Constitutive formation of an RXFP1-signalosome: a novel paradigm in GPCR function and regulation

The classical second messenger cAMP is important in diverse physiological processes, where its spatial and temporal compartmentalization allows precise control over multiple cellular events. Within this context, G-protein-coupled receptors (GPCRs) govern specialized pools of cAMP, which are functionally specific for the unique cellular effects attributed to a particular system. The relaxin receptor, RXFP1, is a GPCR that exerts pleiotropic physiological effects including a potent anti-fibrotic response, increased cancer metastases, and has efficacy as a vasodilator in heart failure. On a cellular level, relaxin stimulation of RXFP1 results in the activation of multiple G-protein pathways affecting cAMP accumulation. Specificity and diversity in the cAMP signal generated by RXFP1 is controlled by differential G-protein coupling dependent upon the background of cellular expression, and cAMP compartmentalization. Further complexity in cAMP signalling results from the constitutive assembly of an RXFP1-signalosome, which specifically responds to low concentrations of relaxin, and activates a distinct cAMP pathway. The RXFP1-signalosome is a higher-order protein complex that facilitates receptor sensitivity to attomolar concentration of peptide, exhibits constitutive activity and dual coupling to G-proteins and β-arrestins and reveals a concentration-biased agonism mediated by relaxin. The specific and directed formation of GPCR-centered signalosomes allows an even greater spatial and temporal control of cAMP, thus rationalizing the considerable physiological scope of this ubiquitous second messenger.     

Release of proteolytic activity following reduction in therapeutic human serum albumin containing products: Detection with a new neoepitope endopeptidase immunoassay

Botulinum type A toxin (BoNT/A) is defined by its specific endopeptidase cleavage of SNAP25 between Gln¹⁹⁷ and Arg¹⁹⁸ under reducing conditions. The neurotoxin is widely used for therapeutic or cosmetic purposes, but should not contain other toxin serotypes or unwanted protease activities. Using a neoepitope endopeptidase immunoassay, additional cleavage between Arg¹⁹⁸ and Ala¹⁹⁹ was detected with a range of therapeutic BoNT/A products confirming an earlier report of an unidentified proteolytic component. By developing the assay and making it insensitive to BoNT/C1, any activity due to the type C1 toxin was excluded. Therapeutic preparations consist of ng quantities of toxin protein which are typically stabilised by 0.125–30 mg of HSA. An excellent correlation (R² = 0.993) between HSA content per vial and measured activity was obtained within the therapeutic BoNT/A products tested. No activity was detected in any of the non-albumin formulated preparations, thereby identifying HSA as the source of the unknown protease for the first time. To investigate the cause of this activity, either as an intrinsic molecular activity of albumin or due to an albumin-associated purification contaminant, further studies on a variety of commercial plasma-derived HSA products or recombinant HSA materials free from potential plasma contaminants were carried out. The measured proteolytic levels were highly consistent amongst preparations, and could all be partially inhibited by the presence of zinc and blocked by PKSI-527 and aprotinin. By contrast, the data did not support the role of plasmin, kallikrein, trypsin, α₂-antiplasmin–plasmin complexes or HSA purification contaminants, PKA (prekallikrein activator) or kallikrein-like activity. Taken together, these findings indicate a new intrinsic proteolytic activity of the albumin molecule revealed under reducing conditions as the source of the unexpected Arg–Ala cleaving activity.     

Potential for a Simple GPS-Based Binary Logit Model to Predict Fishing Effort in a Vertical Hook-and-Line Reef Fish Fishery

Accurate fishing effort information is fundamental to the successful management of fisheries resources. Automated, independent, and reliable methods for quantifying fishing effort are needed. The use of vessel speed from Global Positioning System (GPS) data to identify fishing activity has worked well for trawl fisheries but has been less successful in stationary fisheries. Therefore, five trips on four vessels from a vertical hook-and-line reef fish fishery were used to examine the efficacy of GPS (speed and time) and electronic video monitoring (EVM) sensor (drum and video) data to corroborate an observer’s account of effort using binary logistic regression classification (logit) models as well as a simple speed and time filter (filter). One minute was the minimum data collection interval examined that documented 100% of fishing events. As no fishing occurred at night, opportunistically defined as the 7 h between 2200 and 0500 hours, these records were excluded from analyses. During the day, vessels spent on average 45.2% of the time fishing. Classification success of the approaches examined ranged from 82.4% to 89.5%. Models that included both GPS and EVM sensor data outperformed the filter and GPS-only models. In general, the filter and most model results can be used as a proxy for observer effort data, at least for the trips examined here. The GPS-based speed + time logit model was chosen as the preferred approach because of its discriminatory power compared with the filter and the existing widespread use and lower costs of GPS data collection relative to EVM systems and sensors. The speed + time logit model outlined here may have broad utility in this and similar vertical-line fisheries, including the offshore marine recreational fishing sector.

Received September 9, 2015; accepted December 15, 2015     

A randomized,phase III,double-blind,placebo-controlled trial of intrapleural instillation of methylprednisolone acetate in the management of malignant pleural effusion

STUDY OBJECTIVES: To determine if intrapleural administration of methylprednisolone acetate (MA) after therapeutic thoracentesis for symptomatic malignant pleural effusion improved time to repeat thoracentesis for symptom control, quality of life (QOL), and dyspnea. DESIGN: Double-blind, randomized, placebo-controlled trial. SETTING: A tertiary care cancer treatment center in Edmonton, AB, Canada. Patient selection: Patients with symptomatic pleural effusions secondary to disseminated malignancy requiring therapeutic thoracentesis for symptom control. INTERVENTIONS: Sixty-seven patients underwent ultrasound-guided therapeutic thoracentesis for management of symptomatic malignant pleural effusion. Patients were randomly and blindly assigned to either 160 mg (8 mL) of MA or 8 mL of saline solution instilled into the pleural space. Patients were followed up for 6 weeks to determine the time to repeat therapeutic thoracentesis. All patients completed the Functional Assessment of Cancer Therapy-General (FACT-G) QOL questionnaire and a dyspnea visual analog scale (VAS) at baseline and again 2 weeks later. Measurements and results: Thirty-three patients received MA, and 34 patients received placebo; baseline characteristics for the two groups were similar, apart from a slightly higher use of concurrent systemic therapy in the placebo group. At 6 weeks follow-up, 50% of MA-treated patients required repeat thoracentesis compared to 56% of placebo-treated patients (not significant [NS]). The mean of the individual FACT-G change scores (2 weeks - baseline) was similar in the two groups (NS). VAS scores improved for both groups over the 2-week period, but the mean change scores (2 weeks - baseline) were not statistically different. CONCLUSION: Despite previous case series describing benefit from intrapleural MA in malignant pleural effusion, this controlled study of intrapleural MA instillation did not delay reaccumulation of symptomatic pleural effusion compared to placebo, nor were differences in QOL or dyspnea observed.     

Radionuclide ventriculography in severely underweight anorexia nervosa patients before and during refeeding therapy

Congestive heart failure is a well-recognized complication of refeeding therapy in underweight patients with anorexia nervosa but there are few data describing cardiac function during the critical refeeding period. This prospective study examined left ventricular function with conventional electrocardiographic-gated radionuclide ventriculography (RVG) in severely underweight anorexia nervosa patients both before and during refeeding therapy. Eight patients underwent rest and exercise RVG at admission and after regaining approximately 5% to 10% of their ideal body weight. With the admission study serving as a control, the left ventricular ejection fraction and regional wall motion analysis were analyzed before and after refeeding and weight gain. Resting left ventricular ejection fractions were not significantly different between the first and second RVGs (64 +/- 11% vs. 62 +/- 8%, respectively; P greater than .05). Likewise, the left ventricular ejection fraction with maximal exercise did not significantly differ when comparing the first or the second RVG (74 +/- 10% vs. 72 +/- 8%, P greater than .05). During the baseline RVG, the left ventricular ejection fraction increased from 64 +/- 11% (rest) to 74 +/- 10% (maximal exercise) (P less than .001). During the second RVG, the ejection fraction increased from 62 +/- 8% (rest) to 72 +/- 8% (maximal exercise) (P = .003). However, the left ventricular exercise ejection fraction in the second RVG in one patient increased only by one absolute percentage point. Four of the eight patients had regional wall motion abnormalities detected during resting and/or exercise RVG. Abnormal cardiac function occurs in asymptomatic patients with anorexia nervosa undergoing refeeding therapy.     

A new finding in the radiographic diagnosis of Achilles tendon rupture

The authors describe a new radiographic sign of rupture of the Achilles tendon system. It is a fracture, with separation through an osteophyte at the insertion of this tendon. Previously reported signs are also discussed as well as the present case report.     

Gray-scale ultrasonography of hepatic amoebic abscesses.

Retrospective analysis of the ultrasonograms of 42 hepatic amoebic abscesses in 34 patients was performed. All lesions were less echogenic than normal liver. All but 1 were contiguous with the liver capsule and had slight distal sonic enhancement. Twenty-three were predominantly homogeneous with fine, low-level echoes. This pattern is highly suggestive of hepatic amoebic abscess. Nineteen abscesses did not show this pattern and could not be diagnosed based on ultrasonographic criteria.     

Gastrobiliary fistula: pre- and postoperative assessment with 99mTc-PIPIDA

Angiographic visualization of a vein or artery partly or completely encircling small adenomas of islet cell or parathyroid origin is an occasional helpful localizing sign, particularly when the overlying pancreas or thyroid glands are intensely stained. Three examples (two islet cell; one parathyroid) are presented.