Evaluation of Normothermic Machine Perfusion of Porcine Livers as a Novel Preclinical Model to Predict Biliary Clearance and Transporter-Mediated Drug-Drug Interactions Using Statins.

There is a lack of translational preclinical models that can predict hepatic handling of drugs. In this study, we aimed to evaluate the applicability of normothermic machine perfusion (NMP) of porcine livers as a novel ex vivo model to predict hepatic clearance, biliary excretion, and plasma exposure of drugs. For this evaluation, we dosed atorvastatin, pitavastatin, and rosuvastatin as model drugs to porcine livers and studied the effect of common drug-drug interactions (DDIs) on these processes. After 120 minutes of perfusion, 0.104 mg atorvastatin (n = 3), 0.140 mg pitavastatin (n = 5), or 1.4 mg rosuvastatin (n = 4) was administered to the portal vein, which was followed 120 minutes later by a second bolus of the statin coadministered with OATP perpetrator drug rifampicin (67.7 mg). After the first dose, all statins were rapidly cleared from the circulation (hepatic extraction ratio > 0.7) and excreted into the bile. Presence of human-specific atorvastatin metabolites confirmed the metabolic capacity of porcine livers. The predicted biliary clearance of rosuvastatin was found to be closer to the observed biliary clearance. A rank order of the DDI between the various systems upon coadministration with rifampicin could be observed: atorvastatin (AUC ratio 7.2) > rosuvastatin (AUC ratio 3.1) > pitavastatin (AUC ratio 2.6), which is in good agreement with the clinical DDI data. The results from this study demonstrated the applicability of using NMP of porcine livers as a novel preclinical model to study OATP-mediated DDI and its effect on hepatic clearance, biliary excretion, and plasma profile of drugs. SIGNIFICANCE STATEMENT: This study evaluated the use of normothermic machine perfusion (NMP) of porcine livers as a novel preclinical model to study hepatic clearance, biliary excretion, plasma (metabolite) profile of statins, and OATP-mediated DDI. Results showed that NMP of porcine livers is a reliable model to study OATP-mediated DDI. Overall, the rank order of DDI severity indicated in these experiments is in good agreement with clinical data, indicating the potential importance of this new ex vivo model in early drug discovery.

Single puncture versus standard double needle arthrocentesis for the management of temporomandibular joint disorders: A systematic review.

The aim of this systematic review was to investigate the current evidence in order to assess the efficacy of single puncture arthrocentesis vs standard double needle arthrocentesis in the management of temporomandibular joint(TMJ) disorders. An electronic search of the PubMed, Scopus, Cochrane CENTRAL and Google Scholar databases was performed to identify English studies published up until October 2017. Eligible studies were selected based on inclusion criteria and included randomised controlled trials(RCTs) comparing single puncture arthrocentesis and standard double needle arthrocentesis for the management TMJ disorders. The initial screening identified 984 records, of which only 5 fulfilled the inclusion criteria. A high degree of heterogeneity was found in the 5 studies with each reporting different sample selection and arthrocentesis protocol. All 5 studies reported no difference in reduction in pain intensity and improvement in maximal mouth opening between the single puncture technique and standard double needle technique. This review provides some evidence that single puncture arthrocentesis is clinically as efficacious as standard double needle arthrocentesis. There is a need of well-designed RCT with standard protocol of arthrocentesis comparing different single puncture techniques and standard double needle technique for the management of TMJ disorders.

Comparative study of immediate functional loading and immediate non-functional loading of monocortical implants.

BACKGROUND: Attempts to shorten the overall length of treatment have focused on immediate loading, subsequent to implant placement. Prosthetic rehabilitation immediately after implant placement can be either functional or non-functional in nature. There is paucity of literature on the comparative evaluation of immediate functional and immediate non-functional loading of implants. This in-vivo study was undertaken to comparatively evaluate Immediate Functional Loading and Immediate Non-Functional Loading of monocortical implants with a follow-up period of 18 months. METHODS: 50 partially edentulous cases were selected for the study. The cases were divided into two groups. In first group (Group-1), 25 implants were subjected to immediate functional loading. In second group (Group-2), 25 implants were subjected to immediate non-functional loading. The crestal bone loss, clinical stability and degree of osseointegration of these two groups were comparatively evaluated. RESULTS: The crestal bone loss in both groups was within acceptable limits. The implant stability, which is a reflection of the status of bone-to-implant interface, was comparable in both the groups at different time intervals. Although, the ISQ values in Group-2 were slightly higher than those in Group-1, the results were not statistically significant. Radiodensity indicating degree of osseointegration at different time intervals in both groups was also comparable. CONCLUSION: Both the IFL and INFL protocols can be undertaken satisfactorily in rehabilitation using endosseous implants; however, the main factors for success in IFL and INFL are case selection, meticulous treatment planning and the precision of technique.

Incidence of Facial Nerve Injury and Sialocele Formation Following Mandibular Condylar and Sub-condylar Fracture Fixation.

Purpose: To study the incidence of sialocele formation in the parotid gland and to study the incidence of facial nerve affliction following treatment of mandibular condylar and sub-condylar fractures. Materials and methods: The present study is a retrospective study conducted on a total of 82 patients with 107 sub-condylar and condylar fractures treated in this centre from August 2008 to August 2020. The surgical approaches used to treat the fractures were considered, and the occurrence of sialocele, salivary fistula and facial nerve paralysis was noted. The facial nerve function was analysed using House-Brackmann system of classification. Results: The incidence of sialocele formation was seen in 15.87% of cases, and the incidence was seen more commonly during a preauricular approach (52.94%) followed by retromandibular (41.17%) followed by anterior parotid transmassetric approach (11.76%). The incidence of facial nerve affliction was seen in 17.57% of cases with majority of them showing temporal branch involvement in 21.05% of cases. Conclusion: During the treatment of condylar and sub-condylar fractures, the facial nerve is at considerable risk of damage; however, understanding the anatomy of the nerve is of importance to avoid such complications. Sialocele formation is also an undesirable complication of such surgeries, a prompt diagnosis and early treatment is mandatory to overcome further unwanted sequel.

Karyotype heterogeneity and unclassified chromosomal abnormalities.

In a departure from traditional gene-centric thinking with regard to cytogenetics and cytogenomics, the recently introduced genome theory calls upon a re-focusing of our attention on karyotype analyses of disease conditions. Karyotype heterogeneity has been demonstrated to be directly involved in the somatic cell evolution process which is the basis of many common and complex diseases such as cancer. To correctly use karyotype heterogeneity and apply it to monitor system instability, we need to include many seemingly unimportant non-specific chromosomal aberrations into our analysis. Traditionally, cytogenetic analysis has been focused on identifying recurrent types of abnormalities, particularly those that have been linked to specific diseases. In this perspective, drawing on the new framework of 4D-genomics, we will briefly review the importance of studying karyotype heterogeneity. We have also listed a number of overlooked chromosomal aberrations including defective mitotic figures, chromosome fragmentation as well as genome chaos. Finally, we call for the systematic discovery/characterization and classification of karyotype abnormalities in human diseases, as karyotype heterogeneity is the common factor that is essential for somatic cell evolution.

Validity of IL-6 and Arthrocentesis in the Cause and Management of Internal Derangement of TMJ.

Purpose: The aim of this clinical study was to evaluate the level of Interleukine-6 (IL-6), pre and post arthrocentesis to validate it as a biomarker in the Internal Derangement (ID) of TMJ. Material and Methods: This study included 30 patients (20 females and 10 males) of Temporo-Mandibular Dysfunction (TMD) with Disc displacement without reduction (DDwoR) Wilkes stage III, who were refractory to conservative management. Arthrocentesis was performed as a therapeutic modality. Synovial fluid aspirates were obtained prior to arthrocentesis and post arthrocentesis with 300 ml of Ringer Lactate solution into the superior joint compartment for the assessment of level of IL-6. The clinical parameters used for correlating the level of IL-6 were degree of pain (VAS I), chewing ability (VAS II), Maximal Mouth Opening (MMO) in both pre and post op phase with the follow-up period of 01 day, 01 week, 01 month, 03 month and 06 month and the results were compared. ELISA was performed to analyze the levels of IL-6 in the aspirates. The clinical parameters and the level of IL-6 were recorded and analyzed statistically. Results: The study showed ID of TMJ (Wilkes stage III) s are more prevalent in females especially in the fourth decades of life with the mean age of 38.4 years. The post operative assessment in terms of pain, maximum mouth opening, lateral movements of the mandible and the levels of IL-6 were found to be statistically significant with a P value <0.01. Conclusion: This study validates the role of IL-6 as a definitive biomarker for the pathogenesis of ID of TMJ Wilkes stage III and arthrocentesis proved to be a minimally invasive therapeutic modality for its management.

A Retrospective Study of Complications in Cranioplasty: 7-Year Period.

OBJECTIVE: To discuss the intraoperative and postoperative complications of cranioplasty and management during a 7-year period. METHOD: Retrospective study of 7-year period of 63 patients including both male and female. RESULTS: Highest experienced complications were seizures and dural tear, i.e., 6%, followed by EDH in 3% patients, hydrocephalus and pneumocephalus combined 3%, 1.6% CSF collection and flap necrosis each. All the complications were managed successfully. CONCLUSION: Complications of cranioplasty can be managed by following sound surgical principles. Serious complications like meningitis, air embolism and death are rare.

Efficacy of Platelet-Rich Plasma in Enhancing the Osteogenic Potential of Bone Graft in Oral and Maxillofacial Region.

BACKGROUND: Platelet-rich plasma (PRP) has been a breakthrough in the stimulation and acceleration of bone and soft tissue healing. It represents a relatively new biotechnology that is part of the growing interest in tissue engineering and cellular therapy. METHODS: A prospective study was carried out in 50 patients. The cases were selected randomly in the age group of 8-50 years who needed bone grafts for alveolar cleft defects and surgical defects following removal of osteolytic jaw lesions. They were divided into study group with autologous PRP and control group without PRP. Bone density was calculated as per Hounsfield scale preoperatively and post-operatively for both the groups. RESULTS: There was significant difference in the Hounsfield units at 06 months and 12 months post-operatively in both the groups showing good amount of bone regeneration. The preoperative volume of the defect and the post-operative volume of the regenerated bone were statistically analysed. The mean V2 was 0.7652 cc for the study group, whereas for control group, it was 0.4840 cc. The volume ratio for study group was 0.9070 and for control group was 0.6740. This showed greater bone regeneration in the study group. The results were statistically significant for both the groups. CONCLUSION: PRP is a new application of tissue engineering and a developing area of interest for clinicians and researchers. It is a storage vehicle for growth factors, especially PDGF and TGF-b, both of which influence bone regeneration, and also eliminates the concerns about immunogenic reactions and disease transmission. PRP does enhance the healing of bone grafts in the maxillofacial region as shown by the increase in the density of bone.

Fractures in the Maxillofacial Region: A Four Year Retrospective Study.

INTRODUCTION: The incidence of maxillofacial injuries is on the rise due to motor vehicle accidents and increased incidence of violence in recent times. The aim of this retrospective study was to determine the incidence, aetiology, the pattern of fractures, their management with open reduction and internal fixation (ORIF) and complications, if any. METHODS: A retrospective analysis of 621 fractures in 361 patients managed by ORIF over a four year period was carried out. RESULT: The average age of patients was 24.3 years with a male to female ratio of 21.2:1. Panfacial fractures comprised 4.7%, frontal bone fractures 8.9%, orbital fractures 0.7%, naso-orbito-ethmoid complex (NOE) fractures 0.7%, zygomatic complex fractures 23.5%, fracture maxilla 11.5% and mandibular fractures 52.2% of all facial fractures. All the cases were successfully managed by ORIF under general anaesthesia (GA). Complications were noticed in 6.8% of cases in the form of reactive implants in 3.6%, deranged occlusion in 1% and infection at operated site in 1% cases which were managed satisfactorily. CONCLUSION: The findings of this study reveal sharp annual increase in the number of cases of maxillofacial trauma. Road traffic accidents (RTA) were the commonest cause and the age group most affected was between 20-25 years. ORIF of these fractures was chosen for its obvious advantages of direct anatomical reduction, early return to function and minimal complications.

Application of Navigation Surgery in Temporomandibular Joint Ankylosis Case and Review of Literature.

The aim of this article is to develop a navigation-guided oral and maxillofacial surgery including surgical planning, simulation and navigation in temporomandibular joint ankylosis case practiced in tertiary care hospital. After getting the computed tomographic angiography of head and neck, the special software of Brain lab® is used to mark the arteries and veins of the patient, which was in close approximation to the mandibular condyle. Brain lab® navigation system was used during the surgery to ascertain the middle meningeal artery location, and osteotomy cut was given. Navigation technology use in the neurosurgery is not new. But its use in the subcontinent in the field of maxillofacial surgery is quite rare. First time, it has been used scientifically in temporomandibular joint ankylosis case. Navigation technology use in the maxillofacial surgery requires a holistic imaginative/creative approach to make the surgeries more predictive and safe.

Clinical and MRI Evaluation of Orthodontic Mini-Screws for Disc Repositioning in Internal Derangement of TMJ: A Prospective Study.

PURPOSE: The purpose of this study was to assess the efficacy of orthodontic mini-screws as a modified suture anchor for disc repositioning in cases of internal derangement of the temporomandibular joint. PATIENTS AND METHODS: A prospective evaluation of ten patients was undertaken for a period of 6 months using this modified approach from Jan 2014 to Jun 2016. Symptomatic patients with clinical and MRI features suggestive of internal derangement of TMJ and willing to undergo surgical repositioning of articular disc to alleviate symptoms of temporomandibular dysfunction were taken up for the study. Post-operatively, functional outcomes were assessed in terms of reduction in pain, joint movement and absence of joint noise and clicking sounds. Post-operative MRI was used to assess the disc position and morphological changes in the disc and arthritic changes in the condyle. RESULTS: All patients underwent a surgical repositioning of the anteriorly displaced disc by the modified orthodontic suture anchor. Patients were post-surgically followed up at intervals of 1, 3 and 6 months. Immediate complications in terms of pain, restriction of functional motion and transient facial nerve palsy were noted. Late complications include temporal nerve palsy in one case. All patients experienced significant improvement with good functional outcomes and stable repositioning of disc was noticed at the end of 6 months. CONCLUSION: The modified disc repositioning using an orthodontic screw via a mini preauricular approach provided a good functional outcome in all patients as assessed over a period of 6 months. However, the long-term functional sequel of the procedure and changes in the articular disc needs to be assessed.

Secondary and Delayed Bone Grafting in Alveolar and Anterior Palatal Clefts.

BACKGROUND: Surgical repair with suitable bone graft / substitutes plays an important role in rehabilitation of individuals with residual anterior palatal or alveolar defects associated with an oronasal fistula. METHODS: 52 cases were treated by secondary or delayed bone grafting of the alveolar defects in isolation or associated with defects of the anterior palate from July 2002 - Nov 2004. Dimension and the extent of the defects were assessed with the help of radiographs & maxillofacial CT. Cephalometric analysis, presurgical orthodontics and dentofacial orthopedics preceded surgical repair with cancellous graft from the iliac crest, followed by post surgical orthodontics and prosthetic rehabilitation. RESULT: Satisfactory results were achieved in 49 cases with three cases showing failure of graft acceptance. Postoperative clinical and radiological evaluation for all the patients was done at an interval of one, three and six months. Bone density and trabeculation was comparable to the adjacent bone within six months. CONCLUSION: Recreating the bony continuity of the maxillary arch followed by orthodontic correction of dental discrepancies achieves a comprehensive orthosurgical correction.

Distraction Osteogenesis in Management of Mandibular Deformities.

BACKGROUND: The concept of Distraction Osteogenesis is applied in Maxillofacial surgery extensively in the recent past, revolutionizing the concept of management of facial deformities specially the mandible. METHODS: This article describes intra oral mandibular distractors in managing mandibular deformities in 9 cases. CONCLUSION: Surgical intervention is possible in very young patients. Distraction Osteogenosis does not reduce the need for orthodontic intervention. A multifarious approach to the problem, should form the basis of treatment of mandibular deformities.

Fingerprinting proteins coupled with polymers by mass spectrometry: Investigation of polyethylene glycol-conjugated superoxide dismutase.

Matrix-assisted laser desorption-ionization (MALDI) mass spectrometry was investigated as a method for the rapid determination of the extent of polymer coupling in polyethylene glycol- (PEG) conjugated superoxide dismutase (SOD). PEG-conjugated SOD, an antioxidant with an extended in vivo circulation lifetime compared to that of superoxide dismutase, is being evaluated as an effective therapeutic agent for the treatment of injuries and arthritis. The mass spectra of a standard batch of PEG-conjugated bovine SOD showed the presence of identifiable and well resolved peaks that correspond to 0-7 PEG molecules attached to bovine SOD. The area of each of the peaks provides a determination of the amount of PEG-conjugated SOD with a given number of bound PEG groups. SOD is a noncovalent dimer of two identical subunits that dissociates in MALDI. The information obtained in the mass spectra thus corresponds to a monomer of SOD. Each SOD monomer contains 10 lysines, which are the sites of PEG-conjugation. Multiple MALDI determinations of two batches of samples indicated good reproducibility for routine determination of the extent of polymer content. The amount of PEG-conjugated SOD that contained a given number of PEG molecules, determined by MALDI, was compared with the value deduced from the amount of PEG-conjugation at each attachment site measured by a peptide mapping method. Agreement between the data obtained in the two techniques (MALDI and peptide mapping) indicates that MALDI may be used to obtain quantitative information on PEG-conjugated SOD to determine the amounts of PEG-conjugated protein each with a different number of PEG groups attached. Measurement of several batches of samples stored at a higher temperature showed a lower extent of PEG-conjugation in PEG-conjugated SOD. This reduction in the PEG content resulted from the PEG-deconjugation of PEG-conjugated SOD at a higher temperature. Thus, MALDI can be used to examine the stability of PEG-conjugated SOD. The high sensitivity, relatively straightforward data interpretation, speed of analyses, and good reproducibility in measurements make this technique a useful analytical tool for fingerprinting PEG-conjugated SOD as well as potentially other polymer-conjugated proteins.

Origin and removal of adducts (molecular mass = 98 u) attached to peptide and protein ions in electrospray ionization mass spectra.

Electrospray ionization of peptides and proteins often produces intense adduct ions resulting from the attachment of a moeity with mass 98 u. The formation of these adduct ions results in a substantial reduction in the mass spectrometric sensitivity and an undesirable increase in the complexity of the mass spectra. In the present study it was shown that the removal of the attached adducts from peptide and protein ions can be affected by collisional activation and that the adducts arise from the attachment of sulfuric acid or phosphoric acid to peptide and protein ions. When sulfate and phosphate ions are removed from the samples by chemical means, adduct free ions are obtained from proteins yielding spectra with improved quality and sensitivity.

Production of selenomethionine-labeled recombinant human neutrophil collagenase in Escherichia coli.

Molecular analogs of amino acids can be incorporated into proteins. The amino acid analog selenomethionine (SeMet) has been shown to be efficiently incorporated into the proteins of growing Escherichia coli. SeMet-containing proteins are known to produce sufficiently strong anomalous scatter permitting the solution of the selenomethionyl crystal structure by multiwavelength anomalous diffraction (MAD) techniques. The recombinant protein chosen for these studies is mature, truncated neutrophil collagenase (rmNC-t). The rmNC-t protein is a monomer of 163 amino acid residues featuring one active site and two Met residues. We developed a T7 polymerase expression system allowing incorporation of SeMet into rmNC-t protein produced in E. coli. Substitution of Met with SeMet was accomplished by culturing E. coli DL41(DE3), a SeMet-tolerant strain with metA lesion, in a defined medium containing SeMet as the sole source of Met. The SeMet-labeled rmNC-t was isolated from inclusion bodies by solubilizing in urea, purified by anion column chromatography, and then refolded in the presence of Ca2+ and Zn2+. Analysis of SeMet-labeled rmNC-t demonstrated that Met replacement was 100%. Enzymatic characterization revealed no obvious differences in activity or inhibitor binding between rmNC-t and the SeMet-labeled product. We have produced pure, active SeMet-labeled rmNC-t in sufficient quantities for macromolecular crystallography studies.

Activities of mitochondrial oxidative phosphorylation enzymes in cultured amniocytes.

Amniocytes represent a population of foetal cells that can be used for prenatal diagnosis in families with suspected mitochondrial oxidative phosphorylation (OXPHOS) defects. In this paper, we present a complex protocol for evaluation of the function of mitochondrial OXPHOS enzymes in cultured amniocytes using three independent and complementary methods: (a) spectrophotometry as a tool for determination of the capacities of mitochondrial respiratory-chain enzymes (NADH ubiquinone oxidoreductase, succinate- and glycerophosphate cytochrome c reductase, cytochrome c oxidase and citrate synthase); (b) polarography as a tool for the evaluation of mitochondrial OXPHOS enzyme functions in situ using digitonin-permeabilised amniocytes (rotenone-sensitive oxidation of pyruvate+malate, antimycin A-sensitive oxidation of succinate, KCN-sensitive oxidation of cytochrome c, ADP-activated substrate oxidation) and (c) cytofluorometric determination of tetramethyl rhodamine methyl ester (TMRM) fluorescence in digitonin-permeabilised amniocytes as a sensitive way to determine the mitochondrial membrane potential under steady-state conditions (state 4 with succinate). These protocols are presented together with reference control values using 9-22 independent cultures of amniocytes.

Interrelationship of tryptophan pyrrolase with tyrosinase in melanogenesis of Bufo melanostictus.

Tryptophan pyrrolase, a microsomal enzyme responsible for the break-down of tryptophan, has been detected in Bufo melanostictus. The enzyme has been found to be deactivated under influence of antivitiligo drug psoralene and activated by hydroquinone, an inhibitor of tyrosinase. Tryptophan pyrrolase has been found to have an antagonistic relationship with tyrosinase in Bufo melanostictus. The implication of the results has been discussed in relation to melanogenesis in vitiligo.

An LC/MS/MS method for the quantitation of MTIC (5-(3-N-methyltriazen-1-yl)-imidazole-4-carboxamide), a bioconversion product of temozolomide, in rat and dog plasma.

A sensitive and selective HPLC/electrospray ionization tandem mass spectrometric (LC/ESI/MS/MS) method for the quantitative determination of MTIC (5-(3-N-methyltriazen-1-yl)-imidazole-4-carboxamide), a pharmacologically active hydrolysis product of temozolomide, was developed and validated over a linear range from 10 to 400 ng ml(-1) in dog plasma and from 10 to 500 ng ml(-1) in rat plasma. This HPLC method utilized small plasma volumes (70 microl), rapid sample processing, and isocratic elusion conditions to achieve sensitive and selective MS/MS detection. Samples were processed and analyzed one at a time every 4.5 min in order to compensate for the inherent instability of MTIC. Both MTIC and the internal standard DTIC [5-(3,3'-N,N'-dimethyltriazen-1-yl)-imidazole-4-carboxamide] were quantitated in the positive ion, selected reaction monitoring (SRM) mode. The lower limit of quantitation (LLOQ) was 10 ng ml(-1) in the plasma from both species. Inter-assay accuracy and precision of all calibration standards and quality control (QC) samples were within +/- 11 and 12%, respectively, with the exception of the LLOQ in rat plasma (17%). The validated method was used to determine the time dependent plasma concentration of MTIC in rats and dogs following a single oral dose of temozolomide. The standard curve and the quality control data indicate that the method performed acceptably throughout the sample analysis period.