CRISPR/Cas9 unveils the dynamics of the endogenous µ‐opioid receptors on neuronal cells under continuous opioid stimulation

Long-term opioid use develops tolerance and attenuates analgesic effects. Upon activation, µ-opioid receptors (MOPs) are internalized and directed to either recycling or degradation pathway. Ligand stimulation also promotes de novo MOP synthesis. These processes collaboratively regulate MOP expression and play critical roles in tolerance development. However, there is limited understanding of how the endogenous MOP expression changes after prolonged opioid administration because previous analyses have focused on individual processes using overexpression systems, which ignored physiological regulation. Another fundamental problem is the unavailability of commercial antibodies to detect the low expression of endogenous MOP in neuronal systems. Here, we established a neuronal cell line to detect endogenous MOP with sufficient sensitivity using CRISPR/Cas9 technology. We incorporated the hemagglutinin sequence into the MOP gene of the SH-SY5Y cell. The genome-editing did not significantly impair MOP functions such as MOP internalization or the downstream signaling. The clone was differentiated into a state similar to the primary culture undergoing treatment with all-trans retinoic acid, followed by brain-derived neurotrophic factor. Upon continuous stimulation with MOP ligands, endogenous MOP constantly decreased up to 48 h. The expression level was maintained at a certain level following this period, depending on the ligand properties. DAMGO reduced MOP from the cell surface by about 70%, while morphine did so by 40%. Our results indicate that even a few days of opioid administration could significantly reduce the MOP expression level. Our cell line could be a potential tool to investigate the molecular mechanisms underlying the problems caused by long-term opioid use

Invasive Respiratory or Vasopressor Support and/or Death as a Proposed Composite Outcome Measure for Perioperative Care Research

BACKGROUND: There is a need for a clinically relevant and feasible outcome measure to facilitate clinical studies in perioperative care medicine. This large-scale retrospective cohort study proposed a novel composite outcome measure comprising invasive respiratory or vasopressor support (IRVS) and death. We described the prevalence of IRVS in patients undergoing major abdominal surgery and assessed the validity of combining IRVS and death to form a composite outcome measure. METHODS: We retrospectively collected perioperative data for 2776 patients undergoing major abdominal surgery (liver, colorectal, gastric, pancreatic, or esophageal resection) at Kyoto University Hospital. We defined IRVS as requirement for mechanical ventilation for ≥24 hours postoperatively, postoperative reintubation, or postoperative vasopressor administration. We evaluated the prevalence of IRVS within 30 postoperative days and examined the association between IRVS and subsequent clinical outcomes. The primary outcome of interest was long-term survival. Multivariable Cox proportional regression analysis was performed to adjust for the baseline patient and operative characteristics. The secondary outcomes were length of hospital stay and hospital mortality. RESULTS: In total, 85 patients (3.1%) received IRVS within 30 postoperative days, 15 of whom died by day 30. Patients with IRVS had a lower long-term survival rate (1- and 3-year survival probabilities, 66.1% and 48.5% vs 95.2% and 84.0%, respectively; P < .001, log-rank test) compared to those without IRVS. IRVS was significantly associated with lower long-term survival after adjustment for the baseline patient and operative characteristics (adjusted hazard ratio, 2.72; 95% confidence interval, 1.97–3.77; P < .001). IRVS was associated with a longer hospital stay (median [interquartile range], 65 [39–326] vs 15 [12–24] days; adjusted P < .001) and a higher hospital mortality (24.7% vs 0.5%; adjusted P < .001). Moreover, IRVS was adversely associated with subsequent clinical outcomes including lower long-term survival (adjusted hazard ratio, 1.78; 95% confidence interval, 1.21–2.63; P = .004) when the analyses were restricted to 30-day survivors. CONCLUSIONS: Patients with IRVS can experience ongoing risk of serious morbidity and less long-term survival even if alive at postoperative day 30. Our findings support the validity of using IRVS and/or death as a composite outcome measure for clinical studies in perioperative care medicine

Myocardial sympathetic denervation prevents chamber-specific alteration of beta-adrenergic transmembrane signaling in rabbits with heart failure

Objectives.The purpose of this study was to assess the effect of myocardial sympathetic denervation on the chamber-specific alteration of beta-adrenergic signaling in left ventricular failure in rabbits.Background.Local abnormalities in sympathetic nerve terminals, including the neuronal reuptake of norepinephrine, are thought to be responsible for the chamber-specific regulation of beta-adrenergic signaling in heart failure.Methods.Sixteen rabbits were given 6-hydroxydopamine, 25 mg/kg body weight intravenously on days 1 and 2 and 50 mg/kg intravenously on days 7 and 8. Another 16 rabbits received vehicle. Aortic regurgitation was induced in eight of the 6-hydroxydopamine—treated and eight of the vehicle-treated rabbits on day 14. Another eight of the 6-hydroxydopamine—treated and eight of the vehicletreated rabbits underwent a sham operation. The hearts were excised for biochemical analysis on day 21.Results.Hemodynamic characteristics on day 21 showed left ventricular failure in both the aortic regurgitation groups. The plasma norepinephrine concentration on day 21 was higher in both the aortic regurgitation groups than in the sham groups. The beta-adrenoceptor densities and isoproterenol plus 5′guanylylimidodiphosphate-, 5′-guanylylimidodiphosphate- and sodium fluoride-stimulated adenylyl cyclase activities were decreased only in the failing left ventricle of the vehicle-pretreated aortic regurgitation group, but in both ventricles of the 6-hydroxydopamine-pretreated aortic regurgitation group. The basal and forskolin-stimulated adenylyl cyclase activities were similar in both the aortic regurgitation groups and in the sham groups.Conclusions.Sympathetic denervation prevented chamberspecific alterations in beta-adrenergic signaling in acute left ventricular failure. Local loss of sympathetic nerve endings, and especially the defective neuronal norepinephrine reuptake, are likely to be responsible for the chamber-specific alteration of the beta-adrenoceptor-G protein-adenylyl cyclase system in heart failure in rabbits

Risk Factor Analysis for C5 Palsy after Double-Door Laminoplasty for Cervical Spondylotic Myelopathy

Study DesignA retrospective comparative study.PurposeTo clarify the risk factors related to the development of postoperative C5 palsy through radiological studies after cervical double-door laminoplasty (DDL).Overview of LiteratureAlthough postoperative C5 palsy is generally considered to be the result of damage to the nerve root or segmental spinal cord, the associated pathology remains controversial.MethodsA consecutive case series of 47 patients with cervical spondylotic myelopathy treated by DDL at our institution between April 2008 and April 2015 were reviewed. Postoperative C5 palsy occurred in 5 of 47 cases after DDL. We investigated 9 radiologic factors that have been reported to be risk factors for C5 palsy in various studies, and statistically examined these between the two groups of palsy and the non-palsy patients.ResultsWe found a significant difference between patients with and without postoperative C5 palsy with regards to the posterior shift of spinal cord at C4/5 (p=0.008). The logistic regression analyses revealed posterior shift of the spinal cord at C4/5 (odds ratio, 12.066; p=0.029; 95% confidence interval, 1.295–112.378). For the other radiologic factors, there were no statistically significant differences between the two groups.ConclusionsIn the present study, we showed a significant difference in the posterior shift of the spinal cord at C4/5 between the palsy and the non-palsy groups, indicating that the "tethering phenomenon" was likely a greater risk factor for postoperative C5 palsy

Antisynthetase syndrome: Pulmonary computed tomography findings of adult patients with antibodies to aminoacyl-tRNA synthetases

AbstractObjectivesTo describe the pulmonary CT findings in patients with anti-ARS-antibody-positive interstitial lung disease (anti-ARS-ILD)MethodsThe CT findings of 64 patients with anti-ARS-ILD were retrospectively reviewed. The images were retrospectively reviewed independently by 2 chest radiologists, and the final decision on the CT findings was made by a third chest radiologist.ResultsThere were 16 male and 48 female patients, aged 54.2±13.4 years. Sixteen patients had anti Jo-1, 24 had anti-EJ, 9 had anti-PL-7, 7 had anti-PL-12, 5 had anti-KS, and 3 had anti-OJ antibodies. Overall, 63 patients (98.4%) had CT findings predominantly in the lower lobe; 61 patients (95.3%) showed peripheral opacities, and 47 patients (73.4%) showed peribronchovascular opacities. Ground-glass attenuation, consolidation, and reticulation showed similar distribution patterns. Regarding detailed CT findings, 89.1% of patients had lower volume loss, 76.6% had interlobular septal thickening, and 67.2% had thickening of bronchovascular bundles. The final radiologic diagnoses were as follows: inconsistent with usual interstitial pneumonia (UIP) in 63 patients (98.4%), which included nonspecific interstitial pneumonia (NSIP) in 35 patients (55.6%), organizing pneumonia (OP) in 4 patients (6.3%), and OP with fibrosis in 22 patients (34.9%).ConclusionsThe characteristic CT findings of patients with anti-ARS-ILD were areas of ground-glass attenuation and reticulation, predominantly distributed as lower and peribronchovascular lesions, which is compatible with NSIP. One-third of patients showed OP with fibrosis

The effect of once-yearly zoledronic acid on hip structural and biomechanical properties derived using computed tomography (CT) in Japanese women with osteoporosis

The effects of zoledronic acid on hip structural and biomechanical properties were evaluated in Japanese patients with osteoporosis by computed tomography (CT). The subjects included in this study were a subset of female subjects (zoledronic acid group, 49 subjects; placebo group, 53 subjects) in the phase 3 trial (ZONE study) who were available for multi-detector row CT (MDCT) scanning. Eligible subjects were those diagnosed with primary osteoporosis based on the Diagnostic Criteria for Primary Osteoporosis (2000) by the Japanese Society for Bone and Mineral Research and who had between one and four fractured vertebrae located between the fourth thoracic vertebra and the fourth lumbar vertebra. The subjects received a once-yearly intravenous infusion of zoledronic acid 5 mg or placebo for two years. CT data were obtained at baseline and at 12 and 24 months later and analyzed under blinded conditions. The results demonstrated that once-yearly intravenous infusion of zoledronic acid improved volumetric bone mineral density (vBMD), cortical bone geometry parameters, and CT-derived biomechanical parameters at the femoral neck, intertrochanteric region, and shaft; particularly at the intertrochanteric region, significant improvements in cortical bone geometry parameters and CT-derived biomechanical parameters, compared with those in the placebo group, were detectable early, at 12 months. The present data suggest that zoledronic acid has a possibility to reduce the risk of hip fractures in Japanese patients with osteoporosis

Common Variants in a Novel Gene, FONG on Chromosome 2q33.1 Confer Risk of Osteoporosis in Japanese

Osteoporosis is a common disease characterized by low bone mass, decreased bone quality and increased predisposition to fracture. Genetic factors have been implicated in its etiology; however, the specific genes related to susceptibility to osteoporosis are not entirely known. To detect susceptibility genes for osteoporosis, we conducted a genome-wide association study in Japanese using ∼270,000 SNPs in 1,747 subjects (190 cases and 1,557 controls) followed by multiple levels of replication of the association using a total of ∼5,000 subjects (2,092 cases and 3,114 controls). Through these staged association studies followed by resequencing and linkage disequilibrium mapping, we identified a single nucleotide polymorphism (SNP), rs7605378 associated with osteoporosis. (combined P = 1.51×10−8, odds ratio = 1.25). This SNP is in a previously unknown gene on chromosome 2q33.1, FONG. FONG is predicted to encode a 147 amino-acid protein with a formiminotransferase domain in its N-terminal (FTCD_N domain) and is ubiquitously expressed in various tissues including bone. Our findings would give a new insight into osteoporosis etiology and pathogenesis

Transient acute kidney injury after major abdominal surgery increases chronic kidney disease risk and 1-year mortality

[Purpose] We conducted a retrospective cohort study to determine incidences of transient and persistent acute kidney injury (AKI) after major abdominal surgery and their impacts on long-term outcome. [Materials and methods] We enrolled 3751 patients undergoing major abdominal surgery. Postoperative AKI was classified as transient or persistent based on the return of serum creatinine to the non-AKI range within 7 days post-surgery. Primary outcome was mortality within 1 year. We used multivariable Cox proportional hazard regression analysis to assess independent associations between AKI type and mortality. [Results] Most patients with AKI were classified as transient (84%). Compared to patients without AKI, both patients with transient and persistent AKI demonstrated elevated 1-year mortality rates [adjusted hazard ratio (95% confidence interval): 2.01 (1.34–2.93); P = 0.001, and 6.20 (3.00–11.43); P < 0.001, respectively] and greater risk of chronic kidney disease progression at 1 year [adjusted odds ratio (95% confidence interval): 3.87 (2.12–7.08) and 23.70 (9.64–58.22), respectively; both P < 0.001]. [Conclusions] Although most AKI cases after major abdominal surgery recover completely within 7 days, even these patients with transient AKI are at higher risk for 1-year mortality and chronic kidney disease progression compared to patients without AKI