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Byline: Y.C. Liu, S. Dost, S. Grenier, N. Audet, J. Haas This article presents the results of a three-dimensional numerical simulation study carried out for a three-zone refiner system. Firstly, an overall thermal analysis was performed to study the effects of changes in the size of the ZR system. A complete mass, heat, and momentum transport model was then developed for this system, and the fluid flow and impurity concentration distributions in a single molten zone were studied. A parametric study was also carried out to obtain the most favourable freezing interfaces and molten zone sizes. The flow field in the molten zone was studied to determine its effect on impurity transport.

Objective: To report a case of Langerhans cell histiocytosis of the temporal bone presenting with cerebrospinal fluid fistula. Patient: A Caucasian woman presented to a tertiary care centre in Quebec, Canada, with a new onset of cerebrospinal fluid fistula. She had a significant destructive lesion of the temporal bone, and was diagnosed with Langerhans cell histiocytosis on biopsy. Interventions: The patient underwent surgical resection with reconstruction of the posterior fossa and tegmen. She suffered a relapse less than one year after surgery, and was finally treated with chemotherapy. Main outcome and results: The patient was free of disease at three-year follow up. No recurrence of the cerebrospinal fluid leak was observed after treatment. Conclusion: Langerhans cell histiocytosis of the temporal bone with intra-cranial involvement is rare in adults, with only two cases previously reported. Eleven paediatric cases have been reported. To our knowledge, this patient represents the first report of cerebrospinal fluid fistula as the initial presentation of the disease. [ABSTRACT FROM AUTHOR]

Introduction: The routine assessment of intraoperative margins has long been the standard of care for oral cavity cancers. However, there is a controversy surrounding the best method for sampling surgical margins. The aim of our study is to determine the precision of a new technique for sampling tumor bed margins (TBMs), to evaluate the impact on survival and the rate of free flap reconstructions., Methods: This retrospective cohort study involved 156 patients with primary cancer of the tongue or floor of the mouth who underwent surgery as initial curative treatment. Patients were separated into 2 groups: one using an oriented TBM derived from Mohs' technique, where the margins are taken from the tumor bed and identified with Vicryl sutures on both the specimen and the tumor bed, and the other using a specimen margins (SMs) driven technique, where the margins are taken from the specimen after the initial resection. Clinicopathologic features, including margin status, were compared for both groups and correlated with locoregional control. Precision of per-operative TBM sampling method was obtained., Results: A total of 156 patients were included in the study, of which 80 were in TBM group and 76 were in SM group. Precision analysis showed that the oriented TBM technique pertained a 50% sensitivity, 96.6% specificity, 80% positive predictive value, and an 87.5% negative predictive value. Survival analysis revealed nonstatistically significant differences in both local control (86.88% vs 83.50%; P  = .81) as well as local-regional control (82.57% vs 72.32%; P  = .21). There was a significant difference in the rate of free flap-surgeries between the 2 groups (30% vs 64.5%; P  < .001)., Conclusion: Our described oriented TBM technique has demonstrated reduced risk of free flap reconstructive surgery, increased precision, and similar prognostic in terms of local control, locoregional control, and disease-free survival when compared to the SM method., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Different factors can affect the quality of life of patients treated for head and neck cancer undergoing major surgical intervention. However, it remains unclear which specific factors and what possible interventions could have the greatest influence on quality of life postoperatively for patients undergoing surgical resection with free flap reconstruction. The objective of our systematic review was to identify which factors, at the time of surgical treatment, are associated with a worse postoperative quality of life for patients undergoing surgical resection with free flap reconstruction for head and neck cancer., Methods: We performed a systematic review of MEDLINE, Embase, CINAHL, Web of Science, and the Cochrane Central Register of Controlled Trials (CENTRAL), from their inception through November 2021. We included peer reviewed studies that evaluated the impact of specific factors on quality of life for adult patients who underwent surgery with free flap reconstruction for head and neck cancer. Two reviewers independently screened citations for eligibility and extracted data. Risk of bias of each study was evaluated using the New-Castle Ottawa Scale. Vote counting and qualitative review were used to synthesize results. All relevant findings were reported., Results: We initially identified 1971 articles. We included 22 articles in our systematic review, totaling 1398 patients. There was a high level of variability for factors evaluated throughout studies and many studies presented small sample sizes. However, some factors were associated with worse long-term quality of life, including older age, radiotherapy, higher tumor stage, dysphagia, anxiety as well as depressive symptoms. Very few articles analyzed their data for specific tumor subsites and the impact of psychosocial factors was rarely evaluated throughout studies., Conclusions: For patients with head and neck cancer requiring free flap reconstruction, some specific factors may correlate with changes in quality of life. However, these findings are based on very few and mostly underpowered studies. A better understanding of factors affecting quality of life could allow a more personalized and overall better quality of care for patients., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article

Purpose: Tracheostomy is a surgical procedure that is commonly performed in patients admitted to the intensive care unit (ICU). It is frequently required in patients with moderate to severe traumatic brain injury (TBI), a subset of patients with prolonged altered state of consciousness that may require a long period of mechanical respiratory assistance. While many clinicians favour the use of early tracheostomy in TBI patients, the evidence in favour of this practice remains scarce. The aims of our study were to evaluate the potential clinical benefits of tracheostomy versus prolonged endotracheal intubation, as well as whether the timing of the procedure may influence outcome in patients with moderate to severe TBI., Methods: We conducted a retrospective multicentre cohort study based on data from the provincial integrated trauma system of Quebec (Québec Trauma Registry). The study population was selected from adult trauma patients hospitalized between 2013 and 2019. We included patients 16 yr and older with moderate to severe TBI (Glasgow Coma Scale score < 13) who required mechanical ventilation for 96 hr or longer. Our primary outcome was 30-day mortality. Secondary outcomes included hospital and ICU mortality, six-month mortality, duration of mechanical ventilation, ventilator-associated pneumonia, ICU and hospital length of stay as well as orientation of patients upon discharge from the hospital. We used propensity score covariate adjustment. To overcome the effect of immortal time bias, an extended Cox shared frailty model was used to compare mortality between groups., Results: From 2013 to 2019, 26,923 patients with TBI were registered in the Québec Trauma Registry. A total of 983 patients who required prolonged endotracheal intubation for 96 hr or more were included in the study, 374 of whom underwent a tracheostomy and 609 of whom remained intubated. We observed a reduction in 30-day mortality (adjusted hazard ratio, 0.33; 95% confidence interval, 0.21 to 0.53) associated with tracheostomy compared with prolonged endotracheal intubation. This effect was also seen in the ICU as well as at six months. Tracheostomy, when compared with prolonged endotracheal intubation, was associated with an increase in the duration of mechanical respiratory assistance without any increase in the length of stay. No effect on mortality was observed when comparing early vs late tracheostomy procedures. An early procedure was associated with a reduction in the duration of mechanical respiratory support as well as hospital and ICU length of stay., Conclusion: In this multicentre cohort study, tracheostomy was associated with decreased mortality when compared with prolonged endotracheal intubation in patients with moderate to severe TBI. This effect does not appear to be modified by the timing of the procedure. Nevertheless, the generalization and application of these results remains limited by potential residual time-dependent indication bias., (© 2023. The Author(s).)

Objective: The optimal timing of tracheostomy in nonneurologically injured mechanically ventilated critically ill adult patients is uncertain. We conducted a systematic review of randomized controlled trials to evaluate the effect of early versus late tracheostomy or prolonged intubation in this population., Data Sources: We searched MEDLINE, Embase, CENTRAL, CINAHL, and Web of science databases for randomized controlled trials comparing early tracheostomy (<10 d of intubation) with late tracheostomy or prolonged intubation in adults., Data Selection: We selected trials comparing early tracheostomy (defined as being performed less than 10 d after intubation) with late tracheostomy (performed on or after the 10th day of intubation) or prolonged intubation and no tracheostomy in nonneurologically injured patients. The primary outcome was overall mortality. Secondary outcomes included ventilator-associated pneumonia, duration of mechanical ventilation, ICU, and hospital length of stay., Data Extraction: Two reviewers screened citations, extracted data, assessed the risk of bias, and classification of Grading of Recommendations, Assessment, Development, and Evaluation independently., Data Synthesis: Our search strategy yielded 8,275 citations, from which nine trials (n = 2,457) were included. We did not observe an effect on the overall mortality of early tracheostomy compared with late tracheostomy or prolonged intubation (risk ratio, 0.91, 95% CI, 0.82-1.01; I2 = 18%). Our results were consistent in all subgroup analyses. No differences were observed in ICU and hospital length of stay, duration of mechanical ventilation, incidence of ventilator-acquired pneumonia, and complications. Our trial sequential analysis showed that our primary analysis on mortality was likely underpowered., Conclusion: In our systematic review, we observed that early tracheostomy, as compared with late tracheostomy or prolonged intubation, was not associated with a reduction in overall mortality. However, we cannot exclude a clinically relevant reduction in mortality considering the level of certainty of the evidence. A well-designed trial is needed to answer this important clinical question., Competing Interests: Drs. Villemure-Poliquin and Lessard Bonaventure are recipients of training awards from the Fonds de la Recherche du Québec—Santé (FRQS). Dr. Lauzier is a recipient of a research salary support award from FRQS. Dr. Zarychanski is supported by the Lyonel G. Israels Research Chair in Hematology, University of Manitoba. Dr. Turgeon is the chairholder of the Canada Research Chair in Critical Care Neurology and Trauma. Dr. Audet disclosed that she is a consultant for Merck Canada. Dr. Gagnon disclosed work for hire. Dr. Turgeon’s institution received funding from the Canadian Institutes of Health Research. The remaining authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2022 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved.)

Background: The traditional management of papillary thyroid cancer (PTC) is thyroidectomy (total or partial removal of the thyroid). Active surveillance (AS) may be considered as an alternative option for small, low risk PTC. AS involves close follow-up (including regularly scheduled clinical and radiological assessments), with the intention of intervening with surgery for disease progression or patient preference., Methods: This is a protocol for a prospective, observational, long-term follow-up multi-centre Canadian cohort study. Consenting eligible adults with small, low risk PTC (< 2cm in maximal diameter, confined to the thyroid, and not immediately adjacent to critical structures in the neck) are offered the choice of AS or surgery for management of PTC. Patient participants are free to choose either option (AS or surgery) and the disease management course is thus not assigned by the investigators. Surgery is provided as usual care by a surgeon in an institution of the patient's choice. Our primary objective is to determine the rate of 'failure' of disease management in respective AS and surgical arms as defined by: i) AS arm - surgery for progression of PTC, and ii) surgical arm - surgery or other treatment for disease persistence or progression after completing initial treatment. Secondary outcomes include long-term thyroid oncologic and treatment outcomes, as well as patient-reported outcomes., Discussion: The results from this study will provide long-term clinical and patient reported outcome evidence regarding active surveillance or immediate surgery for management of small, low risk PTC. This will inform future clinical trials in disease management of small, low risk papillary thyroid cancer., Registration Details: This prospective observational cohort study is registered on clinicaltrials.gov (NCT04624477), but it should not be considered a clinical trial as there is no assigned intervention and patients are free to choose either AS or surgery., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sawka, Ghai, Tomlinson, Baxter, Corsten, Imran, Bissada, Lebouef, Audet, Brassard, Zhang, Gupta, Nichols, Morrison, Johnson-Obeski, Prisman, Anderson, Chandarana, Ghaznavi, Jones, Gafni, Matelski, Xu, Goldstein and the Canadian Thyroid Cancer Active Surveillance Study Group.)

The signalling functions of many G protein-coupled receptors (GPCRs) expressed in the myocardium are incompletely understood. Among these are the endothelin receptor (ETR) family and α 1 -adrenergic receptor (α 1 -AR), which are thought to couple to the G protein Gαq. In this study, we used transcriptome analysis to compare the signalling networks downstream of these receptors in primary neonatal rat cardiomyocytes. This analysis indicated increased expression of target genes of cAMP responsive element modulator (CREM) after 24 h treatment with the α 1 -AR agonist phenylephrine, but not the ETR agonist endothelin-1, suggesting a specific role for the α 1 -AR in promoting cAMP production in cardiomyocytes. To validate the difference observed between these two GPCRs, we used heterologous expression of the receptors and genetically encoded biosensors in HEK 293 cell lines. We validated that both α 1A - and α 1B -AR subtypes were able to lead to the accumulation of cAMP in response to phenylephrine in both the nucleus and cytoplasm in a Gαs-dependent manner. However, the ETR subtype ETA did not affect cAMP levels in either compartment. All three receptors were coupled to Gαq signalling as expected. Further, we showed that activation of PKA in different compartments was α 1 -AR subtype specific, with α 1B -AR able to activate PKA in the cytoplasm and nucleus and α 1A -AR only able to in the nucleus. We provide evidence for a pathway downstream of the α 1 -AR, and show that distinct pools of a receptor lead to differential activation of downstream effector proteins dependent on their cellular compartment., (Copyright © 2018 Elsevier Inc. All rights reserved.)

There is significant evidence that internal pools of G protein-coupled receptors (GPCRs) exist and may be affected by both endogenous signaling molecules and hydrophobic pharmaceutical ligands, once assumed to only affect cell surface versions of these receptors. Here, we discuss evidence that the biology of nuclear GPCRs in particular is complex, rich, and highly interactive with GPCR signaling from the cell surface. Caging existing GPCR ligands may be an excellent means of further stratifying the phenotypic effects of known pharmacophores such as β-adrenergic, angiotensin II, and type B endothelin receptor ligands in the cardiovascular system. We describe some synthetic strategies we have used to design ligands to go from in cellulo to in vivo experiments. We also consider how surface and intracellular GPCR signaling might be integrated and ways to dissect this. If they could be selectively targeted, nuclear GPCRs and their associated nucleoligands would represent a completely novel area for exploration by Pharma.

GPCRs form signalling complexes with other receptors as part of dimers, G proteins and effector partners. A proteomic screen to identify proteins that associate with the β 2 -adrenergic receptor (β 2 AR) identified many of components of the Endoplasmic-Reticulum-Associated Degradation (ERAD) quality control system [1], including the valosin-containing protein (VCP/p97). Here, we validated the interaction of VCP with co-expressed FLAG-β 2 AR, demonstrating, using an inducible expression system, that the interaction of FLAG-β 2 AR and VCP is not an artifact of overexpression of the β 2 AR per se. We knocked down VCP and noted that levels of FLAG-β 2 AR were increased in cells with lower VCP levels. This increase in the level of FLAG-β 2 AR did not lead to an increase in the level of functional receptor observed at the cell surface. Similarly, inhibition of the proteasome lead to a dramatic increase in the abundance of TAP-β 2 AR, while cellular responses again remained unchanged. Taken together, our data suggests that a substantial proportion of the β 2 AR produced is non-functional and VCP plays a key role in the maturation and trafficking of the β 2 AR as part of the ERAD quality control process., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: The conventional technique for cancer resection margin analysis studies only 0.1% of the surgical margins. Complete frozen section margins - also known as Mohs margins - allows for analysis of 100% of the surgical margins., Methods: The objective of our study is to compare oral cavity cancer loco-regional recurrence rates when treated by total frozen sections technique (Total Mohs margins) versus conventional margins. We conducted a multicenter retrospective cohort chart review. Loco-regional oral cancer recurrence rates were compared between patients treated with total Mohs margins (2007-2013) and patients treated with conventional margins techniques (2002-2007)., Results: After applying inclusion criteria, a total of 60 patients treated by total Mohs margins and 57 patients with conventional margins were identified. Patients had similar baseline cancer stages, pathological types, past head and neck cancers and comorbidities (all p > 0.05). One-year recurrence rate was lower (10.0% vs 21.1%, p = 0.019) in favor of Mohs total margins and stayed significantly lower at 5 years of follow-up. When adjusted for T grade with N0 disease, Mohs technique was still beneficial in loco-regional recurrence for Tis-T4N0 up to 2 years (10.5% vs 25.7%, z-score 1.849, p = 0.032). The Number Needed to Treat at 2 years of follow-up for this subgroup of patients (Tis-T4N0) is 6.6. Margins had to be retaken more often intra-operatively in Mohs technique (68.3% vs 12.3%, p < 0.0001), mainly for positive deep margins (48.6% of all margins, p = 0.028). Duration of surgery was not increased with Mohs vs conventional technique (380 min vs 475 min respectively, p = 0.025)., Conclusions: Mohs total margins may result in a significant reduction in cancer recurrence rate at 5 years compare to conventional surgery. Moreover, duration of surgery was not increased when using Mohs technique when judiciously performed.

The use of biosensors either individually or as part of panels has now become a common technique to capturing signaling events in living cells. Such biosensors have become particularly important for probing biased signaling and allostery in G protein-coupled receptor drug screening efforts. However, assumptions about the portability of such biosensors between cell types may lead to misinterpretation of drug effects on specific signaling pathways in a given cellular context. Further, the output of a particular biosensor may be different depending on where it is localized in a cell. Here, we discuss strategies to mitigate these concerns which should feed into future biosensor design and usage., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: The purpose of this study was to assess the utility of the pectoralis major muscle flap (PMMF) in the prevention of pharyngocutaneous fistula for total laryngectomy after radiotherapy (RT) METHODS: We conducted a retrospective review of 166 patients who underwent a total laryngectomy after RT between 1998 and 2012 at the CHU de Québec., Results: One hundred fifteen patients underwent a total laryngectomy with primary pharyngeal closure alone and 51 patients received an onlay PMMF. The incidence of pharyngocutaneous fistula in the PMMF group was 14% compared to 36% when only primary closure was done (p = .004). However, the PMMF did not influence the treatment needed for the healing of this complication (p = 1.00). The development of pharyngocutaneous fistula increased the length of stay from 19 to 50 days (p < .0001) and delayed the initiation of oral diet from 15 to 25 days (p = .03)., Conclusion: Nonirradiated tissue coverage should be routine in total laryngectomy after RT. PMMF is a good adjunct to prevent pharyngocutaneous fistula., (© 2014 Wiley Periodicals, Inc.)

According to the standard model of G protein-coupled receptor (GPCR) signaling, GPCRs are localized to the cell membrane where they respond to extracellular signals. Stimulation of GPCRs leads to the activation of heterotrimeric G proteins and their intracellular signaling pathways. However, this model fails to accommodate GPCRs, G proteins, and their downstream effectors that are found on the nuclear membrane or in the nucleus. Evidence from isolated nuclei indicates the presence of GPCRs on the nuclear membrane that can activate similar G protein-dependent signaling pathways in the nucleus as at the cell surface. These pathways also include activation of cyclic adenosine monophosphate, calcium and nitric oxide synthase signaling in cardiomyocytes. In addition, a number of distinct heterotrimeric and monomeric G proteins have been found in the nucleus of various cell types. This review will focus on understanding the function of nuclear G proteins with a focus on cardiac signaling where applicable.

It has become clear in recent years that the Gβγ subunits of heterotrimeric proteins serve broad roles in the regulation of cellular activity and interact with many proteins in different subcellular locations including the nucleus. Protein affinity purification is a common method to identify and confirm protein interactions. When used in conjugation with mass spectrometry it can be used to identify novel protein interactions with a given bait protein. The tandem affinity purification (TAP) technique identifies partner proteins bound to tagged protein bait. Combined with protocols to enrich the nuclear fraction of whole cell lysate through sucrose cushions, TAP allows for purification of interacting proteins found specifically in the nucleus. Here we describe the use of the TAP technique on cytosolic and nuclear lysates to identify candidate proteins, through mass spectrometry, that bind to Gβ1 subunits.

This study assessed how conformational information encoded by ligand binding to δ-opioid receptors (DORs) is transmitted to Kir3.1/Kir3.2 channels. Human embryonic kidney 293 cells were transfected with bioluminescence resonance energy transfer (BRET) donor/acceptor pairs that allowed us to evaluate independently reciprocal interactions among signaling partners. These and coimmunoprecipitation studies indicated that DORs, Gβγ, and Kir3 subunits constitutively interacted with one another. GαoA associated with DORs and Gβγ, but despite being part of the complex, no evidence of its direct association with the channel was obtained. DOR activation by different ligands left DOR-Kir3 interactions unmodified but modulated BRET between DOR-GαoA, DOR-Gβγ, GαoA-Gβγ, and Gβγ-Kir3 interfaces. Ligand-induced BRET changes assessing Gβγ-Kir3.1 subunit interaction 1) followed similar kinetics to those monitoring the GαoA-Gβγ interface, 2) displayed the same order of efficacy as those observed at the DOR-Gβγ interface, 3) were sensitive to pertussis toxin, and 4) were predictive of whether a ligand could evoke channel currents. Conformational changes at the Gβγ/Kir3 interface were lost when Kir3.1 subunits were replaced by a mutant lacking essential sites for Gβγ-mediated activation. Thus, conformational information encoded by agonist binding to the receptor is relayed to the channel via structural rearrangements that involve repositioning of Gβγ with respect to DORs, GαoA, and channel subunits. Further, the fact that BRET changes at the Gβγ-Kir3 interface are predictive of a ligand's ability to induce channel currents points to these conformational biosensors as screening tools for identifying GPCR ligands that induce Kir3 channel activation.

Opioid tendency to generate analgesic tolerance has been previously linked to biased internalization. Here, we assessed an alternative possibility; whether tolerance of delta opioid receptor agonists (DORs) could be related to agonist-specific recycling. A first series of experiments revealed that DOR internalization by DPDPE and SNC-80 was similar, but only DPDPE induced recycling. We then established that the non-recycling agonist SNC-80 generated acute analgesic tolerance that was absent in mice treated with DPDPE. Furthermore, both agonists stabilized different conformations, whose distinct interaction with Gβγ subunits led to different modalities of β-arrestin2 (βarr2) recruitment. In particular, bioluminescence resonance energy transfer (BRET) assays revealed that sustained activation by SNC-80 drew the receptor C terminus in close proximity of the N-terminal domain of Gγ2, causing βarr2 to interact with receptors and Gβγ subunits. DPDPE moved the receptor C-tail away from the Gβγ dimer, resulting in βarr2 recruitment to the receptor but not in the vicinity of Gγ2. These differences were associated with stable DOR-βarr2 association, poor recycling, and marked desensitization following exposure to SNC-80, while DPDPE promoted transient receptor interaction with βarr2 and effective recycling, which conferred protection from desensitization. Together, these data indicate that DORs may adopt ligand-specific conformations whose distinct recycling properties determine the extent of desensitization and are predictive of analgesic tolerance. Based on these findings, we propose that the development of functionally selective DOR ligands that favor recycling could constitute a valid strategy for the production of longer acting opioid analgesics.

Objective: To establish whether outpatient thyroid surgery is safe., Design: Prospective, observational cohort., Setting: Two tertiary care centres., Methods: Although there is currently a trend toward ambulatory surgery in many domains, thyroidectomy has traditionally remained an inpatient procedure. We present our 200-case experience of total and partial thyroidectomy in an outpatient setting. Consecutive patients were prospectively recruited between May 2009 and October 2010. Surgeries were performed by four surgeons. A postoperative parathyroid hormone (PTH) level was obtained in the recovery room and used to guide the prescription of calcium and calcitriol oral supplements according to our institutional protocol., Main Outcome Measures: Postoperative complications, admission and readmission rates, patients' satisfaction, and feeling of security on a 10-point scale., Results: A total of 171 patients, for a total of 200 surgical procedures: 100 hemithyroidectomies, 34 completion hemithyroidectomies, and 66 total thyroidectomies (including 14 with central compartment dissection). Immediate admission was decided in 12% of cases due to peroperative findings (15 patients), anesthetic considerations (7 patients), bilateral vocal fold paralysis noted in the recovery room (1 patient), and surgery late in the afternoon (1 patient). Two patients were readmitted for surgical site infections and one due to hypocalcemia. Temporary symptomatic hypocalcemia or decreased PTH level occurred in 10% (20 patients). On average, patients' satisfaction and feeling of security reached 9.3 on a 10-point scale., Conclusion: Outpatient thyroid surgery is a safe and desirable option.

Bioluminescence energy transfer (BRET) has become a powerful tool to study protein-protein interactions and conformational changes among interacting proteins. In particular, BRET assays performed in living cells have revealed that heptahelical receptors (7TMRs), heterotrimeric G proteins and their proximal effectors form constitutive signalling complexes. BRET technology has also allowed us to demonstrate that these multimeric protein arrays remain intact throughout initial stages of receptor signalling, thus providing a platform for direct transmission of conformational information from activated receptors to downstream signalling partners. A clear example of the latter are the distinct intermolecular re-arrangements undergone by 7TMRs and G protein subunits following activation of the receptor by different ligands. Here we present protocols describing the type of BRET assay that has been used to reveal the existence of constitutive signalling arrays formed by 7TMRs and proximal signalling partners as well as the ability of complex components to undergo ligand-specific conformational changes.

Opiates are the most effective analgesics available for the treatment of severe pain. However, their clinical use is restricted by unwanted side effects such as tolerance, physical dependence and respiratory depression. The strategy to develop new opiates with reduced side effects has mainly focused on the study and production of ligands that specifically bind to different opiate receptors subtypes. However, this strategy has not allowed the production of novel therapeutic ligands with a better side effects profile. Thus, other research strategies need to be explored. One which is receiving increasing attention is the possibility of exploiting ligand ability to stabilize different receptor conformations with distinct signalling profiles. This newly described property, termed functional selectivity, provides a potential means of directing the stimulus generated by an activated receptor towards a specific cellular response. Here we summarize evidence supporting the existence of ligand-specific active conformations for two opioid receptors subtypes (delta and mu), and analyze how functional selectivity may contribute in the production of longer lasting, better tolerated opiate analgesics. double dagger.

Objective: To obtain completely negative margins of 1 to 5 mm at the time of surgery for oral tongue squamous cell carcinoma by using a Mohs-like technique., Study Design: Case series of 12 patients (4 T1, 5 T2, 2 T3, 1 T4) and a review of the literature., Results: For the first six cases, complete, colored for precise orientation, frozen margins of high quality were obtained in a relatively short time (20-75 minutes). Four levels were evaluated within 1 to 2 mm of the line of resection. Obtaining complete free margins for a thickness of 5 mm was done for the last six cases. The time was longer (70-120 minutes) but did not exceed the time necessary to perform the neck dissection, except for one patient. The technique using the scalpel and scissors implied slightly more bleeding, which was never a problem. We have observed no recurrence for these 12 patients (follow-up 12-34 months)., Conclusion: The review of the literature demonstrates that invaded and close margins confer a higher recurrence rate. We have obtained 1 to 2 mm (first six patients) and 5 mm (last six patients) thick, complete, oriented, and free frozen margins with success and no recurrence, but the follow-up was short. We prefer to obtain a 5 mm thick margin when possible. The delay to obtain the pathologic result is reasonable. This approach should reduce dramatically the problem of positive and close margins at the final pathology and, consequently, the rate of local control.

Objective: Positive margins for squamous cell carcinoma of the oral tongue on final pathology are a poor prognostic factor associated with a higher likelihood of local recurrence. Obtaining margin status in a relatively short time by using complete frozen sections, such as Mohs margins for skin cancer, would lower the recurrence rate. The goal of this study was to compare, on pig tongue, the efficacy of different techniques used to obtain complete frozen sections on histologic glass slides of optimal quality., Study Design: We compared the quality of frozen section glass slides on fresh pig tongues. The partial glossectomy was executed with either a sharp instrument (scalpel and scissors), electrocautery in the cutting mode, or electrocautery at the coagulation mode. For each of the three methods, we also compared the frozen section, obtained on the line of resection and for a thickness of 1 to 1.3 mm, using either the cryostat or isopentane for the freezing phase. The percentage and quality of epithelium and muscle present on histologic glass slides were assessed by a pathologist independently., Results: Complete frozen margins of high quality were obtained in a relatively short time for all techniques (28-38 minutes). Sharp dissection showed better results: a shorter processing time for the specimens and better quality for the histologic glass slides. Using cryostat or isopentane for the freezing phase did not show any significant difference., Conclusion and Significance: Complete frozen margins (Mohs margins) of high quality are feasible. Histologic glass slides of very good quality are obtained when using a sharp dissection technique. For the freezing period, both isopentane and cryostat offer very good results. This approach is appropriate on animals and needs further study in clinical situations. The human experience will be presented in the next article which is: Complete frozen section margins (with measurable 1 or 5 mm thick free margin) for cancer of the oral tongue (Clinical experience Part 2 [CSO2008]).

Abstract An important limitation in the clinical use of opiates is progressive loss of analgesic efficacy over time. Development of analgesic tolerance is tightly linked to receptor desensitization. In the case of delta opioid receptors (DOR), desensitization is especially swift because receptors are rapidly internalized and are poorly recycled to the membrane. In the present study, we investigated whether Src activity contributed to this sorting pattern and to functional desensitization of DORs. A first series of experiments demonstrated that agonist binding activates Src and destabilizes a constitutive complex formed by the spontaneous association of DORs with the kinase. Src contribution to DOR desensitization was then established by showing that pre-treatment with Src inhibitor PP2 (20 microM; 1 hr) or transfection of a dominant negative Src mutant preserved DOR signalling following sustained exposure to an agonist. This protection was afforded without interfering with endocytosis, but suboptimal internalization interfered with PP2 ability to preserve DOR signalling, suggesting a post-endocytic site of action for the kinase. This assumption was confirmed by demonstrating that Src inhibition by PP2 or its silencing by siRNA increased membrane recovery of internalized DORs and was further corroborated by showing that inhibition of recycling by monensin or dominant negative Rab11 (Rab11S25N) abolished the ability of Src blockers to prevent desensitization. Finally, Src inhibitors accelerated recovery of DOR-Galphal3 coupling after desensitization. Taken together, these results indicate that Src dynamically regulates DOR recycling and by doing so contributes to desensitization of these receptors.