The patient pathway for overactive bladder management:A quantitative analysis

PURPOSE: We aimed to explore the pathways followed by patients with overactive bladder (OAB) from referral to the urologist to final treatment. METHODS: This was a single-center, retrospective cohort study of female patients diagnosed with OAB in a large Dutch nonacademic teaching hospital. The number, sequence, and duration of treatment steps offered were analyzed, and the effectiveness, reasons for discontinuation, and possible case-mix variables influencing OAB treatment were studied. RESULTS: In total, 120 patients were enrolled and required a median of 2 steps (range, 1-6) of treatment over a median total duration of 28 weeks (range, 5-256). Treatment typically started with drug therapy, including antimuscarinics (38%; 95% CI, 30%-47%), antimuscarinics plus pelvic floor muscle therapy (21%; 95% CI, 15%-29%), or mirabegron (11%; 95% CI, 6%-18%). However, 52% of patients required further treatment, with botulinum toxin A (BoNT-A) injections being most effective (67%; 95% CI, 42%-85%), followed by antimuscarinics plus percutaneous tibial nerve stimulation (50%; 95% CI, 25%-75%), and antimuscarinics plus pelvic floor muscle therapy (36%; 95% CI, 21%-54%). Other therapies showed lower effectiveness. Common reasons for discontinuation were insufficient response and side effects. Overall, 22 patients were lost to follow-up. CONCLUSION: Most patients try at least two treatments before they experience satisfactory symptom relief, with treatment evaluations requiring time because therapeutic onsets differ by patient and treatment. Our data can help to manage expectations among urologists and patients when seeking treatment for OAB

Radiolysis of NaCl at high and low temperatures: development of size distribution of bubbles and colloids

New experimental results are presented on low temperature irradiation (18 °C) of rock-salt samples which had been exposed to initial doses up to 320 GRad at 100 °C. Differential scanning calorimetry (DSC) shows that the latent heat of melting (LHM) of sodium colloids decreases during subsequent low-temperature irradiation, whereas the stored energy (SE) increases slowly, indicating that the process of radiolysis continues. The decrease of the LHM is due to dissolution of large colloids, because the intensities of the melting peaks decrease during the second stage irradiation at low temperature. The model is formulated to describe the nucleation kinetics and the evolution of the size distribution of chlorine precipitates and sodium colloids in NaCl under high dose irradiation. It is shown that the mechanism of dissolution of large Na colloids during low temperature irradiation can be related to melting of sodium colloids.

Ribosomal Protein Mutations Induce Autophagy through S6 Kinase Inhibition of the Insulin Pathway

Mutations affecting the ribosome lead to several diseases known as ribosomopathies, with phenotypes that include growth defects, cytopenia, and bone marrow failure. Diamond-Blackfan anemia (DBA), for example, is a pure red cell aplasia linked to the mutation of ribosomal protein (RP) genes. Here we show the knock-down of the DBA-linked RPS19 gene induces the cellular self-digestion process of autophagy, a pathway critical for proper hematopoiesis. We also observe an increase of autophagy in cells derived from DBA patients, in CD34+ erythrocyte progenitor cells with RPS19 knock down, in the red blood cells of zebrafish embryos with RP-deficiency, and in cells from patients with Shwachman-Diamond syndrome (SDS). The loss of RPs in all these models results in a marked increase in S6 kinase phosphorylation that we find is triggered by an increase in reactive oxygen species (ROS). We show that this increase in S6 kinase phosphorylation inhibits the insulin pathway and AKT phosphorylation activity through a mechanism reminiscent of insulin resistance. While stimulating RP-deficient cells with insulin reduces autophagy, antioxidant treatment reduces S6 kinase phosphorylation, autophagy, and stabilization of the p53 tumor suppressor. Our data suggest that RP loss promotes the aberrant activation of both S6 kinase and p53 by increasing intracellular ROS levels. The deregulation of these signaling pathways is likely playing a major role in the pathophysiology of ribosomopathies

A robust post-insertion method for the preparation of targeted siRNA LNPs

Targeted delivery of nucleic acids is gaining momentum due to improved efficacy, selectivity, increased circulation time and enhanced tissue retention in target cells. Using nucleic acid-based therapies previously undruggable targets have proven now to be amenable for treatment. Currently, several methods for preparing targeted or labelled delivery vehicles for nucleic acids are based on liposomal formulations. Lipid nanoparticles (LNPs) are structurally different from liposomes and these methods should therefore be evaluated before being translated to siRNA LNPs preparation protocols. Here, we describe a robust and facile method for the preparation of targeted or fluorescently labelled siRNA LNPs. Using a copper free strain-promoted azide-alkyne cycloaddition (SPAAC) we demonstrate that post-insertion of ligand-lipid conjugates into preformed LNPs is superior to direct-surface modification because it preserves the physicochemical parameters of the LNPs. We found that the time point of solvent removal by dialysis is critical and affects the hydrodynamic diameter of the LNPs; post-insertion after dialysis shows the smallest increase in hydrodynamic diameter and polydispersity index (PDI). The post-insertion of ligand-lipid conjugates also proceeded with rapid kinetics and high efficacy over a wide temperature range. Using this optimised protocol, we generated siRNA LNPs containing both targeting and fluorescent tracking ligands allowing us to monitor siRNA LNP uptake kinetics in dependence of the targeting ligand. In aggregate, we describe a robust approach for the generation of targeted and labelled siRNA LNPs that allows their controlled and facile decoration with ligand combinations

Mechanistic Study on the Degradation of Hydrolysable Core-Crosslinked Polymeric Micelles

Core-crosslinked polymeric micelles (CCPMs) are an attractive class of nanocarriers for drug delivery. Two crosslinking approaches to form CCPMs exist: either via a low-molecular-weight crosslinking agent to connect homogeneous polymer chains with reactive handles or via cross-reactive handles on polymers to link them to each other (complementary polymers). Previously, CCPMs based on methoxy poly(ethylene glycol)- b-poly[ N-(2-hydroxypropyl) methacrylamide-lactate] (mPEG- b-PHPMAmLac n ) modified with thioesters were crosslinked via native chemical ligation (NCL, a reaction between a cysteine residue and thioester resulting in an amide bond) using a bifunctional cysteine containing crosslinker. These CCPMs are degradable under physiological conditions due to hydrolysis of the ester groups present in the crosslinks. The rapid onset of degradation observed previously, as measured by the light scattering intensity, questions the effectiveness of crosslinking via a bifunctional agent. Particularly due to the possibility of intrachain crosslinks that can occur using such a small crosslinker, we investigated the degradation mechanism of CCPMs generated via both approaches using various analytical techniques. CCPMs based on complementary polymers degraded slower at pH 7.4 and 37 °C than CCPMs with a crosslinker (the half-life of the light scattering intensity was approximately 170 h versus 80 h, respectively). Through comparative analysis of the degradation profiles of the two different CCPMs, we conclude that partially ineffective intrachain crosslinks are likely formed using the small crosslinker, which contributed to more rapid CCPM degradation. Overall, this study shows that the type of crosslinking approach can significantly affect degradation kinetics, and this should be taken into consideration when developing new degradable CCPM platforms

EV-Elute: a universal platform for enrichment of functional surface marker-defined extracellular vesicle subpopulations

Intercellular communication via extracellular vesicles (EVs) has been identified as a vital component of a steadily expanding number of physiological and pathological processes. To accommodate these roles, EVs are equipped with specific proteins, lipids, and RNA molecules by EV-secreting cells. Consequently, EVs have highly heterogeneous molecular compositions. Given that surface molecules on EVs determine their interactions with their environment, it is conceivable that EV functionality differs between subpopulations with varying surface compositions. However, it has been technically challenging to examine such functional heterogeneity due to a lack of non-destructive methods to separate EV subpopulations based on their surface markers. Here, we used Design-of-Experiments methodology to rapidly optimize a protocol, which we name ‘EV-Elute’, to elute intact EVs from commercially available Protein G-coated magnetic beads. We captured EVs from various cell types on these beads using antibodies against CD9, CD63, CD81 and a custom-made protein binding phosphatidylserine (PS). When applying EV-Elute, over 70% of bound EVs could be recovered from the beads in a pH– and incubation time-dependent fashion. EV subpopulations were found to be devoid of co-isolated protein contaminants observed in whole EV isolates and showed intact morphology by electron microscopy. Proteinase K protection assays showed a mild and reversible decrease of EV membrane integrity during elution. Depending on the type of capturing antibody used, some antibodies remained EV-associated after elution. EV subpopulations showed uptake patterns similar to whole EV isolates in co-cultures of peripheral blood mononuclear cells and endothelial cells. However, in Cas9/sgRNA delivery assays, CD63+ EVs showed a lower capacity to functionally deliver cargo as compared to CD9+, CD81+ and PS+ EVs. Taken together, we developed a novel, easy-to-use platform to isolate and functionally compare surface marker-defined EV subpopulations. Importantly, this platform does not require specialized equipment or reagents and is universally applicable to any capturing antibody and EV source. Hence, EV-Elute can open new opportunities to study EV functionality at the subpopulation level

Dibutyltin Disrupts Glucocorticoid Receptor Function and Impairs Glucocorticoid-Induced Suppression of Cytokine Production

BACKGROUND: Organotins are highly toxic and widely distributed environmental chemicals. Dibutyltin (DBT) is used as stabilizer in the production of polyvinyl chloride plastics, and it is also the major metabolite formed from tributyltin (TBT) in vivo. DBT is immunotoxic, however, the responsible targets remain to be defined. Due to the importance of glucocorticoids in immune-modulation, we investigated whether DBT could interfere with glucocorticoid receptor (GR) function. METHODOLOGY: We used HEK-293 cells transiently transfected with human GR as well as rat H4IIE hepatoma cells and native human macrophages and human THP-1 macrophages expressing endogenous receptor to study organotin effects on GR function. Docking of organotins was used to investigate the binding mechanism. PRINCIPAL FINDINGS: We found that nanomolar concentrations of DBT, but not other organotins tested, inhibit ligand binding to GR and its transcriptional activity. Docking analysis indicated that DBT inhibits GR activation allosterically by inserting into a site close to the steroid-binding pocket, which disrupts a key interaction between the A-ring of the glucocorticoid and the GR. DBT inhibited glucocorticoid-induced expression of phosphoenolpyruvate carboxykinase (PEPCK) and tyrosine-aminotransferase (TAT) and abolished the glucocorticoid-mediated transrepression of TNF-alpha-induced NF-kappaB activity. Moreover, DBT abrogated the glucocorticoid-mediated suppression of interleukin-6 (IL-6) and TNF-alpha production in lipopolysaccharide (LPS)-stimulated native human macrophages and human THP-1 macrophages. CONCLUSIONS: DBT inhibits ligand binding to GR and subsequent activation of the receptor. By blocking GR activation, DBT may disturb metabolic functions and modulation of the immune system, providing an explanation for some of the toxic effects of this organotin