There has been considerable progress in recent years in addressing the clinical and pharmacological limitations of hydrogels for drug delivery applications but substantial challenges remain. Here we ...discuss recent progress in overcoming these challenges, particularly with regards to effectively delivering hydrogels inside the body without implantation, prolonging the release kinetics of drugs from hydrogels, and expanding the nature of drugs which can be delivered using hydrogel-based approaches.
In classical pharmacology, bioassay data are fit to general equations (e.g. the dose response equation) to determine empirical drug parameters (e.g. EC
and E
), which are then used to calculate ...chemical parameters such as affinity and efficacy. Here we used a similar approach for kinetic, time course signaling data, to allow empirical and chemical definition of signaling by G-protein-coupled receptors in kinetic terms. Experimental data are analyzed using general time course equations (model-free approach) and mechanistic model equations (mechanistic approach) in the commonly-used curve-fitting program, GraphPad Prism. A literature survey indicated signaling time course data usually conform to one of four curve shapes: the straight line, association exponential curve, rise-and-fall to zero curve, and rise-and-fall to steady-state curve. In the model-free approach, the initial rate of signaling is quantified and this is done by curve-fitting to the whole time course, avoiding the need to select the linear part of the curve. It is shown that the four shapes are consistent with a mechanistic model of signaling, based on enzyme kinetics, with the shape defined by the regulation of signaling mechanisms (e.g. receptor desensitization, signal degradation). Signaling efficacy is the initial rate of signaling by agonist-occupied receptor (k
), simply the rate of signal generation before it becomes affected by regulation mechanisms, measurable using the model-free analysis. Regulation of signaling parameters such as the receptor desensitization rate constant can be estimated if the mechanism is known. This study extends the empirical and mechanistic approach used in classical pharmacology to kinetic signaling data, facilitating optimization of new therapeutics in kinetic terms.
Rainbow trout fry syndrome (RTFS) is a disease caused by the Gram-negative bacterium Flavobacterium psychrophilum, responsible for significant economic losses in salmonid aquaculture worldwide. The ...diversity of F. psychrophilum isolates and the inherent difficulties in vaccinating juvenile fish has hampered the development of a vaccine for RTFS. Disease episodes tend to occur between 10-14 °C with necrotic lesions often seen on the skin surrounding the dorsal fin and tail. At present no commercial vaccines are available for RTFS in the UK, leaving antibiotics as the only course of action to control disease outbreaks. The current work was performed as a pilot study to assess the efficacy of a polyvalent, whole cell vaccine containing formalin-inactivated F. psychrophilum, to induce protective immunity in rainbow trout fry. Duplicate groups of 30 trout (5 g) were immersed in 1 L of the vaccine for 30 s. Samples were taken 4 h, day 2 and 7 post-vaccination (pv) of skin mucus, tissues for histology and gene expression analysis; serum and histology samples were taken 6 weeks pv. A booster vaccination was given at 315 degree days (dd) also by immersion. Challenge was by immersion with a heterologous isolate of F. psychrophilum 630 dd post primary vaccination. The vaccine provided significant protection to the trout fry with a RPS of 84% (p < 0.0001). Detection of increased numbers of IgT positive cells in systemic organs, up-regulation of IgT expression in hind-gut and an increase in total IgT in serum was observed in vaccinated fish; however a functional role of IgT in the observed protection remains to be demonstrated.
The kinetics/dynamics of signaling are of increasing value for G-protein-coupled receptor therapeutic development, including spatiotemporal signaling and the kinetic context of biased agonism. ...Effective application of signaling kinetics to developing new therapeutics requires reliable kinetic assays and an analysis framework to extract kinetic pharmacological parameters. Here we describe a platform for measuring arrestin recruitment kinetics to GPCRs using a high quantum yield, genetically encoded fluorescent biosensor, and a data analysis framework to quantify the recruitment kinetics. The sensor enabled high temporal resolution measurement of arrestin recruitment to the angiotensin AT
and vasopressin V
receptors. The analysis quantified the initial rate of arrestin recruitment (k
), a biologically-meaningful kinetic drug efficacy parameter, by fitting time course data using routine curve-fitting methods. Biased agonism was assessed by comparing k
values for arrestin recruitment with those for Gq signaling via the AT
receptor. The k
ratio values were in good agreement with bias estimates from existing methods. This platform potentially improves and simplifies assessment of biased agonism because the same assay modality is used to compare pathways (potentially in the same cells), the analysis method is parsimonious and intuitive, and kinetic context is factored into the bias measurement.
Hematopoietic stem cell transplantation (HSCT) is an increasingly common treatment for children with a range of hematological disorders. Conditioning with cytotoxic chemotherapy and total body ...irradiation leaves patients severely immunocompromised. T‐cell reconstitution can take several years due to delayed restoration of thymic output. Understanding T‐cell reconstitution in children is complicated by normal immune system maturation, heterogeneous diagnoses, and sparse uneven sampling due to the long time spans involved. We describe here a mechanistic mathematical model for CD4 T‐cell immune reconstitution following pediatric transplantation. Including relevant biology and using mixed‐effects modeling allowed the factors affecting reconstitution to be identified. Bayesian predictions for the long‐term reconstitution trajectories of individual children were then obtained using early post‐transplant data. The model was developed using data from 288 children; its predictive ability validated on data from a further 75 children, with long‐term reconstitution predicted accurately in 81% of the patients.
1. The resolution of direct conflict between humans and elephants in Africa has become a serious local political issue in recent years, and a continental conservation problem. `Problem elephants' ...damage crops, food stores and water sources, and sometimes threaten human life. 2. Eighty per cent of the African elephant's range lies outside formally protected areas, and inadequate management of human-elephant conflict is frequently a precursor to further decline in the numbers and distribution of elephants. Conflict appears to be increasing in an assortment of African ecosystems as the agricultural interface with elephant range expands. 3. The present study recorded incidents by problem elephants in small subdivisions of a 15 000 km2elephant range. The level of problem elephant activity over 3 years showed huge variation and could not be explained by elephant density, proximity of a protected area, area of human settlement, human density or local rainfall. 4. It is proposed that the irregular and unpredictable nature of human-elephant conflict incidents in the study area mainly depended upon the behavioural ecology of individual elephant bulls. 5. This study proposes a statistic to quantify problem elephant activity in Africa which can be used to compare the intensity of problem incidents between different ecosystems at different times: `elephant incidents per square kilometre of human settlement area per year'. Spatial analyses of appropriate data at the human-elephant interface may yield a more predictive understanding of the conflict process.
Neurons integrate inputs over different time and space scales. Fast excitatory synapses at boutons (ms and μm), and slow modulation over entire dendritic arbors (seconds and mm) are all ultimately ...combined to produce behavior. Understanding the timing of signaling events mediated by G-protein-coupled receptors is necessary to elucidate the mechanism of action of therapeutics targeting the nervous system. Measuring signaling kinetics in live cells has been transformed by the adoption of fluorescent biosensors and dyes that convert biological signals into optical signals that are conveniently recorded by microscopic imaging or by fluorescence plate readers. Quantifying the timing of signaling has now become routine with the application of equations in familiar curve fitting software to estimate the rates of signaling from the waveform. Here we describe examples of the application of these methods, including (1) Kinetic analysis of opioid signaling dynamics and partial agonism measured using cAMP and arrestin biosensors; (2) Quantifying the signaling activity of illicit synthetic cannabinoid receptor agonists measured using a fluorescent membrane potential dye; (3) Demonstration of multiplicity of arrestin functions from analysis of biosensor waveforms and quantification of the rates of these processes. These examples show how temporal analysis provides additional dimensions to enhance the understanding of GPCR signaling and therapeutic mechanisms in the nervous system.
Corticotropin-releasing factor (CRF) receptor antagonists are under preclinical and clinical investigation for stress-related disorders. In this study the impact of receptor-ligand binding kinetics ...on CRF₁ receptor antagonist pharmacology was investigated by measuring the association rate constant (k₁), dissociation rate constant (k₋₁), and kinetically derived affinity at 37°C. Three aspects of antagonist pharmacology were reevaluated: comparative binding activity of advanced compounds, in vivo efficacy, and structure-activity relationships. Twelve lead compounds, with little previously noted difference of affinity, varied substantially in their kinetic binding activity with a 510-fold range of kinetically derived affinity (k₋₁/k₁), 170-fold range of k₋₁, and 13-fold range of k₁. The k₋₁ values indicated previous affinity measurements were not close to equilibrium, resulting in compression of the measured affinity range. Dissociation was exceptionally slow for three ligands (k₋₁ t(1/2) of 1.6-7.2 h at 37°C). Differences of binding behavior were consistent with in vivo pharmacodynamics (suppression of adrenocorticotropin in adrenalectomized rats). Ligand concentration-effect relationships correlated with their kinetically derived affinity. Two ligands that dissociated slowly (53 and 130 min) produced prolonged suppression, whereas only transient suppression was observed with a more rapidly dissociating ligand (16 min). Investigating the structure-activity relationship indicated exceptionally low values of k₁, approaching 100,000-fold less than the diffusion-limited rate. Retrospective interpretation of medicinal chemistry indicates optimizing specific elements of chemical structure overcame kinetic barriers in the association pathway, for example, constraint of the pendant aromatic orthogonal to the ligand core. Collectively, these findings demonstrate receptor binding kinetics provide new dimensions for understanding and potentially advancing the pharmacology of CRF₁ receptor antagonists.
Although Nile tilapia (Oreochromis niloticus) is a well-established aquaculture species globally, there are a limited number of commercial vaccines available or are used for this species. The ...majority of diseases affecting farmed tilapia are bacterial, with antibiotics frequently used to treat fish. The current study was performed to optimise the use of mucosal vaccines for tilapia by adapting an existing bacterin vaccine against Francisella noatunensis subsp. orientalis (Fno) as a proof of concept. This vaccine has previously provided excellent protection by injection, however, the preference for tilapia farmers would be to vaccinate fish by immersion or orally, due to the lower cost and ease of application. These vaccination routes, however, are often less efficacious probably due to the lack of adjuvants in immersion and oral vaccines. The aims of this study, therefore, were to optimise the formulation and dose of the Fno vaccine with mucosal adjuvants for oral and immersion delivery. Tilapia fry (av. 6 g) were given three concentrations (high, medium, low; i.e. 1×109, 1×108 and 1×107 CFU mL-1) of antigen combined with the oral adjuvant by oral gavage, to optimise the dose needed to induce an immune response to Fno, and the immune response obtained compared with fish vaccinated by immersion (with and without an immersion adjuvant). Fry were boosted by the same route at 420 degree days (DD), and samples (serum, mucus ) taken at 840 DD for specific antibody responses measured by ELISA and western blotting. Specific IgM titres were significantly elevated in serum and mucus of fish given the high dose adjuvanted vaccine by gavage. In addition, by western blotting with serum, a significant immunogenic reaction was evident between 20 and 37 kDa in the fish given the high dose oral vaccine by gavage. As protection against Fno provided by the injection vaccine was correlated with specific antibody responses these findings suggest the oral vaccine also has potential to provide protection. Further studies are needed to optimise delivery of the vaccine via feed.
•Tilapia given an oral vaccine had significantly higher levels of anti-Fno IgM in serum and mucus than un-vaccinated fish.•An immunogenic reaction was seen at 20–37 kDa in orally vaccinated tilapia, also observed after intraperitoneal vaccination.•As specific antibodies are linked to protection against Fno, the oral vaccine should be developed for in-feed application.
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