Author Correction: Structural insights into µ-opioid receptor activation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Indocyanine green angiography guidance for vascular preservation in skin and nipple sparing mastectomy.

PURPOSE: The skin and/or nipple-sparing approach has become an oncologically sound and desirable choice for women choosing mastectomy. Indocyanine green (ICG) perfusion imaging has been shown to reduce ischemic complications in mastectomy skin flaps. Immediate reconstruction requires a well-vascularized skin flap capable of tolerating full expansion. Identification of the perforating subcutaneous vessels to the skin envelope may allow for better and more consistent blood vessel preservation and flap perfusion. METHODS: The authors conducted an institutional review board-approved prospective study with 41 patients to assess the feasibility of using ICG perfusion imaging to visualize, cutaneously map, and preserve the vessels that supply the skin flap and nipple-areolar complex. For each patient, the number of vessels initially mapped, the number of vessels preserved, the extent to which each vessel was preserved, and the proportion of the flap with adequate perfusion (as defined by the SPY-Q > 20% threshold) was recorded and analyzed. RESULTS: Vessels were able to be identified and marked in a high majority of patients (90%). There was a moderate linear relationship between the number of vessels marked and the number preserved. Successful mapping of vessels was associated with lower rates of wound breakdown (p = 0.036). Mapping and preserving at least one vessel led to excellent flap perfusion (> 90%). No increase in complications was observed from utilizing ICG angiography preoperatively. CONCLUSION: This prospective study using preoperative ICG perfusion mapping demonstrated safety, feasibility, and good prognostic outcomes. LEVEL OF EVIDENCE: III.

Safety and utility of panniculectomy in renal transplant candidates and end stage renal disease patients.

BACKGROUND: As the obesity crisis in the United States continues, some renal transplantation centers have liberalized their BMI criteria necessary for transplant eligibility. More individuals with larger body-habitus related comorbidities with End-Stage Renal Disease (ESRD) now qualify for renal transplantation (RT). Surgical modalities from other fields also interact with this patient population. METHODS: In order to assess surgical outcomes of panniculectomy in the context of renal transplantation and ESRD, the authors performed a systematic review following the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) 2020 guidelines. Due to a paucity of existing primary studies, we retrospectively collected data on patients with ESRD undergoing panniculectomy from the American College of Surgeons' National Surgical Quality Improvement Program (NSQIP) to evaluate outcomes of body contouring in this patient population. RESULTS: From the systematic review, a total of 783 ESRD patients underwent panniculectomy among the studies identified. Of these, 91 patients underwent panniculectomy simultaneously to RT while 692 had their pannus resected prior to kidney transplant. The most common complication was hematoma followed by wound dehiscence. From the NSQIP database, 24 868 patients met the inclusion criteria for analysis. In the setting of renal transplant status, patients with diabetes, hypertension requiring medication, and requiring dialysis were more likely to suffer postoperative complications (OR 1.31, 1.15, and 2.2, respectively). However, upon sub-analysis of specific types of complications, the only retained association was between diabetes and wound complication. CONCLUSION: Preliminary data show that panniculectomy in ESRD patients appears to be safe, though with a nominal increased risk for complications. Pannus resection does not appear to impact post-transplantation outcomes, including long-term allograft survival. Larger, higher powered, randomized studies are needed to confirm the safety, utility, and medical benefit of panniculectomy in the context of renal transplantation.

Ominous Causes of Headache.

PURPOSE OF REVIEW: While primary headaches like migraines or cluster headaches are prevalent and often debilitating, it's the secondary headaches-those resulting from underlying pathologies-that can be particularly ominous. This article delves into the sinister causes of headaches, underscoring the importance of a meticulous clinical approach, especially when presented with red flags. RECENT FINDINGS: Headaches, one of the most common complaints in clinical practice, span a spectrum from benign tension-type episodes to harbingers of life-threatening conditions. For the seasoned physician, differentiating between these extremes is paramount. Headache etiologies covered in this article will include subarachnoid hemorrhage (SAH), cervical artery dissection, cerebral venous thrombosis, meningitis, obstructive hydrocephalus, and brain tumor.

Obesity Portends Increasing Rates of Superficial Surgical Site Infection Following Pediatric Reduction Mammoplasty: A National Surgical Database Analysis.

INTRODUCTION: Pediatric reduction mammoplasty has become increasingly common due to the obesity epidemic. While obesity remains the leading cause of macromastia leading to surgery, it may also be a risk factor for postoperative complications. This study examines the safety of pediatric reduction mammoplasty and the risk of obesity for complications following this procedure. METHODS: The American College of Surgeons National Surgical Quality Improvement Program Pediatrics was queried to obtain all reduction mammoplasty cases from 2012 to 2020. Univariate and multivariate logistic regression analyses controlling for confounders were carried out to assess the relationship between body mass index (BMI) and rates of complication. RESULTS: One thousand five hundred eighty-nine patients with the primary Current Procedural Terminology code 19318 were included in the final analysis. The mean age was 16.6 (SD, 1.1) years, and the mean BMI was 30.5 (SD, 6.2) lb/in2. Notably, 49% of the patients were obese, and 31% were overweight, while only 0.4% were underweight. Forty-three patients (2.7%) sustained a superficial surgical site infection (SSI) postoperatively. Other complications were less prevalent, including deep SSI (4 patients, 0.3%), dehiscence (11, 0.7%), reoperation (21, 1%), and readmission (26, 1.6%).Independent variables analyzed included age, sex, BMI, diabetes mellitus, American Society of Anesthesiologists (ASA) class, and operative time, of which only BMI and ASA class were found to be significantly associated with SSI on univariate analysis. On multivariate logistic regression while controlling for ASA class and the false discovery rate, there was a strong association between increasing rates of superficial SSI and increasing BMI (unit odds ratio, 1.05; 95% confidence interval, [1.01, 1.09]; P = 0.02). The OR indicates that for each 1-unit increase in BMI, the odds of SSI increase by 5%. CONCLUSIONS: Complications following pediatric reduction mammoplasty are uncommon, demonstrating the safety of this procedure. High BMI was found to have a significantly higher risk for superficial SSI. Increased caution and infection prophylaxis should be taken when performing this operation on obese patients.

Prevalence of Craniofacial Injuries in the Elderly Population.

INTRODUCTION: Recognizing and understanding risk factors for craniofacial injury in the elderly is of paramount importance in prevention. This research aims to investigate the prevalence of craniofacial injuries in connection with extrinsic preventable factors, particularly identifying common household products that pose the greatest risk for such injuries. MATERIALS AND METHODS: This study was done with the utilization of the 2013 to 2022 National Electronic Injury Surveillance System (NEISS). Data gathered included patient age, injury type, cause of injury, and year of incidence. "Elderly" was defined as an individual of 65 years of age or older. RESULTS: There was a total of 9,703,688 estimated national cases of elderly craniofacial injury from 2013 to 2022. In all, 5,888,112 (60.68%) of these occurred in females. In descending order, the 5 most common items responsible for craniofacial injury in the elderly are floors/flooring Materials (3,741,706, 30.92%), beds/bed frames (1,250,396, 10.33%), stairs/steps (907,92, 7.50%), chairs (546,697, 4.52%), and tables (453,989, 3.75%). These top 5 account for roughly 57% of all cases. The 5 most common presenting diagnoses were internal injury (2,957,095, 40.21%), lacerations (1,435,926, 19.53%), ABR (1,191,008, 16.20%), fracture (568,842, 7.74%), and hematoma (355,871, 4.84%). CONCLUSIONS: Out of the roughly 10 million cases of craniofacial injury in the last decade, ~three-fifths have happened to women. The majority of injuries occur in a home setting. The overwhelming majority of cases were related to the product code 1807-floors or flooring materials, and the largest diagnosis was internal injury by a wide margin. Evidently, there is a large population of elderly patients who suffer from craniofacial injuries related to objects and items that permeate within their living residences. The elimination of excess elderly craniofacial injury can be achieved by reducing fall risk factors in the immediate vicinity of the elderly.

The risk factors for radial artery and saphenous vein graft occlusion are different.

Objectives. To determine risk factors for radial artery and saphenous vein graft occlusion during long-term follow-up after coronary artery bypass grafting (CABG). Methods: From a cohort of 119 patients who had received a radial artery graft, 76 - of whom 55 also had at least one saphenous vein graft - underwent a preplanned direct angiography and anthropometric, biochemical, and endothelial function assessment 7.6-12.1 (mean 8.9) years after CABG. Comorbidity, medication, and smoking habits were also recorded. The association between these parameters and conduit longevity was analyzed in univariable and multivariable logistic regression models. Results: Radial artery graft occlusions were associated with higher plasma levels of high-sensitive C-reactive protein and patency was best among patients with pharmacologically treated hypertension. The sole independent risk factor identified for saphenous vein graft occlusion was tobacco smoking 8-12 years postoperatively. Conclusion: Our data support the contention that the pathogenesis of radial artery graft failure is distinct from vein graft disease and is related to hypertension status and systemic inflammation. These risk factors are potential targets for preventive measures. Accordingly, the study supports the eventual design of personalized secondary prevention regimens.Clinical registration number: ISRCTN23118170.

Structural insights into µ-opioid receptor activation.

Activation of the µ-opioid receptor (µOR) is responsible for the efficacy of the most effective analgesics. To shed light on the structural basis for µOR activation, here we report a 2.1 Å X-ray crystal structure of the murine µOR bound to the morphinan agonist BU72 and a G protein mimetic camelid antibody fragment. The BU72-stabilized changes in the µOR binding pocket are subtle and differ from those observed for agonist-bound structures of the ß2-adrenergic receptor (ß2AR) and the M2 muscarinic receptor. Comparison with active ß2AR reveals a common rearrangement in the packing of three conserved amino acids in the core of the µOR, and molecular dynamics simulations illustrate how the ligand-binding pocket is conformationally linked to this conserved triad. Additionally, an extensive polar network between the ligand-binding pocket and the cytoplasmic domains appears to play a similar role in signal propagation for all three G-protein-coupled receptors.

Direct angiography demonstrates equal 8-12 years patency rates of radial artery and saphenous vein grafts.

Objectives: The benefits of coronary artery bypass surgery depend on lasting graft patency. To aid rational graft selection, the relative long-term merits of radial artery and saphenous vein grafts need to be determined by a gold standard method and with minimal clinically driven selection bias. Methods: The patency rates of various conduits were determined by direct angiography in 76 patients from a cohort of 119 undergoing coronary artery bypass grafting 7.6-12.1 (mean 8.9) years before. Results: 14 out of 76 radial artery and 10 out of 61 saphenous vein grafts were occluded (rates 0.18 and 0.16, respectively). Conclusion: The high long-term patency rate of saphenous vein grafts does not support a preferential use of the radial artery as a coronary artery bypass conduit. Clinical registration number: ISRCTN23118170.

Cigarette smoking is associated with adverse survival among women with ovarian cancer: Results from a pooled analysis of 19 studies.

Cigarette smoking is associated with an increased risk of developing mucinous ovarian tumors but whether it is associated with ovarian cancer survival overall or for the different histotypes is unestablished. Furthermore, it is unknown whether the association between cigarette smoking and survival differs according to strata of ovarian cancer stage at diagnosis. In a large pooled analysis, we evaluated the association between various measures of cigarette smoking and survival among women with epithelial ovarian cancer. We obtained data from 19 case-control studies in the Ovarian Cancer Association Consortium (OCAC), including 9,114 women diagnosed with ovarian cancer. Cox regression models were used to estimate adjusted study-specific hazard ratios (HRs), which were combined into pooled hazard ratios (pHR) with corresponding 95% confidence intervals (CIs) under random effects models. Overall, 5,149 (57%) women died during a median follow-up period of 7.0 years. Among women diagnosed with ovarian cancer, both current (pHR = 1.17, 95% CI: 1.08-1.28) and former smokers (pHR = 1.10, 95% CI: 1.02-1.18) had worse survival compared with never smoking women. In histotype-stratified analyses, associations were observed for mucinous (current smoking: pHR = 1.91, 95% CI: 1.01-3.65) and serous histotypes (current smoking: pHR = 1.11, 95% CI: 1.00-1.23; former smoking: pHR = 1.12, 95% CI: 1.04-1.20). Further, our results suggested that current smoking has a greater impact on survival among women with localized than disseminated disease. The identification of cigarette smoking as a modifiable factor associated with survival has potential clinical importance as a focus area to improve ovarian cancer prognosis.

Insulin Degludec 200 Units/mL Is Associated With Lower Injection Frequency and Improved Patient-Reported Outcomes Compared With Insulin Glargine 100 Units/mL in Patients With Type 2 Diabetes Requiring High-Dose Insulin.

IN BRIEF Many patients with type 2 diabetes require high basal insulin doses, necessitating multiple injections, increasing patient burden, and resulting in reduced treatment adherence. This randomized, controlled, crossover trial compared the efficacy, safety, and patient-reported outcomes for a concentrated formulation of insulin degludec (200 units/mL) to those of insulin glargine in patients requiring high doses of basal insulin. By offering equivalent glycemic control while reducing the rate of confirmed hypoglycemia and the number of injections required for administration, insulin degludec 200 units/mL may be preferred by patients with type 2 diabetes who require high basal insulin doses.

Effect of Insulin Degludec vs Insulin Glargine U100 on Hypoglycemia in Patients With Type 1 Diabetes: The SWITCH 1 Randomized Clinical Trial.

IMPORTANCE: Hypoglycemia, common in patients with type 1 diabetes, is a major barrier to achieving good glycemic control. Severe hypoglycemia can lead to coma or death. OBJECTIVE: To determine whether insulin degludec is noninferior or superior to insulin glargine U100 in reducing the rate of symptomatic hypoglycemic episodes. DESIGN, SETTING, AND PARTICIPANTS: Double-blind, randomized, crossover noninferiority trial involving 501 adults with at least 1 hypoglycemia risk factor treated at 84 US and 6 Polish centers (January 2014-January 12, 2016) for two 32-week treatment periods, each with a 16-week titration and a 16-week maintenance period. INTERVENTIONS: Patients were randomized 1:1 to receive once-daily insulin degludec followed by insulin glargine U100 (n = 249) or to receive insulin glargine U100 followed by insulin degludec (n = 252) and randomized 1:1 to morning or evening dosing within each treatment sequence. MAIN OUTCOMES AND MEASURES: The primary end point was the rate of overall severe or blood glucose-confirmed (<56 mg/dL) symptomatic hypoglycemic episodes during the maintenance period. Secondary end points included the rate of nocturnal symptomatic hypoglycemic episodes and proportion of patients with severe hypoglycemia during the maintenance period. The noninferiority criterion for the primary end point and for the secondary end point of nocturnal hypoglycemia was defined as an upper limit of the 2-sided 95% CI for a rate ratio of 1.10 or lower; if noninferiority was established, 2-sided statistical testing for superiority was conducted. RESULTS: Of the 501 patients randomized (mean age, 45.9 years; 53.7% men), 395 (78.8%) completed the trial. During the maintenance period, the rates of overall symptomatic hypoglycemia were 2200.9 episodes per 100 person-years' exposure (PYE) in the insulin degludec group vs 2462.7 episodes per 100 PYE in the insulin glargine U100 group for a rate ratio (RR) of 0.89 (95% CI, 0.85-0.94; P < .001 for noninferiority; P < .001 for superiority; rate difference, -130.31 episodes per 100 PYE; 95% CI, -193.5 to -67.16). The rates of nocturnal symptomatic hypoglycemia were 277.1 per 100 PYE in the insulin degludec group vs 428.6 episodes per 100 PYE in the insulin glargine U100 group, for an RR of 0.64 (95% CI, 0.56-0.73; P < .001 for noninferiority; P < .001 for superiority; rate difference, -61.94 episodes per 100 PYE; 95% CI, -83.85 to -40.03). A lower proportion of patients in the insulin degludec than in the insulin glargine U100 group experienced severe hypoglycemia during the maintenance period (10.3%, 95% CI, 7.3%-13.3% vs 17.1%, 95% CI, 13.4%-20.8%, respectively; McNemar P = .002; risk difference, -6.8%; 95% CI, -10.8% to -2.7%). CONCLUSIONS AND RELEVANCE: Among patients with type 1 diabetes and at least 1 risk factor for hypoglycemia, 32 weeks' treatment with insulin degludec vs insulin glargine U100 resulted in a reduced rate of overall symptomatic hypoglycemic episodes. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT02034513.

Trends in all-cause five-year mortality after head and neck cancers diagnosed over a period of 33 years. Focus on estimated degree of association with human papillomavirus.

BACKGROUND: Factors influencing survival after head and neck cancer (HNC) include among others stage, age, and sex. More recently, human papillomavirus (HPV) positivity has been described as a favorable prognostic factor in relation to some HNCs. MATERIAL AND METHODS: In this nationwide register-based cohort study of all 20 925 individuals diagnosed with squamous cell carcinoma of the head and neck (HNSCC) in Denmark 1978-2010, we investigate secular trends in all-cause five-year mortality after HNSCC according to the anticipated degree of association with HPV using a Cox proportional hazards model. Furthermore, we examine whether any trend over time differed according to sex, stage, and age at diagnosis. RESULTS: All-cause five-year mortality after HNSCC has decreased over time. The greatest decrease was seen in the last decade (2000-2010) among patients with HNSCC at sites estimated to be strongly associated with HPV, i.e. the base of the tongue and the tonsils, where a 28% decrease per five years (e.g. HRbase of tongue/tonsils=0.72; 95% CI 0.64-0.81) was observed. When examining sex- and age-specific time trends, the decrease in mortality was most pronounced among male patients and patients below 60 years at diagnosis. In contrast, no clear pattern was observed when examining five-year all-cause mortality trends according to stage. CONCLUSION: All-cause five-year mortality after HNSCC has decreased significantly in Denmark from 1978 to 2010, especially for HNSCCs at sites anticipated to be strongly associated with HPV, possibly due to an increasing proportion of HPV-positive HNSCCs.

Mortality Among Women With Cervical Cancer During or Shortly After a Pregnancy in Denmark 1968 to 2006.

OBJECTIVE: Cervical cancer diagnosed in relation to a pregnancy is rare; however, the current trend to have children later in life increases the risk of pregnancy and cervical cancer coinciding. We investigated the mortality of women diagnosed with cervical cancer during or in relation to a pregnancy. MATERIALS AND METHODS: From the nationwide Danish Cancer Registry, we identified women diagnosed with a primary cervical cancer at ages 15 to 44 years during 1968 to 2006 born after April 1, 1935. The women were linked to several Danish national registries to obtain information on births and abortions. In addition, linkage was made to the Cause of Death Register. Overall and cause-specific hazards ratios (HRs) were assessed by Cox proportional hazards regression with adjustment for age, calendar year, and extent of disease. RESULTS: A total of 6135 cervical cancers were identified. Among these, 126 women were diagnosed with cervical cancer during pregnancy, 1856 were diagnosed with cervical cancer 0 to 4 years after a pregnancy, and 4153 were diagnosed with cervical cancer more than 30 days before or 5 years or more after a pregnancy or had no known pregnancies. The latter group was used as reference. The adjusted HR for death due to cervical cancer was 1.77 (95% confidence interval, 1.21-2.60) among women diagnosed with cervical cancer during pregnancy compared with that in the reference group, while the corresponding HR among women with cervical cancer 0 to 4 years after pregnancy was 0.96 (95% confidence interval, 0.83-1.10) compared with that in the reference group. CONCLUSIONS: Our results suggest an increased mortality for women diagnosed with cervical cancer during pregnancy, but not among those diagnosed shortly after pregnancy. This finding should be explored further in larger populations.

A nationwide analysis of upper extremity injuries in martial arts.

Objective: Given the rise of martial arts within the fitness and recreational industry, it is important to study trends in injury and note that practicing martial arts comes with a risk of injury, particularly to the upper extremities. By studying consumer product-related injuries to the upper extremities and addressing the current gap in the literature, future martial arts equipment can come with increased safety features, better provide information to healthcare providers treating such injuries, and improve risk mitigation through the Consumer Product Safety Commission. Methods: The National Electronic Injury Surveillance System Database was analyzed to collect data on martial arts equipment-related injuries on the upper extremities within the last 10 years. Results: Over the most recent span of 10 years (2013-2022), there were approximately 78,680 injuries reported to the ED. The majority of them took place in 2013 (12.8%). Men were 2.4 times more likely to get injured than women. Injuries peaked in the age group 10-19-year-olds (33%) and particularly age 12 (4.8%). The upper extremity most commonly affected was the shoulder (29.1%), and the most common diagnosis group was a fracture (29.2%). Most injuries presented to the emergency department were not hospitalized (98.7%). Conclusions: This study highlights the occurrence of upper extremity injuries due to martial arts within the last 10 years and provides new information on the prevalence of such injuries. The results highlight that these injuries are usually non-severe and most commonly affect adolescent males; however, future research should explore performance-based recovery post-injury and long-term pain.

Racial disparities in subjective cognitive decline and its implications among Alzheimer's caretakers.

BACKGROUND/OBJECTIVE: Alzheimer's disease is a prominent neurodegenerative disorder characterized by cognitive decline and memory loss. Variations in subjective cognitive decline among Alzheimer's patients, often reported by caregiver, may stem from cultural, socioeconomic, healthcare access, and genetic factors. This study investigates racial disparities in subjective cognitive decline reported by caregivers and their implications. METHODS: In this study, data from 12,627 Alzheimer's caretakers from the CDC's Alzheimer's Disease and Healthy Aging Data Portal were analyzed using JMP software. Caregivers reported patients' cognitive decline for various racial categories: Asian/Pacific Islander, Black, Hispanic, Native American/Native Alaskan, and White. Fit model tests and distribution analyses were employed to assess disparities in symptom severity. The study focused on four key questions regarding symptom prevalence and healthcare communication to assess the degree of symptoms the patients were experiencing. RESULTS: Significant disparities in symptom severity reported by Alzheimer's caretakers were observed among the racial groups analyzed. The symptom severity ranked from least to most severe is the following: White, Asian/Pacific Islander, Black, Native American/Native Alaskan, and Hispanic patients. There was variance when it came to communication with healthcare providers, as the Asian population had the lowest communication rates. These findings underscore the need for targeted interventions considering cultural differences. It is important that tailoring healthcare approaches for different racial backgrounds is happening as a remedy to this gap in communication. CONCLUSION: Due to cultural, socioeconomic, genetic factors, and others, there were significant observed disparities. Tailoring interventions to these diverse populations is crucial to address these inequities.

Immediate Nipple Reconstruction in Skin-sparing Mastectomy with A Modified Wise-pattern Design.

This article discusses a novel approach to immediate nipple reconstruction during skin-sparing mastectomy with Wise-pattern design, a common procedure in direct-to-implant breast reconstruction. Traditionally, nipple reconstruction is performed as a second procedure, incurring additional costs and potential complications. This novel technique involves a simple modification to the Wise-pattern flap during the initial mastectomy, allowing for one-step reconstruction. The procedure includes preoperative markings of the Wise-pattern design, with a U-shaped flap added at the top angle to create the nipple position. This U-shaped flap, containing both skin and subcutaneous tissue, is then folded over itself and sutured, resulting in an inverted T-shaped incision with the reconstructed nipple at the apex. This article emphasizes the avoidance of a secondary operation for nipple reconstruction, avoiding additional costs and potential complications associated with flap loss, especially in postradiation patients. The technique was applied to five consecutive cases, with patient satisfaction reported as very high. Follow-up at 6 months showed no venous congestion or flap loss, and in patients without adjuvant radiation, the reconstructed nipple maintained almost all its initial postoperative height. Minor loss of nipple protrusion over time was comparable to traditional nipple reconstruction (eg, skate flap), and the construction of the new nipple required only an average of 10 extra minutes. Although larger-scale studies are needed for a comprehensive evaluation, the preliminary results suggest promising outcomes and encourage larger-scale safety and outcomes studies of this technique in Wise-pattern skin-sparing reconstructions.

Small-molecule positive allosteric modulation of homomeric kainate receptors GluK1-3: development of screening assays and insight into GluK3 structure.

The kainate receptors GluK1-3 (glutamate receptor ionotropic, kainate receptors 1-3) belong to the family of ionotropic glutamate receptors and are essential for fast excitatory neurotransmission in the brain, and are associated with neurological and psychiatric diseases. How these receptors can be modulated by small-molecule agents is not well understood, especially for GluK3. We show that the positive allosteric modulator BPAM344 can be used to establish robust calcium-sensitive fluorescence-based assays to test agonists, antagonists, and positive allosteric modulators of GluK1-3. The half-maximal effective concentration (EC50) of BPAM344 for potentiating the response of 100 µm kainate was determined to be 26.3 µm for GluK1, 75.4 µm for GluK2, and 639 µm for GluK3. Domoate was found to be a potent agonist for GluK1 and GluK2, with an EC50 of 0.77 and 1.33 µm, respectively, upon co-application of 150 µm BPAM344. At GluK3, domoate acts as a very weak agonist or antagonist with a half-maximal inhibitory concentration (IC50) of 14.5 µm, in presence of 500 µm BPAM344 and 100 µm kainate for competition binding. Using H523A-mutated GluK3, we determined the first dimeric structure of the ligand-binding domain by X-ray crystallography, allowing location of BPAM344, as well as zinc-, sodium-, and chloride-ion binding sites at the dimer interface. Molecular dynamics simulations support the stability of the ion sites as well as the involvement of Asp761, Asp790, and Glu797 in the binding of zinc ions. Using electron microscopy, we show that, in presence of glutamate and BPAM344, full-length GluK3 adopts a dimer-of-dimers arrangement.

Protein interacting with C kinase 1 (PICK1) reduces reinsertion rates of interaction partners sorted to Rab11-dependent slow recycling pathway.

The scaffolding protein PICK1 (protein interacting with C kinase 1) contains an N-terminal PSD-95/Discs large/ZO-1 (PDZ) domain and a central lipid-binding Bin/amphiphysin/Rvs (BAR) domain. PICK1 is thought to regulate trafficking of its PDZ binding partners but different and even opposing functions have been suggested. Here, we apply ELISA-based assays and confocal microscopy in HEK293 cells with inducible PICK1 expression to assess in an isolated system the ability of PICK1 to regulate trafficking of natural and engineered PDZ binding partners. The dopamine transporter (DAT), which primarily sorts to degradation upon internalization, did not form perinuclear clusters with PICK1, and PICK1 did not affect DAT internalization/recycling. However, transfer of the PICK1-binding DAT C terminus to the ß(2)-adrenergic receptor, which sorts to recycling upon internalization, led to formation of PICK1 co-clusters in Rab11-positive compartments. Furthermore, PICK1 inhibited Rab11-mediated recycling of the receptor in a BAR and PDZ domain-dependent manner. In contrast, transfer of the DAT C terminus to the δ-opioid receptor, which sorts to degradation, did not result in PICK1 co-clusters or any change in internalization/recycling. Further support for a role of PICK1 determined by its PDZ cargo was obtained for the PICK1 interaction partner prolactin-releasing peptide receptor (GPR10). GPR10 co-localized with Rab11 and clustered with PICK1 upon constitutive internalization but co-localized with the late endosomal marker Rab7 and did not cluster with PICK1 upon agonist-induced internalization. Our data suggest a selective role of PICK1 in clustering and reducing the recycling rates of PDZ domain binding partners sorted to the Rab11-dependent recycling pathway.

Identification of a small-molecule inhibitor of the PICK1 PDZ domain that inhibits hippocampal LTP and LTD.

Proteins containing PSD-95/Discs-large/ZO-1 homology (PDZ) domains play key roles in the assembly and regulation of cellular signaling pathways and represent putative targets for new pharmacotherapeutics. Here we describe the first small-molecule inhibitor (FSC231) of the PDZ domain in protein interacting with C kinase 1 (PICK1) identified by a screening of approximately 44,000 compounds in a fluorescent polarization assay. The inhibitor bound the PICK1 PDZ domain with an affinity similar to that observed for endogenous peptide ligands (K(i) approximately 10.1 microM). Mutational analysis, together with computational docking of the compound in simulations starting from the PDZ domain structure, identified the binding mode of FSC231. The specificity of FSC231 for the PICK1 PDZ domain was supported by the lack of binding to PDZ domains of postsynaptic density protein 95 (PSD-95) and glutamate receptor interacting protein 1 (GRIP1). Pretreatment of cultured hippocampal neurons with FSC231 inhibited coimmunopreciptation of the AMPA receptor GluR2 subunit with PICK1. In agreement with inhibiting the role of PICK1 in GluR2 trafficking, FSC231 accelerated recycling of pHluorin-tagged GluR2 in hippocampal neurons after internalization in response to NMDA receptor activation. FSC231 blocked the expression of both long-term depression and long-term potentiation in hippocampal CA1 neurons from acute slices, consistent with inhibition of the bidirectional function of PICK1 in synaptic plasticity. Given the proposed role of the PICK1/AMPA receptor interaction in neuropathic pain, excitotoxicity, and cocaine addiction, FSC231 might serve as a lead in the future development of new therapeutics against these conditions.