Uncertain diagnosis and prognosis in advanced melanoma: a qualitative study of the experiences of bereaved carers in a time of immune and targeted therapies.

BACKGROUND: Recent advances in advanced melanoma therapies are associated with improved survival for some patients. However, how patients with diagnoses of advanced disease and their carers experience this expanding treatment paradigm is not well understood. OBJECTIVES: To explore bereaved carers' accounts of the trajectory of advanced melanoma involving treatment by immune or targeted therapies, to build an understanding of their experiences of care relating to diagnosis and prognosis. METHODS: A qualitative exploratory design, using methods drawn from grounded theory, was adopted. Analyses drew on in-depth interviews with 20 bereaved carers from three metropolitan melanoma treatment centres in Australia. A flexible interview guide and structured approach to concurrent data collection and analysis were applied. RESULTS: Carers described qualities of the experience, including the shock of diagnosis after a sometimes-innocuous presentation with vague symptoms. They reported an unclear prognosis with complexity arising from interplay between an uncertain disease trajectory and often ambiguous expectations of outcomes of emerging immune and targeted therapies. Uncertainty dominated carers' experiences, increasing the complexity of care planning. CONCLUSIONS: Effective communication of an advanced melanoma diagnosis and prognosis is critical. Recognition of the uncertainty inherent in the benefit of immune and targeted therapies in a constructive manner may facilitate more timely and effective care-planning conversations between patients, carers and medical specialists.

Metabolism and disposition of the anticancer quinolone derivative vosaroxin, a novel inhibitor of topoisomerase II.

Background Vosaroxin is a first-in-class anticancer quinolone derivative that is being investigated for patients with relapsed or refractory acute myeloid leukemia (AML). The primary objective of this study was to quantitatively determine the pharmacokinetics of vosaroxin and its metabolites in patients with advanced solid tumors. Methods This mass balance study investigated the pharmacokinetics (distribution, metabolism, and excretion) of vosaroxin in cancer patients after a single dose of 60 mg/m2 14C-vosaroxin, administered as short intravenous injection. Blood, urine and feces were collected over 168 h after injection or until recovered radioactivity over 24 h was less than 1% of the administered dose (whichever was earlier). Total radioactivity (TRA), vosaroxin and metabolites were studied in all matrices. Results Unchanged vosaroxin was the major species identified in plasma, urine, and feces. N-desmethylvosaroxin was the only circulating metabolite detected in plasma, accounting for <3% of the administered dose. However, in plasma, the combined vosaroxin + N-desmethylvosaroxin AUC0-∞ was 21% lower than the TRA AUC0-∞ , suggesting the possible formation of protein bound metabolites after 48 h when the concentration-time profiles diverged. The mean recovery of TRA in excreta was 81.3% of the total administered dose; 53.1% was excreted through feces and 28.2% through urine. Conclusions Unchanged vosaroxin was the major compound found in the excreta, although 10 minor metabolites were detected. The biotransformation reactions were demethylation, hydrogenation, decarboxylation and phase II conjugation including glucuronidation.

Prospective Assessment of Radiation in Pediatric Urology: The Pediatric Urology Radiation Safety Evaluation Study.

PURPOSE: Pediatric tissues are exquisitely sensitive to ionizing radiation from diagnostic studies and therapies involving fluoroscopy. We prospectively monitored radiation exposure in our pediatric urology patients during fluoroscopy guided operative procedures with single point dosimeters to quantify radiation dose. MATERIALS AND METHODS: Children undergoing fluoroscopy guided urological procedures were prospectively enrolled in the study from 2013 to 2015. Single point dosimeters were affixed to skin overlying the procedural site for the durations of the procedures to record dosimetry data. Patient demographics, procedural variables and fluoroscopic settings were recorded. RESULTS: A total of 78 patients underwent 96 procedures, including retrograde pyelography, ureteral stent insertion, ureteroscopy and percutaneous nephrolithotomy. Median patient age was 12 years (range 0.3 to 17) and median body mass index percentile for age was 70.7 (1.0 to 99.1). Median skin entrance radiation dose for all procedures performed was 0.56 mGy. Median dosages associated with the 29 diagnostic procedures and 49 definitive interventions were 0.6 mGy (mean 0.8, range 0.1 to 2.2) and 0.7 mGy (1.1, 0.0 to 5.5), respectively. The dose associated with the 18 procedures of temporization was significantly higher by comparison (median 1.0 mGy, mean 2.6, range 0.1 to 10.7, p = 0.02). CONCLUSIONS: Pediatric radiation exposure is not insignificant during urological procedures. Further multi-institutional work would provide context for our findings. Protocols to optimize fluoroscopic settings and minimize patient exposure, and guidelines for radiation based imaging should have a key role in all pediatric radiation safety initiatives.

Balloon tamponade for variceal haemorrhage: a practical approach.

Balloon tamponade with compression tubes is used to stabilise life-threatening variceal bleeds when first-line endotherapy has failed and acts as a bridge to early definitive therapy. We present an overview of the use of compression tubes for variceal haemorrhage with a focus on insertion technique and aftercare.

Augmentation cystoplasty and risk of neoplasia: fact, fiction and controversy.

PURPOSE: We determined if ileal/colonic bladder augmentation performed in patients with congenital bladder abnormalities is an independent risk factor for bladder malignancy. MATERIALS AND METHODS: We reviewed a registry of patients with bladder dysfunction due to neurological abnormalities, exstrophy and posterior urethral valves. Individuals treated with augmentation cystoplasty were matched (1:1) to a control group treated with intermittent catheterization based on etiology of bladder dysfunction, gender and age (±2 years). RESULTS: We evaluated 153 patients with an ileal/colonic cystoplasty and a matched control population. There was no difference (p=0.54) in the incidence of bladder cancer in patients with augmentation cystoplasty (7 patients [4.6%]) vs controls (4 [2.6%]). In addition, there was no difference between the 2 groups regarding age at diagnosis (51 vs 49.5 years, p>0.7), stage (3.4 vs 3.8, p>0.5), mortality rate (5 of 7 [71%] vs 4 of 4 [100%], p>0.4) or median survival (18 vs 17 months, p>0.8). Irrespective of augmentation status patients with a history of renal transplant on chronic immunosuppression had a significantly higher incidence of bladder cancer (3 of 20 [15%]), compared to patients who were not immunosuppressed (8 of 286 [2.8%], p=0.03). CONCLUSIONS: In patients with congenital bladder dysfunction ileal/colonic bladder augmentation does not appear to increase the risk of bladder malignancy over the inherent cancer risk associated with the underlying congenital abnormality. In addition, immunosuppression irrespective of bladder treatment is an independent risk factor for malignancy in this patient population.

Insulin-stimulated hydrolysis of a novel glycolipid generates modulators of cAMP phosphodiesterase.

Insulin action may involve the intracellular generation of low molecular weight substances that modulate certain key enzymes. The production of two substances that regulate the activity of adenosine 3',5'-monophosphate phosphodiesterase was evaluated in cultured myocytes by incorporation of radiolabeled precursors. Insulin caused the rapid hydrolysis of a chemically undefined membrane glycolipid, resulting in the production of two related complex carbohydrates as well as diacylglycerol. Both the glycolipid precursor and the aqueous products were monitored by labeling with radioactive inositol and glucosamine. Depletion of the labeled precursor and the appearance of labeled water-soluble products and diacylglycerol occurred within 30 seconds after hormone treatment and was followed by rapid resynthesis of the precursor. The aqueous products that were radioactively labeled appeared chromatographically and electrophoretically identical to phosphodiesterase modulating activities produced by insulin from the same cells. The purified radiolabeled and bioactive substances had similar chemical properties. Hydrolysis of the glycolipid precursor and subsequent generation of products could be reproduced by incubation of extracted lipids with a phosphatidylinositol-specific phospholipase C. These studies suggest that insulin stimulates an endogenous, selective phospholipase C activity that hydrolyzes a novel glycolipid, resulting in the generation of a complex carbohydrate-phosphate substance containing inositol and glucosamine that may mediate some of the actions of the hormone.

Cardiovascular aspects of glossopharyngeal insufflation and exsufflation.

Breath-hold divers use glossopharyngeal breathing to inhale above total lung capacity (glossopharyngeal insufflation, GI) or exhale below residual volume (glossopharyngeal exsufflation, GE). In these maneuvers, air is moved using glossopharyngeal rather than respiratory muscle activity. Four competitive divers performed several GI and GE maneuvers in sitting or standing position, while cardiovascular parameters were measured with a photoplethysmographic method; echocardiography was also performed during GE. During GI, the divers showed a 48% drop in mean arterial pressure (MAP) to 50 mmHg, with a 88% decrease in pulse pressure (PP), while heart rate (HR) increased by 36% to 103 beats/min and cardiac output (CO) dropped by 79% to 1.3 l/min. The increase in intrathoracic pressure during GI, measured in separate experiments, is probably responsible for these hemodynamic changes, by impeding venous return into the chest. Associated with the drop in MAP during GI were various neurological signs and symptoms, including dizziness, tunnel vision, involuntary twitching of facial muscles and one brief episode of loss of consciousness. During GE, initially MAP and PP increased by 36% and 61%, to 149 and 95 mmHg respectively; later HR decreased by 37% to 45 beats/min and CO dropped by 37% to 4.3 l/min. The early cardiovascular changes of GE may be related to a decrease in intrathoracic pressure, enhancing venous return, as shown by a 6 to 15% increase in end-diastolic diameter; later changes are similar to the responses to apnea at low lung volumes. Because of their hemodynamic effects, these breathing maneuvers should be performed with caution, particularly in the case of GI.

Quantification of vosaroxin and its metabolites N-desmethylvosaroxin and O-desmethylvosaroxin in human plasma and urine using high-performance liquid chromatography-tandem mass spectrometry.

Vosaroxin is a first-in-class anticancer quinolone derivative topoisomerase II inhibitor that is currently in development in combination with cytarabine for the treatment of acute myeloid leukemia (AML). To investigate vosaroxin pharmacokinetics (PK) in patients, liquid chromatography tandem mass spectrometry (LC-MS/MS) assays to quantify vosaroxin and the two metabolites N-desmethylvosaroxin and O-desmethylvosaroxin in human plasma and urine were developed and validated. Immediately after collection the samples were stored at -80°C. Prior to analysis, the plasma samples were subjected to protein precipitation and the urine samples were diluted. For both assays the reconstituted extracts were injected on a Symmetry Shield RP8 column and gradient elution was applied using 0.1% formic acid in water and acetonitrile-methanol (50:50, v/v). Analyses were performed with a triple quadruple mass spectrometer in positive-ion mode. A deuterated isotope of vosaroxin was used as internal standard for the quantification. The validated assays quantify vosaroxin and N-desmethylvosaroxin in the concentration range of 2-500ng/mL in plasma and urine. For O-desmethylvosaroxin the concentration range of 4-500ng/mL in plasma and urine was validated. Dilution integrity experiments show that samples can be diluted 25 fold in control matrix prior to analysis. The expanded concentration range for plasma and urine for vosaroxin and N-desmethylvosaroxin is therefore from 2 to 15,000ng/mL and in plasma for O-desmethylvosaroxin from 4 to 15,000ng/mL.

Thallous ion permeation through the cation-selective channel of the sarcoplasmic reticulum. Anomalous mole fraction dependence.

The thallous ion was found to permeate the cation-selective channel of rabbit sarcoplasmic reticulum and to block current through this channel when present in mixtures with other permeant ions. Channel conductance in pure thallium acetate saturates with increasing concentration, with a maximum limiting conductance of 60 pS. The conductance ratio GK/GTl at 1 M is 3.7, while the permeability ratio is near 0.4 over the concentration range 0.01 to 1 M. Thallium blockade in mixtures can be described by the equation of Neher (Neher, E. (1975) Biochim. Biophys. Acta 401, 540-544).

Single-channel recordings from cultured human retinal pigment epithelial cells.

We have applied patch-clamp techniques to on-cell and excised-membrane patches from human retinal pigment epithelial cells in tissue culture. Single-channel currents from at least four ion channel types were observed: three or more potassium-selective channels with single-channel slope conductances near 100, 45, and 25 pS as measured in on-cell patches with physiological saline in the pipette, and a relatively nonselective channel with subconductance states, which has a main-state conductance of approximately 300 pS at physiological ion concentrations. The permeability ratios, PK/PNa, measured in excised patches were 21 for the 100-pS channels, 3 for the 25-pS channels, and 0.8 for the 300-pS nonselective channel. The 45-pS channels appeared to be of at least two types, with PK/PNa's of approximately 41 for one type and 3 for the other. The potassium-selective channels were spontaneously active at all potentials examined. The average open time for these channels ranged from a few milliseconds to many tens of milliseconds. No consistent trend relating potassium-selective channel kinetics to membrane potential was apparent, which suggests that channel activity was not regulated by the membrane potential. In contrast to the potassium-selective channels, the activity of the nonselective channel was voltage dependent: the open probability of this channel declined to low values at large positive or negative membrane potentials and was maximal near zero. Single-channel conductances observed at several symmetrical KCl concentrations have been fitted with Michaelis-Menten curves in order to estimate maximum channel conductances and ion-binding constants for the different channel types. The channels we have recorded are probably responsible for the previously observed potassium permeability of the retinal pigment epithelium apical membrane.

Emergent transesophageal echocardiography in hemodynamically unstable obstetric patients.

BACKGROUND: The obstetric population has an increasing incidence of comorbid conditions. These, coupled with the possibility of acute embolic events involving air, amniotic fluid, and thrombus, increase the likelihood of hemodynamic instability. Although the utility of transesophageal echocardiography to guide management in cardiac and high-risk, non-cardiac surgical populations has been well established, the emergent use in critically-ill parturients has not been comprehensively evaluated. METHODS: Using our departmental transesophageal echocardiography database of 28 293 examinations, parturients were identified who underwent emergent transesophageal echocardiography for evaluation of hemodynamic instability, including cardiac arrest, between January 1999 and March 2014. Transesophageal echocardiography findings and their impact on patient management were analyzed. RESULTS: Ten peripartum patients were evaluated. Six patients became unstable during dilation and evacuation procedures; one after a forceps delivery; one during and one after cesarean delivery; and one during a postpartum laparotomy. Six patients proceeded to cardiac arrest; however, all women survived their initial operation and resuscitation. Transesophageal echocardiography was instrumental in determining the etiology and guiding resuscitation in all 10 patients including emergent cardiac surgical intervention with cardiopulmonary bypass (n=2). Seven patients survived to hospital discharge, but three died after experiencing neurologic complications. CONCLUSIONS: Severe hemodynamic instability and cardiac arrest can occur in previously healthy parturients in pregnancy. Our data suggest that emergent transesophageal echocardiography is a valuable tool in determining the etiology and directing therapy of refractory hypotension or cardiac arrest in obstetric patients.

Tracking of radiation exposure in pediatric stone patients: The time is now.

BACKGROUND: Despite the increasing incidence of pediatric nephrolithiasis, there is little data quantifying the radiation exposure associated with treatment of this disease. In this study, pediatric patients with nephrolithiasis who were managed at a single institution were identified, and the average fluoroscopy time and estimated radiation exposure associated with their procedures were reported. METHODS: Stone procedures performed on pediatric patients between 2005 and 2012 were retrospectively identified. Procedures were classified as primary ureteroscopy (URS), stent placement prior to ureteroscopy (SURS), percutaneous nephrolithotomy (PCNL), and bilateral ureteroscopy (BLURS). Patient demographic information, stone size, stone location, number of radiographic images, and fluoroscopy times were analyzed. RESULTS: A total of 152 stone procedures were included in the final analysis (92 URS, 38 SURS, eight BLURS and 14 PCNL). Mean patient age at time of stone treatment was 15.94 ± 4.1 years. Median fluoroscopy times were 1.6 (IQR 0.8-2.4), 2.1 (IQR 1.6-3.0), 2.5 (IQR 2.0-2.9), and 11.7 (IQR 5.0-18.5) minutes for URS, SURS, BLURS and PCNL, respectively. There was a moderate correlation between stone size and fluoroscopy time (r = 0.33). When compared with ureteroscopic procedures, PCNL was associated with a significantly higher fluoroscopy time (11.7 vs 2.1 min, P < 0.001). The estimated median effective dose was 3 mSv for ureteroscopic procedures and 16.8 mSv for PCNL. In addition to radiation exposure during treatment, patients in this cohort were exposed to an average of one (IQR1-3) CT scan and three (IQR 1-8) abdominal X-rays. No new malignancies were identified during the limited follow-up period. CONCLUSIONS: Radiation exposure during treatment of pediatric stone disease is not trivial, and is significantly greater when PCNL is performed. Given the recommended maximum effective dose of 50 mSv in any one year, urologists should closely monitor the amount of fluoroscopy used, and consider the potential for radiation exposure when choosing the operative approach. Prospective studies are currently underway to elucidate precise dose measurements and localize sites of radiation exposure in children during stone treatment.

Natural selection for grazer resistance to toxic cyanobacteria: evolution of phenotypic plasticity?

We studied the selection response of the freshwater grazing zooplankter, Daphnia galeata, to increased abundance of cyanobacteria in its environment. Cyanobacteria are a poor-quality and often toxic food. Distinct genotypes of D. galeata were hatched from diapausing eggs extracted from three time horizons in the sediments of Lake Constance, Europe, covering the period 1962 to 1997, a time of change in both the prevalence of planktonic cyanobacteria and levels of phosphorus pollution. We assessed whether the grazers evolved to become more resistant to dietary cyanobacteria by exposing genetically distinct clones to two diets, one composed only of the nutritious green alga, Scenedesmus obliquus (good food), and the other a mixture of S. obliquus and the toxic cyanobacterium Microcvstis aeruginosa (poor food). Genotype performance was measured as the specific rate of weight gain from neonate to maturity (gj). We evaluated evolutionary change in the Daphnia population using an analysis of reaction norms based on relative (log-transformed) changes in gj. Log(gj) is a measure of the proportional effect of dietary cyanobacteria on other fitness components of the Daphnia phenotype. For comparison, we also analyze absolute (i.e., nontransformed) changes in gj and discuss the interpretations of the two approaches. Statistical results using a general linear model demonstrate a significant effect of genotype (showing differences in gj among genotypes), a significant genotype x food-type interaction (showing differences in phenotypic plasticity among genotypes), and, in the case of log-transformed data, a significant sediment-genotype-age x food-type interaction. The latter shows that phenotypic plasticity evolved over the period studied. Two constraints act on response to selection in the D. galeata-Lake Constance system. First, gj on a diet containing poor food is highly correlated with gj on a diet of good food, thus evolving resistance also meant evolving an increase in gj on both diets. Second, because genotypes with a high gj also grow to a large adult body size, which in turn increases Daphnia vulnerability to fish predation, we suggest that selection only acted to favor genotypes possessing a high potential gj after cyanobacteria became prevalent. The presence of cyanobacteria depressed realized gj and led to animals of small adult body size even if their genotypes had the potential for high gj and large size. With realized gj reduced, genotypes with an inherently high value could be selected even in the presence of predatory fish. The joint action of selection by dietary cyanobacteria and vulnerability to fish predation provides an explanation for the observed evolution of resistance to poor food through reduced phenotypic plasticity.

Nonclinical studies addressing the mechanism of action of trastuzumab (Herceptin).

HER2 is a ligand-less member of the human epidermal growth factor receptor or ErbB family of tyrosine kinases. In normal biological systems, HER2 functions as a co-receptor for a multitude of epidermal growth factor-like ligands that bind and activate other HER family members. HER2 overexpression is observed in a number of human adenocarcinomas and results in constitutive HER2 activation. Specific targeting of these tumors can be accomplished with antibodies directed against the extracellular domain of the HER2 protein. One of these antibodies, 4D5, has been fully humanized and is termed trastuzumab (Herceptin; Genentech, San Francisco, CA). Treatment of HER2-overexpressing breast cancer cell lines with trastuzumab results in induction of p27KIP1 and the Rb-related protein, p130, which in turn significantly reduces the number of cells undergoing S-phase. A number of other phenotypic changes are observed in vitro as a consequence of trastuzumab binding to HER2-overexpressing cells. These phenotypic changes include downmodulation of the HER2 receptor, inhibition of tumor cell growth, reversed cytokine resistance, restored E-cadherin expression levels, and reduced vascular endothelial growth factor production. Interaction of trastuzumab with the human immune system via its human immunoglobulin G1 Fc domain may potentiate its antitumor activities. In vitro studies demonstrate that trastuzumab is very effective in mediating antibody-dependent cell-mediated cytotoxicity against HER2-overexpressing tumor targets. Trastuzumab treatment of mouse xenograft models results in marked suppression of tumor growth. When given in combination with standard cytotoxic chemotherapeutic agents, trastuzumab treatment generally results in statistically superior antitumor efficacy compared with either agent given alone. Taken together, these studies suggest that the mechanism of action of trastuzumab includes antagonizing the constitutive growth-signaling properties of the HER2 system, enlisting immune cells to attack and kill the tumor target, and augmenting chemotherapy-induced cytotoxicity.

Phosphorylation of fructose bisphosphate aldolase in Trypanosoma brucei.

Methods were developed for in vivo labelling of trypanosome proteins with inorganic phosphate and the labelling of fructose bisphosphate aldolase and variant surface glycoprotein was investigated. It was found that the large pool size of phosphate in trypanosomes precludes detailed kinetic analyses. Aldolase contains low levels of phosphoserine but the function of this phosphorylation has yet to be determined.

Glycosyl-sn-1,2-dimyristylphosphatidylinositol is the membrane anchor for Trypanosoma equiperdum and T. (Nannomonas) congolense variant surface glycoproteins.

We have analysed the structures of the Trypanosoma (Nannomonas) congolense and T. equiperdum variant surface glycoprotein (VSG) membrane anchors. Myristic acid uptake, phospholipase treatment, and nitrous acid deamination showed that, for each species, the anchor is glycosyl-sn-1,2-dimyristylphosphatidylinositol, as has been previously described for T. brucei. Osmotic lysis of these trypanosomes resulted in the release of soluble VSG, lacking fatty acid. In both species and in T. evansi, an endogenous phospholipase C, which cleaved diacylglycerol from membrane form VSG, was identified.

Neuropeptide-hydrolysing activities in synaptosomal fractions from dog ileum myenteric, deep muscular and submucous plexi. Their participation in neurotensin inactivation.

The mapping of neuropeptidases in synaptosomal fractions prepared from dog ileum myenteric, deep muscular and submucous plexus was established by means of fluorigenic substrates and specific inhibitors. Endopeptidase 24.11, angiotensin-converting enzyme and aminopeptidases were found in all tissues, the highest amounts being recovered in the submucous preparation. Post-proline dipeptidyl aminopeptidase was obtained in high quantities whatever the tissue source while proline endopeptidase was detected in low amounts and pyroglutamyl-peptide hydrolase was never detectable. The above peptidases were examined for their putative participation in the inactivation of neurotensin by monitoring the effect of specific inhibitors on the formation of the metabolites of labeled neurotensin separated by HPLC. Endopeptidases 24.11, 24.15 and 24.16 were respectively responsible for the formation of neurotensin(1-11), neurotensin(1-8) and neurotensin(1-10) that are devoid of biological activity. The secondary attacks occurring on neurotensin degradation products were the following: cleavage of neurotensin(1-10) into neurotensin(1-8) by angiotensin-converting enzyme; conversion of neurotensin(9-13) into neurotensin(11-13) by post-proline dipeptidyl aminopeptidase; hydrolysis of neurotensin(11-13) into free tyrosine by aminopeptidase(s).

An improved method for illuminating pipet tips for fire-polishing.

Shining light down the long axis of a pipet causes the pipet tip and walls to glow, yet leaves the background dark. The resulting contrast, and the appearance of colored bands near the pipet tip, improves visualization of the tip, affording more precise control of fire-polishing. Existing fire-polishing apparatus can be easily modified to incorporate this type of illumination.

Novel omega-conopeptides reduced field potential amplitudes in the rat hippocampal slice.

The synthetic omega-conopeptides SNX-111 (MVIIA), SNX-124 (GVIA), SNX-183 (SVIB), and SNX-185 (TVIA) reduced excitatory postsynaptic potential (EPSP) amplitudes measured in the stratum radiatum of rat hippocampal slices by about 50% at doses at or near 200 nM. These effects were at least partially reversible in most slices. EPSP amplitude was not further reduced at the highest concentrations of SNX-111, SNX-124, and SNX-185 (1.5-10 microM). In contrast, the highest concentrations of SNX-183 (2 and 10 microM) further reduced EPSP amplitude to about 20% of control. SNX-183 reduced EPSP amplitude in slices previously treated with maximally effective doses of SNX-111 or SNX-124. These results suggest that there are at least two classes of omega-conopeptide receptor sites that modulate neurotransmitter release in the rat hippocampus.