guide to the bone. The orthogonal position of the pillars enabled real stability during the osteotomies. In the operator experience, the length of the pillars was too important, and led to incomplete osteotomies, and the whole device was too bulky. However, with adaptations in the size of the pillars and the size of the whole device, this solution could be useful in cutting guide design to avoid impairments due to the soft tissues surrounding the fibula.

Twenty-four years of experience in management of complex mandibular fractures with low cost, custom-made mandibular external fixation: A 65-patient series.

INTRODUCTION: Ginestet introduced the first external device used to fix the mandible in 1936. In 1949, Morris introduced a biphasic fixation device. This "Joe Hall Morris fixation" design led to use of a self-crafted external fixator based on pins connected by a breathing tube filled with dental resin. The objective of this study was to present our surgical results with this device through a 65-patient series. METHODS: This retrospective study included all the patients who benefited from the self-crafted mandibular external fixator at our Oral and Maxillofacial department from 1995 to 2019. Sixty-five patients were allocated into two groups. There were 39 patients in the temporary stabilisation (TS) group and 26 in the bone healing (BH) group. Functional criteria were investigated, including mouth opening limitations and occlusal abnormalities. Aesthetic evaluation focused on skin healing, evaluated by both surgeon and patient. RESULTS: Twenty-three patients exhibited spontaneous bone healing during their immobilisation period and two patients developed a pseudoarthrosis in the BH group. Most TS group patients benefited from secondary management by bone graft, bone free flap, or distraction osteogenesis. Few complications were noted with our technique during the study period. CONCLUSION: Our self-crafted external fixation with Joe Hall Morris fixation style is a valuable option for external stabilisation of the lower third of the face.