Single-incision laparoscopic surgery for benign multicystic mesothelioma of the peritoneum in a young man: A case report.

Benign multicystic peritoneal mesothelioma (BMPM) is a rare condition, particularly in men, and the preoperative diagnosis poses a challenge. Here, we present a case involving single-incision laparoscopic surgery (SILS) for BMPM in a 24-year-old man with a pelvic mass and a history of ulcerative colitis. Pelvic imaging revealed multifocal cysts, prompting the performance of SILS. The tumor was successfully resected with no residual lesions, and pathology confirmed the diagnosis of BMPM. This case represents the first documented instance of SILS being employed for BMPM in a man. BMPM, characterized by pelvic multifocal cysts, is a differential diagnosis, and SILS emerges as a viable option for both diagnosis and treatment.

Genome-wide analysis of DNA methylation in pseudomyxoma peritonei originated from appendiceal neoplasms.

INTRODUCTION: Pseudomyxoma peritonei (PMP) is a disease characterized by progressive accumulation of intraperitoneal mucinous ascites produced by neoplasms in the abdominal cavity. Since the prognosis of patients with PMP remain unsatisfactory, the development of effective therapeutic drug(s) is a matter of pressing concern. Genetic analyses of PMP have clarified the frequent activation of GNAS and/or KRAS. However, the involvement of global epigenetic alterations in PMPs has not been reported. METHODS: To clarify the genetic background of the 15 PMP tumors, we performed genetic analysis using AmpliSeq Cancer HotSpot Panel v2. We further investigated global DNA methylation in the 15 tumors and eight non-cancerous colonic epithelial cells using Methylation EPIC array BeadChip (Infinium 850k) containing a total of 865,918 probes. RESULTS: This is the first report of comprehensive DNA methylation profiles of PMPs in the world. We clarified that the 15 PMPs could be classified into at least two epigenotypes, unique methylation epigenotype (UME) and normal-like methylation epigenotype (NLME), and that genes associated with neuronal development and synaptic signaling may be involved in the development of PMPs. In addition, we identified a set of hypermethylation marker genes such as HOXD1 and TSPYL5 in the 15 PMPs. CONCLUSIONS: These findings may help the understanding of the molecular mechanism(s) of PMP and contribute to the development of therapeutic strategies for this life-threatening disease.

Clinical Effectiveness of Preoperative 18F-FDG PET/CT in Predicting Pathological Tumor Grade in Patients with Pseudomyxoma Peritonei Originating from Appendix: A Retrospective Cohort Study.

BACKGROUND: Cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy is the standard treatment for patients with pseudomyxoma peritonei (PMP). In some malignancies, the standard uptake value of positron emission tomography with 2-deoxy-2-18F-fluoro-D-glucose integrated with computed tomography (18F-FDG PET/CT) is now accepted as a reliable indicator of neoplastic behavior. This study aimed to evaluate the association between the maximum standardized uptake value (SUVmax) and pathological grade in patients with PMP and to investigate the significance of SUVmax in the preoperative assessment of these patients. PATIENTS AND METHODS: In this retrospective single-center study, consecutively enrolled patients diagnosed with PMP of appendiceal origin underwent preoperative 18F-FDG PET/CT. SUVmax was calculated as the highest SUVmax value in the abdomen excluding the primary site. SUVmax was compared with the pathological grade (low or high grade) of PMP tumors according to the World Health Organization classification and further analyzed with respect to the estimated cutoff point, sensitivity, specificity, and receiver operating characteristic. RESULTS: In total, 160 patients were included. CRS was successfully performed in 93 patients and palliative debulking surgery in 67 patients. The pathological grade was high in 45 patients and low in 115. High-grade patients had a higher median SUVmax on 18F-FDG PET/CT than did low-grade patients (3.83 versus 2.34, p < 0.001). The highest area under the curve was 0.81, with a sensitivity of 77.8%, specificity of 72.3%, and cutoff point of 2.63. CONCLUSION: This study suggests that the SUVmax of preoperative 18F-FDG PET/CT is associated with the pathological grade in patients with PMP.

A case of pseudomyxoma peritonei arising from a perforated intraductal papillary mucinous neoplasm that underwent cytoreductive surgery and hyperthermic intraperitoneal chemotherapy.

Pseudomyxoma peritonei (PMP) of pancreatic origin arising from an intraductal papillary mucinous neoplasm (IPMN) is rare. Cytoreductive surgery (CRS) with hyperthermic intraperitoneal chemotherapy (HIPEC) has been established as the optimal treatment for PMP. However, the benefits and safety of CRS with HIPEC for treating PMP of pancreatic origin remain unclear. Herein, we describe a case of PMP of pancreatic origin that was treated with CRS and HIPEC without postoperative complications. A 75-year-old woman was referred to our department. Computed tomography (CT) revealed a multilocular cystic tumor in the pancreatic tail, notable mucinous ascites in the abdominal cavity, and scalloping of the liver and spleen. CT did not reveal the appendix, and the ovaries were normal in size. The patient was diagnosed with PMP of pancreatic origin, and CRS and HIPEC were performed. Intraoperatively, the pancreatic tumor was perforated, and there was a large amount of mucinous ascites. We performed distal pancreatectomy in addition to CRS and HIPEC, with no intraoperative complications. The postoperative course was uneventful, and the patient survived after 6 months without recurrence. CRS with HIPEC may be a feasible treatment option for PMP of pancreatic origin.

Highly Potent and Selective Dopamine D4 Receptor Antagonists Potentially Useful for the Treatment of Glioblastoma.

To better understand the role of dopamine D4 receptor (D4R) in glioblastoma (GBM), in the present paper, new ligands endowed with high affinity and selectivity for D4R were discovered starting from the brain penetrant and D4R selective lead compound 1-(3-(4-phenylpiperazin-1-yl)propyl)-3,4-dihydroquinolin-2(1H)-one (6). In particular, the D4R antagonist 24, showing the highest affinity and selectivity over D2R and D3R within the series (D2/D4 = 8318, D3/D4 = 3715), and the biased ligand 29, partially activating D4R Gi-/Go-protein and blocking ß-arrestin recruitment, emerged as the most interesting compounds. These compounds, evaluated for their GBM antitumor activity, induced a decreased viability of GBM cell lines and primary GBM stem cells (GSC#83), with the maximal efficacy being reached at a concentration of 10 µM. Interestingly, the treatment with both compounds 24 and 29 induced an increased effect in reducing the cell viability with respect to temozolomide, which is the first-choice chemotherapeutic drug in GBM.

Association of intraoperative gross hematuria with acute kidney injury after cytoreductive surgery.

Objectives: Cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (CRS-HIPEC) has been established in the management of peritoneal carcinomatosis. Although it is still necessary to take adequate measures against major postoperative complications including acute kidney injury (AKI), consensus is lacking on how to assess and stratify risk for patients with postoperative AKI after CRS-HIPEC. The aim of this retrospective cohort study was to investigate the association of intraoperative gross hematuria as a surrogate marker of ureter injury with postoperative AKI incidence. Methods: This retrospective cohort study investigated patients without impaired preoperative kidney function who underwent CRS-HIPEC at a single referral center, and evaluated the relationship between intraoperative gross hematuria and incidence of postoperative AKI as defined by the Kidney Disease Improving Global Outcomes practice guidelines. Logistic regression analysis was performed to calculate the odds ratio of intraoperative gross hematuria for AKI, adjusting for confounding factors and other risk factors for AKI. Results: We enrolled 185 patients (males, 37%). Twenty-five patients developed intraoperative gross hematuria. Postoperative AKI occurred in 10 (40%) of 25 patients with hematuria and 28 (17.5%) of 160 patients without hematuria. The crude odds ratio for exposure to hematuria was 3.14 (95% confidence interval, 1.30-7.60; p=0.020) for postoperative AKI. Adjusted odds ratio as estimated by multivariate logistic regression was 4.57 (95% confidence interval, 1.55-13.45; p=0.006). Conclusions: Intraoperative gross hematuria is significantly associated with postoperative AKI incidence after CRS-HIPEC.

Prognostic value of 18F-FDG PET/CT with texture analysis in patients with rectal cancer treated by surgery.

PURPOSE: The aim of this study was to evaluate the ability of texture analysis using pretreatment 18F-FDG PET/CT to predict prognosis in patients with surgically treated rectal cancer. METHODS: We analyzed 94 patients with pathologically proven rectal cancer who underwent pretreatment 18F-FDG PET/CT and were subsequently treated with surgery. The volume of interest of the primary tumor was defined using a threshold of 40% of the maximum standardized uptake value (SUVmax), and conventional (SUVmax, metabolic tumor volume [MTV], total lesion glycolysis [TLG]) and textural PET features were extracted. Harmonization of PET features was performed with the ComBat method. The study endpoints were overall survival (OS) and progression-free survival (PFS), and the prognostic value of PET features was evaluated by Cox regression analysis. RESULTS: In the follow-up period (median 41.7 [interquartile range, 30.5-60.4] months), 21 (22.3%) and 30 (31.9%) patients had cancer-related death or disease progression, respectively. Univariate analysis revealed a significant association of (1) MTV, TLG, and gray-level co-occurrence matrix (GLCM) entropy with OS; and (2) SUVmax, MTV, TLG, and GLCM entropy with PFS. In multivariate analysis including clinical characteristics, GLCM entropy (≥ 2.13) was the only relevant prognostic PET feature for poor OS (hazard ratio [HR]: 4.16, p = 0.035) and PFS (HR: 2.70, p = 0.046). CONCLUSION: GLCM entropy, which indicates metabolic intratumoral heterogeneity, was an independent prognostic factor in patients with surgically treated rectal cancer. Compared with conventional PET features, GLCM entropy has better predictive value and shows potential to facilitate precision medicine.

A case of pseudomyxoma peritonei successfully treated with trifluridine/tipiracil (TAS-102) and bevacizumab after palliative debulking surgery.

Pseudomyxoma peritonei is a rare disease with a reported prevalence of about 1-3 per million people annually. Cytoreductive surgery and perioperative hyperthermic intraperitoneal chemotherapy are considered as treatment options improving disease control or long-term survival. However, for patients with incomplete cytoreduction or debulking surgery, outcomes are significantly poorer compared with patients who have obtained complete or optimal cytoreduction. In cases of high-grade pseudomyxoma peritonei that are considered inoperable and/or unresectable, combination chemotherapy regimen with a neo-angiogenesis inhibitor such as bevacizumab is recommended. In this report, a 62-year-old Japanese man presented with abdominal distention. Examination of ascites demonstrated a jelly-like consistency and peritoneal pseudomyxoma was suspected. To relieve progressive symptoms, palliative debulking surgery with total colectomy was performed. Postoperative pathology confirmed high-grade appendiceal mucinous neoplasm with high-grade pseudomyxoma peritonei. In our case, combination chemotherapy with trifluridine/tipiracil (TAS-102) and bevacizumab was initiated after palliative debulking surgery. As a result, carcinoembryonic antigen level was kept stable and the volume of ascites remained almost the same as at the beginning of treatment for more than 2 years. In conclusion, combination chemotherapy comprising TAS-102 and bevacizumab in patients with palliative debulking could be a useful option for patients with high-grade mucinous appendiceal neoplasm and high-grade pseudomyxoma peritonei.

The National Health Service (NHS) response to the COVID-19 pandemic: a colorectal surgeons experience in the UK.

The UK government was arguably slow to take action against the COVID-19 pandemic. However, since switching their policy from "mitigation" to "suppression", swift changes have been implemented to all aspects of life. In this unprecedented crisis healthcare has been on the battlefront across the globe. Every effort has been made in the UK to stop the National Health Service (NHS) from being overwhelmed, leading to the national slogan: "Stay at home. Protect the NHS. Save lives". In this article, a consultant general and colorectal surgeon in Southampton reports on the NHS response to the COVID-19 pandemic.

Cytoreductive Surgery Combined with Hyperthermic Intraperitoneal Chemotherapy for Peritoneal Metastasis from Colorectal Cancer.

Peritoneal metastasis from colorectal cancer (PM-CRC) is used to be considered a systemic and fatal condition; however, it has been growingly accepted that PM-CRC can still be local disease rather than systemic disease as analogous to liver or lung metastasis. Cytoreductive surgery (CRS) combined with hyperthermic intraperitoneal chemotherapy (HIPEC) is now considered an optimal treatment for PM-CRC with accumulating evidence. There is a good reason that CRS + HIPEC, widely accepted as a standard of care for pseudomyxoma peritonei (PMP), could be a viable option for PM-CRC given a similarity between PM-CRC and PMP. Recent years have also seen that modern systemic chemotherapy with or without molecular targeted agents can be effective for PM-CRC. It is possible that neoadjuvant or adjuvant chemotherapy combined with CRS + HIPEC could further improve outcomes. Patient selection, utilizing modern images and increasingly laparoscopy, is crucial. Particularly, diagnostic laparoscopy is likely to play a significant role in predicting the likelihood of achieving complete cytoreduction and assessing the peritoneal cancer index score.

Immunohistochemical staining as supportive diagnostic tool for pseudomyxoma peritonei arising from intraductal papillary mucinous neoplasm: A report of two cases and literature review.

BACKGROUND/OBJECTIVES: Pseudomyxoma peritonei (PMP) arising from an intraductal papillary mucinous neoplasm of the pancreas (IPMN) is a rare condition. The diagnosis of IPMN as the origin of PMP is mainly inferred from the clinical course and the exclusion of PMP from other organs. The pathological diagnosis has not yet been established. To evaluate the usefulness of immunohistochemical staining for the diagnosis of the primary lesion of PMP as IPMN. METHODS: There are 2 cases of PMP arising from IPMN between March 2010 and December 2019 at National Center for Global Health and Medicine. A PubMed search that reported PMP arising from IPMN identified 16 additional cases. Diagnostic methods and clinicopathological features of 18 cases were compared. RESULTS: Four cases including our two cases used immunohistochemical staining for the diagnosis of PMP arising from IPMN. The correspondence of the immunohistochemical staining between PMP and IPMN was shown in the three cases including previously reported two cases and one of our two cases to identify the primary lesion of PMP as IPMN. In addition, we revealed that the comparison of the immunostaining pattern of PMP with the representative immunostaining pattern of the candidate primary lesions is helpful for the diagnosis of the primary lesion of PMP. CONCLUSIONS: Immunohistochemical staining is helpful to identify the primary lesion of PMP as IPMN.

Pseudomyxoma peritonei: visceral scalloping on CT is a predictor of recurrence after complete cytoreductive surgery.

OBJECTIVES: Pseudomyxoma peritonei (PMP) is characterized by peritoneal dissemination of gelatinous ascites following rupture of a mucinous tumor. Treatment by cytoreductive surgery (CRS) has improved its prognosis. Although visceral scalloping, notably liver scalloping, on computed tomography (CT) is a typical feature of PMP, its prognostic value remains unknown. We aimed to investigate the efficacy of liver scalloping in predicting recurrence in PMP patients. METHODS: Among 159 consecutive patients with PMP who had contrast-enhanced CT between September 2012 and December 2018, 64 treatment-naïve patients who subsequently underwent CRS with complete resection (i.e., completeness of cytoreduction score (CC)-0 or CC-1), were included in analysis. Presence of liver scalloping and maximum thickness of mucin deposition at the liver surface were evaluated on CT. Disease-free survival (DFS) was determined based on the combination of postoperative CT features and tumor marker values. RESULTS: Median follow-up was 24.3 months. CT revealed liver scalloping in 40/64 (63.4%) patients. Kaplan-Meier analysis showed significantly shorter DFS in patients with scalloping than in those without (p = 0.001; hazard ratio, 4.3). In patients with scalloping, greater mucin deposition (thickness ≥ 20 mm) significantly correlated with poorer DFS (p = 0.042). In multivariate Cox proportional hazards regression including CC status, pathologic type, and tumor markers, the presence of scalloping independently and significantly correlated with DFS (p = 0.031). CONCLUSIONS: Liver scalloping was an independent predictor even after adjusting for clinical covariates. The presence of liver scalloping can lead to a high recurrence rate after CRS. KEY POINTS: • The presence of liver scalloping is a prognostic factor independent of histological grade and tumor markers. • Greater mucin deposition (thickness ≥ 20 mm at the liver surface) is associated with higher recurrence rates in patients with liver scalloping.

Control of glutamate release by complexes of adenosine and cannabinoid receptors.

BACKGROUND: It has been hypothesized that heteromers of adenosine A2A receptors (A2AR) and cannabinoid CB1 receptors (CB1R) localized in glutamatergic nerve terminals mediate the integration of adenosine and endocannabinoid signaling involved in the modulation of striatal excitatory neurotransmission. Previous studies have demonstrated the existence of A2AR-CB1R heteromers in artificial cell systems. A dependence of A2AR signaling for the Gi protein-mediated CB1R signaling was described as one of its main biochemical characteristics. However, recent studies have questioned the localization of functionally significant A2AR-CB1R heteromers in striatal glutamatergic terminals. RESULTS: Using a peptide-interfering approach combined with biophysical and biochemical techniques in mammalian transfected cells and computational modeling, we could establish a tetrameric quaternary structure of the A2AR-CB1R heterotetramer. This quaternary structure was different to the also tetrameric structure of heteromers of A2AR with adenosine A1 receptors or dopamine D2 receptors, with different heteromeric or homomeric interfaces. The specific quaternary structure of the A2A-CB1R, which depended on intermolecular interactions involving the long C-terminus of the A2AR, determined a significant A2AR and Gs protein-mediated constitutive activation of adenylyl cyclase. Using heteromer-interfering peptides in experiments with striatal glutamatergic terminals, we could then demonstrate the presence of functionally significant A2AR-CB1R heteromers with the same biochemical characteristics of those studied in mammalian transfected cells. First, either an A2AR agonist or an A2AR antagonist allosterically counteracted Gi-mediated CB1R agonist-induced inhibition of depolarization-induced glutamate release. Second, co-application of both an A2AR agonist and an antagonist cancelled each other effects. Finally, a CB1R agonist inhibited glutamate release dependent on a constitutive activation of A2AR by a canonical Gs-Gi antagonistic interaction at the adenylyl cyclase level. CONCLUSIONS: We demonstrate that the well-established cannabinoid-induced inhibition of striatal glutamate release can mostly be explained by a CB1R-mediated counteraction of the A2AR-mediated constitutive activation of adenylyl cyclase in the A2AR-CB1R heteromer.

Anti-apoptotic effect by the suppression of IRF1 as a downstream of Wnt/ß-catenin signaling in colorectal cancer cells.

Impaired Wnt signaling pathway plays a crucial role in the development of colorectal cancer through activation of the ß-catenin/TCF7L2 complex. Although genes upregulated by Wnt/ß-catenin signaling have been intensively studied, the roles of downregulated genes are poorly understood. Previously, we reported that interferon-induced proteins with tetratricopeptide repeats 2 (IFIT2) was downregulated by the Wnt/ß-catenin signaling, and that the suppressed expression of IFIT2 conferred antiapoptotic property to colorectal cancer (CRC) cells. However, the mechanisms underlying how Wnt/ß-catenin signaling regulates IFIT2 remain to be elucidated. In this study, we have uncovered that the expression of IFIT2 is induced by IRF1, which is negatively regulated by the Wnt/ß-catenin signaling. In addition, we found that downregulation of IRF1 is mediated by its degradation through the ubiquitination-proteasome pathway, and that decreased activity of a deubiquitinase complex containing USP1 and UAF1 is involved in the degradation of IRF1 by Wnt/ß-catenin signaling. These data should provide better understanding of the Wnt signaling pathway and human carcinogenesis.

Hepatectomy for metachronous colorectal liver metastases following complete cytoreductive surgery and hyperthermic intraperitoneal chemotherapy for peritoneal metastases: a report of three cases.

BACKGROUND: Cytoreductive surgery (CRS) with hyperthermic intraperitoneal chemotherapy (HIPEC) for peritoneal metastasis (PM) from colorectal cancer (CRC) has been reported to substantially improve the prognosis and the quality of life of patients in comparison to systemic chemotherapy or palliative approaches. This study aimed to demonstrate the safety and feasibility of hepatectomy for metachronous liver metastases from CRC following CRS and HIPEC for PM on the basis of three case reports. CASE PRESENTATION: We describe three cases involving patients who underwent hepatectomy for metachronous liver metastases from CRC after CRS and HIPEC for PM. All patients underwent CRS and HIPEC after primary tumor resection, and hepatectomy was performed for the metachronous liver metastases after CRS and HIPEC. The hepatectomy procedures for cases 1, 2, and 3 were left hemihepatectomy and partial resection of S5, posterior sectionectomy, and left-lateral sectionectomy and partial resection of S5 and S8, respectively. Although adhesion of surrounding organs to the liver surface was observed on a broad level, dissections and hepatectomy could be performed safely. No recurrence was detected in cases 1 and 2 after hepatectomy. In case 3, liver metastases were detected from the time of the initial diagnosis of the primary tumor, and complete remission was achieved once with systemic chemotherapy. Although we performed hepatectomy for the recurrence of liver metastases after complete remission, early re-recurrence was observed after hepatectomy. CONCLUSIONS: Hepatectomy for metachronous liver metastases after CRS and HIPEC for PM could be a multi-modality treatment option for CRC recurrence.

Development of an MSI-positive colon tumor with aberrant DNA methylation in a PPAP patient.

Polymerase proofreading-associated polyposis (PPAP) is a disease caused by germline variations in the POLE and POLD1 genes that encode catalytic subunits of DNA polymerases. Studies of cancer genomes have identified somatic mutations in these genes, suggesting the importance of polymerase proofreading of DNA replication in suppressing tumorigenesis. Here, we identified a germline frameshift variation in the POLE gene (c.4191_4192delCT, p.Tyr1398*) in a case with multiple adenomatous polyps and three synchronous colon cancers. Interestingly, one of the colon cancers showed microsatellite instability-high (MSI-H) and another microsatellite stable. Immunohistochemical staining revealed that the MSI-H tumor cells lost the expression of MLH1 protein. Whole genome sequencing of the MSI-H tumor did not find pathogenic somatic mutations in mismatch repair genes but found frameshift mutations in the TET genes that catalyze 5-methylcytosine hydroxylation. Bisulfite sequencing of the tumor corroborated an increase in the number of hypermethylated regions including the MLH1 promoter. These data indicate that PPAP patients might develop MSI-positive tumors through epigenetic silencing of MLH1. These findings will contribute to comprehensive understanding of the molecular basis of tumors that involve deficiency of proofreading activity of DNA polymerases.

Impact of a modified peritoneal cancer index using FDG-PET/CT (PET-PCI) in predicting tumor grade and progression-free survival in patients with pseudomyxoma peritonei.

OBJECTIVES: The peritoneal cancer index (PCI) is widely used for assessing pseudomyxoma peritonei (PMP) in surgery. The aim of this study was to evaluate the utility of a modified PCI using 18F-fluorodeoxyglucose (18F-FDG)-PET/CT (PET-PCI) for predicting pathologic grade and progression-free survival (PFS) in patients with PMP. METHODS: Thirty-five patients who underwent 18F-FDG-PET/CT before cytoreductive surgery and/or hyperthermic intraperitoneal chemotherapy were enrolled. PET-PCI was determined by summing up the visually scored 18F-FDG uptake of PMP lesions in 13 specific abdominal-pelvic regions. Uptake score was defined as 0, no lesion or lesion without uptake; 1, slight uptake less than or equivalent to mediastinal blood pool; 2, moderate uptake above mediastinal but below or equal to liver; and 3, intense uptake moderately to markedly higher than liver. SUVmax of the lesion was also evaluated. RESULTS: Pathologic diagnosis revealed 19 patients with low-grade PMP and 16 patients with high-grade PMP. Patients with high-grade PMP showed significantly higher PET-PCI and SUVmax than patients with low-grade PMP (PET-PCI 14.8 vs. 8.7, p = 0.007; SUVmax 3.6 vs. 2.6, p = 0.013). Using a cutoff PET-PCI of 12, Kaplan-Meier analyses showed a significant difference in PFS between patients with high and low PET-PCI (p < 0.001; hazard ratio (HR), 12.4). For SUVmax, the optimal cutoff was 2.7 and the correlation with PFS was also significant (p = 0.008; HR, 4.7). In multivariate Cox proportional-hazards regression, PET-PCI was independently and significantly correlated with PFS. CONCLUSIONS: PET-PCI can reflect histopathologic features and appears useful for predicting recurrence in patients with PMP. KEY POINTS: • Peritoneal cancer index using 18F-FDG-PET/CT (PET-PCI) has great potential for predicting progression-free survival in patients with pseudomyxoma peritonei. • PET-PCI provides higher prognostic performance than maximum standardized uptake value (SUVmax). • PET-PCI shows high correlation with histopathologic grade of pseudomyxoma peritonei.

The Effects of Terminal Tagging on Homomeric Interactions of the Sigma 1 Receptor.

The sigma 1 receptor (σ1R) has been implicated in cancers, neurological disorders, and substance use disorders. Yet, its molecular and cellular functions have not been well-understood. Recent crystal structures of σ1R reveal a single N-terminal transmembrane segment and C-terminal ligand-binding domain, and a trimeric organization. Nevertheless, outstanding issues surrounding the functional or pharmacological relevance of σ1R oligomerization remain, such as the minimal protomeric unit and the differentially altered oligomerization states by different classes of ligands. Western blot (WB) assays have been widely used to investigate protein oligomerizations. However, the unique topology of σ1R renders several intertwined challenges in WB. Here we describe a WB protocol without temperature denaturization to study the ligand binding effects on the oligomerization state of σ1R. Using this approach, we observed unexpected ladder-like incremental migration pattern of σ1R, demonstrating preserved homomeric interactions in the detergent environment. We compared the migration patterns of intact σ1R construct and the C-terminally tagged σ1R constructs, and found similar trends in response to drug treatments. In contrast, N-terminally tagged σ1R constructs show opposite trends to that of the intact construct, suggesting distorted elicitation of the ligand binding effects on oligomerization. Together, our findings indicate that the N-terminus plays an important role in eliciting the impacts of bound ligands, whereas the C-terminus is amenable for modifications for biochemical studies.

Gs- versus Golf-dependent functional selectivity mediated by the dopamine D1 receptor.

The two highly homologous subtypes of stimulatory G proteins Gαs (Gs) and Gαolf (Golf) display contrasting expression patterns in the brain. Golf is predominant in the striatum, while Gs is predominant in the cortex. Yet, little is known about their functional distinctions. The dopamine D1 receptor (D1R) couples to Gs/olf and is highly expressed in cortical and striatal areas, making it an important therapeutic target for neuropsychiatric disorders. Using novel drug screening methods that allow analysis of specific G-protein subtype coupling, we found that, relative to dopamine, dihydrexidine and N-propyl-apomorphine behave as full D1R agonists when coupled to Gs, but as partial D1R agonists when coupled to Golf. The Gs/Golf-dependent biased agonism by dihydrexidine was consistently observed at the levels of cellular signaling, neuronal function, and behavior. Our findings of Gs/Golf-dependent functional selectivity in D1R ligands open a new avenue for the treatment of cortex-specific or striatum-specific neuropsychiatric dysfunction.

Pharmacological profiling of sigma 1 receptor ligands by novel receptor homomer assays.

The sigma 1 receptor (σ1R) is a structurally unique transmembrane protein that functions as a molecular chaperone in the endoplasmic reticulum (ER), and has been implicated in cancer, neuropathic pain, and psychostimulant abuse. Despite physiological and pharmacological significance, mechanistic underpinnings of structure-function relationships of σ1R are poorly understood, and molecular interactions of selective ligands with σ1R have not been elucidated. The recent crystallographic determination of σ1R as a homo-trimer provides the foundation for mechanistic elucidation at the molecular level. Here we report novel bioluminescence resonance energy transfer (BRET) assays that enable analyses of ligand-induced multimerization of σ1R and its interaction with BiP. Haloperidol, PD144418, and 4-PPBP enhanced σ1R homomer BRET signals in a dose dependent manner, suggesting their significant effects in stabilizing σ1R multimerization, whereas (+)-pentazocine and several other ligands do not. In non-denaturing gels, (+)-pentazocine significantly decreased whereas haloperidol increased the fraction of σ1R multimers, consistent with the results from the homomer BRET assay. Further, BRET assays examining heteromeric σ1R-BiP interaction revealed that (+)-pentazocine and haloperidol induced opposite trends of signals. From molecular modeling and simulations of σ1R in complex with the tested ligands, we identified initial clues that may lead to the differed responses of σ1R upon binding of structurally diverse ligands. By combining multiple in vitro pharmacological and in silico molecular biophysical methods, we propose a novel integrative approach to analyze σ1R-ligand binding and its impact on interaction of σ1R with client proteins.