Identification of Gα12-vs-Gα13-coupling determinants and development of a Gα12/13-coupled designer GPCR.

G-protein-coupled receptors (GPCRs) transduce diverse signals into the cell by coupling to one or several Gα subtypes. Of the 16 Gα subtypes in human cells, Gα12 and Gα13 belong to the G12 subfamily and are reported to be functionally different. Notably, certain GPCRs display selective coupling to either Gα12 or Gα13, highlighting their significance in various cellular contexts. However, the structural basis underlying this selectivity remains unclear. Here, using a Gα12-coupled designer receptor exclusively activated by designer drugs (DREADD; G12D) as a model system, we identified residues in the α5 helix and the receptor that collaboratively determine Gα12-vs-Gα13 selectivity. Residue-swapping experiments showed that G12D distinguishes differences between Gα12 and Gα13 in the positions G.H5.09 and G.H5.23 in the α5 helix. Molecular dynamics simulations observed that I378G.H5.23 in Gα12 interacts with N1032.39, S1693.53 and Y17634.53 in G12D, while H364G.H5.09 in Gα12 interact with Q2645.71 in G12D. Screening of mutations at these positions in G12D identified G12D mutants that enhanced coupling with Gα12 and to an even greater extent with Gα13. Combined mutations, most notably the dual Y17634.53H and Q2645.71R mutant, further enhanced Gα12/13 coupling, thereby serving as a potential Gα12/13-DREADD. Such novel Gα12/13-DREADD may be useful in future efforts to develop drugs that target Gα12/13 signaling as well as to identify their therapeutic indications.

Two cases of laparoscopic deroofing of giant liver cysts using indocyanine green fluorescence imaging.

Laparoscopic deroofing (LD) for giant liver cysts using indocyanine green (ICG) fluorescence imaging was performed in two patients: a 53-year-old man with a 26-cm, symptomatic cyst and a 50-year-old woman with a 13-cm, symptomatic cyst. ICG fluorescence imaging can be used to easily identify the boundary between the liver parenchyma and the liver cyst. No postoperative bile leakage was observed in both patients. ICG fluorescence imaging is expected to become a desirable procedure in LD for giant liver cysts to reduce the occurrence of perioperative complications.

Mechanisms of biased agonism by Gαi/o-biased stapled peptide agonists of the relaxin-3 receptor.

The neuropeptide relaxin-3 is composed of an A chain and a B chain held together by disulfide bonds, and it modulates functions such as anxiety and food intake by binding to and activating its cognate receptor RXFP3, mainly through the B chain. Biased ligands of RXFP3 would help to determine the molecular mechanisms underlying the activation of G proteins and ß-arrestins downstream of RXFP3 that lead to such diverse functions. We showed that the i, i+4 stapled relaxin-3 B chains, 14s18 and d(1-7)14s18, were Gαi/o-biased agonists of RXFP3. These peptides did not induce recruitment of ß-arrestin1/2 to RXFP3 by GPCR kinases (GRKs), in contrast to relaxin-3, which enabled the GRK2/3-mediated recruitment of ß-arrestin1/2 to RXFP3. Relaxin-3 and the previously reported peptide 4 (an i, i+4 stapled relaxin-3 B chain) did not exhibit biased signaling. The staple linker of peptide 4 and parts of both the A chain and B chain of relaxin-3 interacted with extracellular loop 3 (ECL3) of RXFP3, moving it away from the binding pocket, suggesting that unbiased ligands promote a more open conformation of RXFP3. These findings highlight roles for the A chain and the N-terminal residues of the B chain of relaxin-3 in inducing conformational changes in RXFP3, which will help in designing selective biased ligands with improved therapeutic efficacy.

[A Case of Secondary Syphilis Mimicking a Penile Cancer with Lymph Node Metastases].

A 42-year-oldman visited our hospital because of gradually worsening penile swelling over 3 weeks. A hard mass on the glans was palpated; however, we were unable to observe it due to severe phimosis. Magnetic resonance imaging of the pelvis revealed enlargement of glans and swelling of bilateral inguinal lymph nodes as both showed a low signal intensity on T2-weightedimaging, a high signal intensity on diffusion-weighted imaging, and a low signal intensity on the apparent diffusion coefficient map. Fluorine- 18-deoxyglucose (FDG) positron emission tomography showed FDG uptake at the external iliac, common iliac, obturator, and cervical lymph nodes besides the glans and inguinal lymph nodes. Although his serum squamous cell carcinoma antigen level was within the normal range, his soluble interleukin-2 receptor concentration was elevated to 2,290 U/ml. Therefore, we diagnosed these lesions as penile cancer with multiple lymph node metastases, with a possible differential diagnosis of malignant lymphoma. We planned a penile needle biopsy; however, the rapid plasma reagin test and treponema pallidum hemagglutination test, which were performed during the preoperative examination, were positive and led to a diagnosis of secondary syphilis. The patient was treated with oral amoxicillin at 1,500 mg/day for 8 weeks. The penile and lymph node swelling subsided after starting medication.

Class B1 GPCR activation by an intracellular agonist.

G protein-coupled receptors (GPCRs) generally accommodate specific ligands in the orthosteric-binding pockets. Ligand binding triggers a receptor allosteric conformational change that leads to the activation of intracellular transducers, G proteins and ß-arrestins. Because these signals often induce adverse effects, the selective activation mechanism for each transducer must be elucidated. Thus, many orthosteric-biased agonists have been developed, and intracellular-biased agonists have recently attracted broad interest. These agonists bind within the receptor intracellular cavity and preferentially tune the specific signalling pathway over other signalling pathways, without allosteric rearrangement of the receptor from the extracellular side1-3. However, only antagonist-bound structures are currently available1,4-6, and there is no evidence to support that biased agonist binding occurs within the intracellular cavity. This limits the comprehension of intracellular-biased agonism and potential drug development. Here we report the cryogenic electron microscopy structure of a complex of Gs and the human parathyroid hormone type 1 receptor (PTH1R) bound to a PTH1R agonist, PCO371. PCO371 binds within an intracellular pocket of PTH1R and directly interacts with Gs. The PCO371-binding mode rearranges the intracellular region towards the active conformation without extracellularly induced allosteric signal propagation. PCO371 stabilizes the significantly outward-bent conformation of transmembrane helix 6, which facilitates binding to G proteins rather than ß-arrestins. Furthermore, PCO371 binds within the highly conserved intracellular pocket, activating 7 out of the 15 class B1 GPCRs. Our study identifies a new and conserved intracellular agonist-binding pocket and provides evidence of a biased signalling mechanism that targets the receptor-transducer interface.

Generation of Gαi knock-out HEK293 cells illuminates Gαi-coupling diversity of GPCRs.

G-protein-coupled receptors (GPCRs) are pivotal cell membrane proteins that sense extracellular molecules and activate cellular responses. The G-protein α subunit i (Gαi) family represents the most common GPCR-coupling partner and consists of eight subunits with distinct signaling properties. However, analyzing the coupling pattern has been challenging owing to endogenous expression of the Gαi subunits in virtually all cell lines. Here, we generate a HEK293 cell line lacking all Gαi subunits, which enables the measurement of GPCR-Gαi coupling upon transient re-expression of a specific Gαi subunit. We profile Gαi-coupling selectivity across 11 GPCRs by measuring ligand-induced inhibitory activity for cAMP accumulation. The coupling profiles are then classified into three clusters, representing those preferentially coupled to Gαz, those to Gαo, and those with unapparent selectivity. These results indicate that individual Gαi-coupled GPCRs fine-tune Gαi signaling by exerting coupling preference at the Gαi-subunit level.

Successful management of ureteral injury after gender-affirming laparoscopic hysterectomy: A case report.

INTRODUCTION AND IMPORTANCE: A two-step process involving ureteral stenting and surgical repair is generally recommended to manage a delayed diagnosis of postoperative ureteral injury; however, retrograde stenting is often difficult. CASE PRESENTATION: A 35-year-old female-to-male transgender person who underwent laparoscopic gender-affirming total hysterectomy with bilateral salpingo-oophorectomy developed right ureteral injury at 2 months postoperatively. Initially, the stenting guidewire could not pass through the stenotic tract and was diverted into the abdominal cavity. Using a 0.014-in. microguidewire and a 2-Fr microcatheter, both of which are designed for angiography, the ureteral stent was ultimately placed. The patient underwent surgical repair using the Boari flap technique. The double J catheter was removed at 1 month postoperatively, and postoperative retrograde pyelography revealed no urinary leakage or ureteral stricture. CLINICAL DISCUSSION: Immediate primary repair is desirable for intraoperative ureteral injuries. However, up to 70 % of ureteral injuries are diagnosed during the postoperative period. For a delayed diagnosis of ureteral injury, urinary diversion with ureteral stent, nephrostomy, or both, followed by delayed repair, is recommended to avoid the inflammatory phase. In this patient, ureteral stenting was difficult on the first attempt. Thin microguidewires designed for angiography could be useful in such difficult situation. CONCLUSION: A ureteral injury at the mid-ureter diagnosed at 2 months postoperatively was successfully managed using a two-step process involving ureteral stenting and surgical repair. A microguidewire and a microcatheter are useful for successful stenting in patients with late-diagnosed, severe ureteral strictures.

Efficacy of cabozantinib therapy for brain metastases from renal cell carcinoma.

Introduction: We report two cases of renal cell carcinoma with brain metastases that showed remarkable responses to cabozantinib. Case presentation: (Case 1) A 70-year-old man with cT3aN0M0 clear cell renal cell carcinoma underwent radical nephrectomy and developed multiple brain metastases 2 months postoperatively. The brain lesions regressed after stereotactic radiotherapy followed by ipilimumab plus nivolumab therapy, but a new brain metastasis that caused hemiplegia developed after 6 months and showed no response to stereotactic radiotherapy. However, complete remission was achieved, and hemiplegia ceased within 2 weeks of cabozantinib therapy. (Case 2) A 63-year-old man with cT3aN0M1 clear cell renal cell carcinoma and brain metastases underwent upfront cytoreductive nephrectomy. The brain lesions progressed rapidly 1 month postoperatively. The lesions disappeared 2 weeks after cabozantinib plus nivolumab therapy. Conclusion: Cabozantinib, alone or in combination with immune checkpoint inhibitors, may be a viable option for clear cell renal cell carcinoma with brain metastases.

A case of clear cell renal cell carcinoma with vena cava thrombus responding to presurgical avelumab, and axitinib.

INTRODUCTION: We report a case of renal cell carcinoma with vena cava thrombus showing a marked reduction with presurgical avelumab plus axitinib, facilitating nephrectomy with thrombectomy. CASE PRESENTATION: A 50-year-old man was taken to emergent care unit due to spontaneous renal rupture and was diagnosed to have left-sided renal cell carcinoma with level IV tumor thrombus. After hemostasis was obtained via transcatheter arterial embolization, avelumab plus axitinib was introduced because upfront surgery was deemed unfeasible due to poor performance status and possible retroperitoneal tumor dissemination. After four treatment cycles, thrombus was reduced to level II, and nephrectomy with thrombectomy was performed. Histological analyses revealed massive CD8+ T cell infiltration in the thrombus, suggesting immunotherapy efficacy. He has remained recurrence-free without any additional treatment for eight months. CONCLUSION: For locally advanced renal cell carcinoma with vena cava thrombus, presurgical combination therapy with avelumab plus axitinib could be an option to facilitate curative surgery.

[Successful Re-Administration of Nivolumab in Patient with Metastatic Renal Cell Carcinoma : A Case Report].

A 46-year-old woman was referred to our hospital with a left-sided renal tumor pointed out by ultrasonography at the time of a medical checkup．Computed tomography revealed a mass measuring 88×77×68 mm on the upper pole of the left kidney. She was diagnosed with cT2aN0M0 clear cell renal cell carcinoma. Laparoscopic left nephrectomy was performed uneventfully. Histopathological diagnosis was clear cell renal cell carcinoma, G2, v1, pT2. Four months after surgery, lung metastases appeared, and systemic therapy was given sequentially as follows ; sunitinib for 2 months, nivolumab for 8 months, axitinib for 17 months, and pazopanib for 2 months．However, metastases progressed, and a re-administration of nivolumab was planned. The nivolumab re-treatment resulted in a marked reduction in multiple lung metastases despite the previous failure by nivolumab treatment. There are few reports on the therapeutic effect of re-administration of nivolumab. We report a case of successful treatment by re-administration of nivolumab.

5-Fluorouracil-based adjuvant chemotherapy improves the clinical outcomes of patients with lymphovascular invasion of upper urinary tract cancer and low expression of dihydropyrimidine dehydrogenase.

Lymphovascular invasion (LVI) by urothelial carcinoma of the upper urinary tract (UC-UUT) is associated with an unfavorable prognosis. However, a high proportion of patients with UC-UUT are unable to receive the recommended doses of cisplatin-based adjuvant chemotherapy due to advanced age or renal dysfunction resulting from nephroureterectomy. Tegafur-uracil is an oral form of 5-fluorouracil whose efficacy is influenced by the activities of enzymes associated with its metabolism, such as dihydropyrimidine dehydrogenase (DPD), orotatephosphoribosyltransferase (OPRT) and thymidylate synthase (TS). The aim of the present study was to investigate the efficacy of adjuvant 5-fluorouracil chemotherapy for UC-UUT with LVI, and to assess the expression of enzymes associated with 5-fluorouracil metabolism as promising biomarkers of therapy efficacy. The present study retrospectively investigated 52 cases of UC-UUT. Following nephroureterectomy, tegafur-uracil was administered to 15 out of 30 patients with LVI who were not eligible for cisplatin-based adjuvant chemotherapy. Levels of DPD, OPRT and TS expression in tumor specimens were determined by reverse transcription-quantitative polymerase chain reaction, and their associations with the efficacy of adjuvant 5-fluorouracil chemotherapy were analyzed. The levels of DPD, OPRT and TS expression were not associated with pathological factors or outcome, although a higher expression of TS was associated with a poorer outcome. Adjuvant 5-fluorouracil chemotherapy significantly improved the outcome of patients with lower DPD expression. However, the levels of OPRT and TS expression did not influence therapeutic efficacy. Adjuvant 5-fluorouracil chemotherapy appears to be effective for lymphovascular-invasive UC-UUT in patients with lower DPD expression.

The Predictive Value of Glycated Hemoglobin and Albumin for the Clinical Course Following Hospitalization of Patients with Febrile Urinary Tract Infection.

BACKGROUND: Diabetes is considered a risk factor for acquisition of febrile urinary tract infection (f-UTI), but information on the association of diabetes with subsequent course of the disease is lacking. Thus, we investigated the clinical variables including diabetic status which determined the clinical course in patients with community-acquired f-UTI. MATERIALS AND METHODS: Patients hospitalized consecutively for f-UTI between February 2016 and January 2018 were used for this single center study. The routine laboratory tests including blood glucose and glycated hemoglobin (HbA1c) were done and empiric treatment with parenteral antibiotics was commenced on admission. The clinical course such as duration of fever (DOF) and length of hospital stay (LOS) were compared among groups classified by the clinical variables. RESULTS: Among the101 patients admitted for f-UTI, 15 patients with diabetes (14.9%) experienced significantly longer febrile period and hospitalization compared to those with hyperglycemia (n = 18, 17.8%) or those without diabetes and hyperglycemia (n = 68, 67.3%). Of the laboratory parameters tested on admission and several clinical factors, the presence of diabetes and risk factors for severe complicated infection (hydronephrosis, urosepsis, and disseminated intravascular coagulopathy) as well as HbA1c and albumin were identified as predictors for LOS by univariate analysis, whereas none of the variables failed to predict DOF. In the subsequent multivariate analysis, HbA1c levels and albumin levels were isolated as independent predictors of LOS. CONCLUSION: Patients with higher HbA1c and lower albumin levels required the longest period of hospitalization. Thus, an evaluation of diabetic and nutritional status on admission will be feasible to foretell the clinical course and better manage the subset of patients at risk of prolonged hospitalization.