Cohorts of 6–8 week old female BALB/c mice were obtained from Charles River Laboratories (St. Constant, QC). All experiments were conducted in accordance with the ethical guidelines by the University of Saskatchewan and the Canadian Council for Animal Care. The mice (n = 12 per group) were given a single immunization by subcutaneous injection on the back with formulations containing 10 μg of PCEP, 20 μg of IDR 1002, 10 μg CpG ODN 10101 as SOL, MP or AQ formulations, with Quadracel®

(Sanofi-Pasteur) diluted to 1 μg of PTd per animal and one group received only phosphate buffered saline pH 7.4 (PBS). The mice were immunized on day 1 and serum was separated from blood collected by tail vein puncture on days 14 and 28 after immunization. Selleckchem VE822 Bleomycin supplier B. pertussis Tohoma-1 strain were streaked onto charcoal agar plates supplemented with 10% sheep blood (CBA) and incubated at 37 °C for 48 h to obtain single colonies. A few single colonies were subsequently spread onto fresh CBA plates and incubated as above. After 48 h, plates were overlaid with 300 μl of 1% casamino acids, bacteria were scraped off into the casamino acid solution and 200 μl of the suspension was used to inoculate fresh CBA plates. These were incubated and harvested as described above and transferred into 2 ml of

SS medium and quantified using a spectrophotometer. Bacterial concentration was adjusted to 5 × 106/20 μl and administered intranasally. After 2 h, 2 animals from each group were humanely euthanized and their lungs were collected and homogenized in 1 ml of SS

medium and 10-fold dilutions were plated on CBA agar plates to determine the number of viable bacteria. Lungs from 5 mice per group were collected at days 3 and 7 after challenge and processed as described above. The lung homogenates were stored in 0.1 mg/ml of PMSF at −20 °C and used to examine MCP-1, TNF-α, IL-12p40, and IFN-γ cytokine production and to evaluate total IgG and IgA antigen-specific antibody responses. Antigen specific total IgG, IgG1, IgG2a and IgA immune responses were determined by end-point ELISA using methods previously described [14]. Briefly, 100 μl of pertussis toxin (PT, Sigma–Aldrich Inc., CA, USA; 0.25 μg/ml) Adenosine in inhibitors carbonate coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) was added to each well. Wells were washed 6 times with Tris-buffered saline pH 7.3 (TBS) containing 0.05% TWEEN™ 20 (TBS-T). Diluted mouse serum samples (for IgG1 and IgG2a) or lung homogenates (IgG and IgA) were added to the wells at 100 μl/well and incubated for 1 h at room temperature. Wells were washed again with TBS-T and biotinylated goat-anti mouse IgG, IgG1, IgG2a, and IgA antibodies (Caltag Laboratories, CA, USA) were added to wells (1/5000) at 100 μl/well and incubated for 1 h at room temperature.

Modular programmes will meet educational objectives for the spectrum of vaccinology deliverables. mTOR target EVRI advanced courses, with a strong hands-on component, will link the best institutions in Europe. EVRI partner institutions will deliver specific training courses focusing on different aspects of vaccinology, which will be validated by a system of credits. Theoretical training should go hand-in-hand with practical training through internships in the vaccine formulation and manufacturing sites of EVRI or its corporate partners. The portfolio content will be adjusted according to participants’ and faculty’s feedback and on the needs expressed by the vaccine community. The development and implementation

of education

and training in vaccinology by EVRI will also involve academic research organisations from different EU Member States which will facilitate the accreditation throughout Europe of the training offered. EVRI will be accessible to the entire European vaccine development community. Partners and users will include (i) academic public sector, and non-profit organisations, (ii) small and medium sized enterprises, (iii) product development partnerships, (iv) vaccine pharmaceutical industry, (v) regulatory agencies, and (vi) patients’ organisations. As EVRI must be sustainable, services will generally be offered on a fee-for-service basis. The fee for academic research groups buy Epacadostat and non-profit organisations will cover operational costs, while corporate fees will include a profit margin. In addition, to ensure potential access to services at no cost, EVRI will make open calls for awards for research and training. These will support projects distinguished by their excellence and high potential. EVRI will ask for a discretionary funding element to support such awards in the first

five years of operation. EVRI will work with partners to establish guidelines for Intellectual Property (IP) rights related to research findings facilitated by EVRI. These guidelines will recognise institutional background IP, promote fair ownership of IP rights, the use and dissemination of IP, access rights and confidentiality. Different rules may apply depending on the nature and degree of EVRI’s contribution to the Montelukast Sodium development and funding of a specific project. An IP agreement will be signed before collaboration begins and the project will be designed to include a case-by-case evaluation. In general, IP generated by EVRI’s services will remain the ownership of the user whereas IP generated by EVRI member organisations during joint research activities will be shared fairly among the different contributors. EVRI will be a de-centralised organisation under a coordinating Secretariat, associating leading vaccine R&D institutions in both human and veterinary vaccines fields, and integrating activities that currently exist in different EU Member States and Associated Modulators Countries.

Slightly more boys were lost to follow-up, those lost to follow up had lower Selleck LY2109761 SES, higher BMI z-score at 11 years and higher parental obesity than those followed up (see Table 2), though differences were small. Prevalence of healthy weight (BMI < 85th centile), Modulators overweight (BMI 85th–94th centile) and obesity (BMI at or above 95th centile) are described in Table 3 for the CiF sample and Table 4 for the entire

cohort. Prevalence of overweight and obesity was similar between the CIF sample and the entire ALSPAC cohort and between boys and girls. There was some differential loss to follow up from 3 to 7 years and 11 to 15 years. Children who were obese at 3 years were slightly more likely to be lost to follow-up at 7 years [25.3% (21/83)] than those overweight at 3 years [23.0% (28/122)] or healthy weight at 3 years [19.5% (165/846)]. Children who were obese at 11 years were slightly more likely to be lost to follow up at 15 years [35.2% (50/142)] see more than children overweight [31.2% (39/125)] or healthy weight [28.5% (170/597)]. From 7 to 11 years, loss to follow up was similar in each group (~ 18%). The incidence of overweight and obesity in the CiF sample from 3 to 7, 7 to 11, and 11 to 15 years is shown in Table 5A. Incidence of overweight and obesity was higher

from 7 to 11 years [overweight 11.8% (76/646); obese 6.7% (43/646)] than 3 to 7 years [overweight 5.3% (36/681); obese 5.1% (35/681)] and 11 to 15 years [overweight 5.6% (24/427); obese 1.6% (7/427)]. There was some differential loss to follow up from 7 to 11 years and 11 to 15 years. Children obese at 7 years were slightly more likely to be lost to follow up at 11 years [28.3% (184/651)] than those overweight at 7 [23.4% (167/714)] or healthy weight at 7 [23.4% (1499/6394)]. Children obese at 11 years were slightly more likely to be lost to follow up at 15 [35.3% (376/1066)] than children who were overweight [32.1% (291/907)] or healthy weight [29.7% (1417/4778)]. Incidence of overweight [11.4% (558/4895)] and obesity [5.0% (243/4895)] from 7 to 11 years in the entire cohort was higher than the incidence

from 11 to 15 years [overweight 6.5% (220/3361); obese 1.4% (47/3361)], see Table 5B. In addition, the incidence of because overweight was slightly higher than the incidence of obesity at each time period. Furthermore, incidence of overweight and obesity from 7 to 11 years and from 11 to 15 years was similar between boys and girls and to the entire ALSPAC cohort (for full results see Supplementary Web Figs. 1 and 2 in Appendix A). In the CiF sample, 47.3% (52/110) of children who were overweight and obese at 3 years were overweight and obese at 15 years compared to 20% (93/465) of children who were healthy weight at 3 years; 70% (77/110) of children who were overweight and obese at 7 years were overweight and obese at 15 years compared to 15.3% (75/491) of children who were healthy weight at 7 years; 67.

It seems surprising that physicians thought parents would most likely forego pneumococcal vaccination if MenB vaccination were introduced, since this is a disease at least as severe as MenB IMD with a higher pre-vaccination incidence [17], but maybe less in the focus of privately practicing than of hospital-based pediatricians [18]. However, the other three vaccines named in this context either protect against diseases that are perceived as less severe (rotavirus, varicella) or with a lower risk of infection than MenB IMD (MenC). Age, sex, region and years spent in pediatric practice had a SKI-606 molecular weight significant effect on some of the responses (Table 2). As age of pediatrician and years

in practice were highly correlated (Pearson’s correlation coefficient = 0.83, p < 0.005), we present results only for the latter. Female physicians and physicians in practice ≥10 years were less likely to fear refusal of other recommended

vaccines if MenB vaccination were introduced, but were more likely to object to simultaneous administration of three vaccines or concomitant MenB vaccination and other vaccines. Correspondingly, female physicians were less likely to prefer Option 1 than their male colleagues, especially females in practice >10 years (see Appendix). Compared to pediatricians from Northern states, pediatricians from Western and Eastern states were more likely and pediatricians from Southern states less likely to Modulators believe that parents would be acceptant of MenB vaccination. Southern pediatricians were also more likely to fear refusal of other vaccines if MenB vaccination were recommended, ZD1839 purchase particularly if in practice <10 years, while those in Eastern states were less likely Histone demethylase to fear this, particularly those in practice ≥20 years (Appendix). This corresponds with a lower uptake of standard vaccines in Southern states than in other parts of Germany [19], possibly explained by a higher percentage of anthroposophists/vaccine-sceptics in their population [20] and [21] and a less positive physicians’ attitude towards

vaccination [14]. In contrast, uptake of standard vaccines is highest in Eastern Germany [19], where pediatricians, particularly female pediatricians (Appendix), were most likely to recommend MenB vaccination. Nonetheless, Eastern pediatricians were also more likely to object to simultaneous administration of 3 vaccines and prefer Option 2. Regional differences among German physicians were also seen in a previous study regarding attitude towards pertussis and measles vaccination [14]. As physicians play a crucial role in the implementation and acceptance of new vaccines, assessment of their views is essential. So far, results from only one other study, conducted in 2012 in France, are available on the attitude of pediatricians and general practitioners towards MenB vaccination [22].

4 ms and 12.5 ms, respectively, with 71% of correlations peaking within 20 ms and 93% within 50 ms (Figure 1E). Distributions of lag times did not differ significantly between inhibitory and excitatory connections (p = 0.24, Student’s t test). Taking into account delays due to action potential propagation (0.1 to 6.5 ms; Figure S3) and postsynaptic response (15.4 ms as measured by Itri et al. [2004]), these results

suggest that a majority of the deduced connections represent direct, fast synaptic interactions and a minority (i.e., those with longer lag times) may arise from polysynaptic interactions, common inputs, or postinhibitory rebound. Because most, if not all, SCN neurons are GABAergic and express GABAA receptors, we tested whether the mapped interactions depend on GABA-mediated signaling using gabazine (Gbz, 100 μM) or bicuculline (200 μM). These Adriamycin concentration GABAA receptor antagonists decreased the number of significant connections

by 90% ± 2% (mean ± SEM) compared to vehicle (p = 0.03; Figure 1F; comparing connections from 120 randomly selected GSK1349572 price neurons in three cultures during vehicle and drug application). This loss in functional connections was not due to a decrease in firing since discharge rates actually increased slightly under GABA blockade (vehicle = 4.06 ± 0.31 Hz; mean ± SEM; GABAA-R antagonists = 5.41 ± 0.38 Hz; p < 0.05). Those few cross-correlations that remained had low Z-scores, suggesting that they could have been weakened by the blockers or could depend on an alternate, weak intercellular signal. Given the 96% hit rate of BSAC, we conclude that at least 93% of all detected interactions in these SCN cultures, both inhibitory and excitatory, depend on GABAA receptor signaling. Specific network topologies have been postulated to underlie coordinated activity in a variety of biological systems including neural networks (Grinstein and Linsker, 2005; Harris et al., 2003; Bonifazi et al., 2009). These

topologies are considered scale-free if their degree distributions follow a power law. To assess the architecture of the GABA-dependent network Amisulpride in the SCN, we measured the number of connections from and to each neuron (out and in node degrees, respectively). As indicated by spatial network maps (e.g., Figures 1D and S2), levels of single-cell connectivity were heterogeneous. Neurons received connections from a median of 4.4% of the network and sent connections to 4.5% of the network; strikingly, some cells (15 of 330 cells) were directly connected to greater than 25% of the whole population and only 1.8% of nodes remained unconnected. Out and in degree distributions did not differ significantly across cultures (p > 0.05 respectively, one-way ANOVAs) and were fit better by first-order exponential decay functions (r2 = 0.95 and 0.87, respectively) than power functions (out: r2 = 0.82, F(1,20) = 51.83, p < 0.0001; in: r2 = 0.81, F(1,15) = 8.13, p = 0.012, extra-sum-of-squares F test).

, 2008). CadN mediated homophilic interactions between pre- and postsynaptic cells are thought to be key for synaptic partner selection. However, it is still unclear how the cell type specific function of CadN is achieved since CadN is widely expressed in the brain ( Lee et al., 2001). In cultured mouse hippocampal neurons, CadN undergoes both constitutive and activity dependent endocytosis and this website recycling, which is critical for synaptic plasticity (

Tai et al., 2007). It has been reported that the CadN expression pattern is highly dynamic during fly eye development but how it is regulated is unknown ( Matthews et al., 2008 and Nern et al., 2008). It is therefore interesting to establish how trafficking of CadN contributes to the dynamic changes of its expression pattern and determines PR cell targeting specificity. Here, we present evidence that Rab6, and its potential GEF protein Rich, control PR cell target selection by regulating CadN. Rab6 is a small GTPase that is present in the Golgi apparatus as well as cytoplasmic vesicles (Del Nery

et al., 2006, Martinez et al., 1994, Opdam et al., 2000 and Utskarpen et al., 2006). Rab6 recruits the dynein motor complex and plays an important role in microtubule dependent retrograde GSK1349572 supplier transport from the endosome to the Golgi and from the Golgi to the ER (Girod et al., 1999, Martinez et al., 1997, Matanis et al., 2002, Short et al., 2002 and White et al., 1999). In addition, Rab6 also directs targeting of secretory vesicles to the plasma membrane (Coutelis and Ephrussi, 2007, Grigoriev et al., 2007 and Januschke et al., 2007).

Like other GTPases, Rab6 alternates between a GTP-bound active form and a GDP-bound inactive form. The GEF proteins unload GDP from Rab GTPases to generate active GTP loaded Rabs (Stenmark, 2009). To date, the only Rab6 GEF that has been characterized is a yeast protein complex comprised of two proteins Ric1p and Rgp1p. The Ric1p-Rgp1p complex binds to Ypt6p, the yeast Rab6 homolog, in a nucleotide-dependent manner. The purified complex stimulates guanine nucleotide exchange on Ypt6p (Siniossoglou et al., 2000). Rab6 is highly conserved in eukaryotes. However, both Ric1 and Rgp1 are only present in budding yeast, although related proteins containing RIC1 domains as well as other domains or Rgp1 domains can be identified in higher whatever eukaryotes. In a forward genetic screen, we isolated a complementation group in which the PR cells display defects in synaptic specificity. We mapped the lesions to a novel gene CG9063 that encodes a protein containing a RIC1 domain as well as other domains. Our data indicate that this protein is required for proper synaptic specificity in the eye and antenna lobe (AL) by regulating CadN trafficking via Rab6. To isolate new genes involved in synapse development and function, we carried out an F1 genetic screen in the Drosophila visual system ( Ohyama et al.

Surprisingly, past work has shown that neuroprosthetic skills rely on similar neural substrates as natural motor learning (Green and Kalaska, 2011) and therefore have similar computational

requirements for rapid and flexible information transfer. Importantly, BMI tasks offer the unique advantage that researchers can define which neuronal ensembles are directly relevant for behavioral output, therefore allowing for an investigation of functional specificity within local populations. Recent theories have proposed that alterations in the pattern of large-scale synchronous activity could serve as the substrate for the flexible neuronal associations necessary to coordinate network activity for performance of both natural and neuroprosthetic behaviors Onalespib price (Womelsdorf et al., 2007 and Canolty et al., 2010). Oscillatory local field potential (LFP) activity reflects rhythmic current flow across cell membranes in local ensembles and is hypothesized to alter the excitability of cell groups across different spatiotemporal LDK378 purchase scales (Buzsáki and Draguhn, 2004, Lakatos et al., 2005 and Fröhlich and McCormick, 2010). Therefore, precise temporal control in neural networks could enhance the efficiency of information transfer in specific populations (Wang et al., 2010 and Tiesinga et al., 2001). It could also serve as a mechanism for synaptic gain control (Zeitler et al., 2008) and influence spike-timing-dependent

plasticity (Huerta and Lisman, 1993 and Harris et al., 2003), as spikes arriving at

excitability peaks will have enhanced efficacy relative to poorly timed spikes. Temporally coordinated activity in ensembles of neurons has been implicated in processes as diverse as perception (Rodriguez et al., 1999), expectation (von Stein et al., 2000), decision making (Pesaran et al., 2008), coordination (Dean et al., 2012), memory (Pesaran et al., 2002 and Siegel et al., 2009), spatial cognition (Colgin et al., 2009), reward processing (van der Meer and Redish, 2011), and attentional shifting (Bollimunta et al., 2011, Lakatos et al., 2008 and Fries et al., 2008). In some cases, this synchrony manifests as spiking in one region, becoming highly coordinated with LFP activity in a separate region (Pesaran et al., 2008). Importantly, many tasks evoke changes in the temporal pattern nearly of spiking without concomitant changes in firing rate, suggesting that synchrony could serve as an additional information channel in neural circuits (Riehle et al., 1997). Alterations in synchrony and LFP dynamics have also been implicated in pathological states such as epilepsy (Bragin et al., 2010) and Parkinson’s disease (Costa et al., 2006), highlighting their importance for normal brain functioning. Despite increasing evidence that changes in synchronous LFP activity are related to changes in behavior during learning (DeCoteau et al.

Computational studies of naturalistic behaviors show that the act of acquiring information—whether it is overt

or remains internal to the brain—may indeed have material value, as it increases the chance of success of a future action (Tatler et al., 2011). However, these studies also show that the processes required to compute information value differ markedly from those that have been so far considered in decision tasks. A salient property of this process is that information value depends critically on the subjects’ uncertainty and, in the Rescorla-Wagner CT99021 equation is more closely related with the right side of the equation—the act of learning or modifying expectations. As a simple illustration of this distinction, consider again the tea-making task in Figure 2B. To prepare

and consume her tea, the subject must make both arm and leg actions, and in the reinforcement equation both actions would be assigned a high value term (V). The subject’s gaze, however, is very selectively allocated to the targets of the arm and not the leg actions. This selectivity GSK126 cannot be explained in terms of action value alone but reflects the fact that the arm movements have higher uncertainty and thus more to gain from new information. Thus, the drive that motivates a shift of gaze is not value per se but the need to learn—i.e., to update one’s predictions through new information. Independent support for a view of attention as a learning mechanism comes from an area of research that has been mostly separate all from the oculomotor field (but see Le Pelley, 2010) but has directly addressed

the cognitive aspects of information selection—namely, the question of how subjects learn from and about sensory cues ( Pearce and Mackintosh, 2010). A central finding emerging from these studies is that subjects estimate the reliability of a sensory stimulus based on their prior experience with that stimulus and use this knowledge to modulate their future learning based on that cue. In the Rescorla-Wagner equation this process is implemented using an associability parameter, α, which is a stimulus-specific learning rate ( Pearce and Mackintosh, 2010): equation(Equation 2) Vt=Vt−1+α∗β∗δVt=Vt−1+α∗β∗δ While, as we have seen above, the standard learning rate β is applied globally to a context or task, associability is a property of an individual cue and can differentially weight the available cues. As I discuss in detail in the following sections, this apparently simple modification entails a complex, hierarchical learning mechanism. It entails an executive process which, having previously learned the predictive validity of a sensory cue, guides the moment by moment information selection—i.e., has in effect learnt how to learn. A final line of evidence for the information-bound nature of eye movement control comes from single-neuron studies of target selection that dissociate shifts of attention from overt shifts of gaze (Gottlieb and Balan, 2010).

Using dominant-negative overexpression and RNAi approaches, we show that signal recognition by μ1A, as well as the whole AP-1 complex and clathrin, are required for sorting of these selleck chemicals llc transmembrane cargoes to the somatodendritic domain. Microscopic imaging shows that sorting involves exclusion of the receptor proteins

from transport carriers destined for the axonal domain at the level of the soma. The neuron-specific glutamate receptor proteins mGluR1, NR2A, and NR2B, but not GluR1 and GluR2, are also sorted to the somatodendritic domain by a similar mechanism. Interference with AP-1-dependent somatodendritic sorting causes morphological changes in dendritic spines and decreases the number of synapses. These findings demonstrate that signal-AP-1 interactions mediate clathrin-dependent sorting of selected transmembrane cargoes to the somatodendritic domain of hippocampal neurons. More generally, they support the notion that AP-1 is a global regulator of polarized sorting RO4929097 order in different cell types. To analyze the mechanisms of somatodendritic sorting in rat hippocampal neurons, we initially used TfR as a model transmembrane protein. TfR is a type II integral membrane protein that functions as an endocytic receptor for iron-loaded

transferrin and that localizes in a polarized manner to the basolateral domain of epithelial cells (Fuller and Simons, 1986) and the somatodendritic domain of neurons (Cameron et al., 1991) by virtue of sorting information contained within its N-terminal cytosolic domain (Figure 1A) (Collawn et al., 1990; Odorizzi and Trowbridge, 1997; West et al., 1997). Confocal fluorescence microscopy of day in vitro 10 (DIV10) neurons expressing TfR tagged at its C-terminal ectodomain with monomeric green fluorescent protein (GFP) (A206K variant) (TfR-GFP) showed that this protein localized

to the dendrites and soma (Figures 1B and 1C) but was largely excluded from the axon (Figure 1B, arrowheads, Figure 1C). Quantification of fluorescence intensity in dendrites versus axons in many cells yielded a polarity index of 9.1 ± 3.0 for this protein (Table 1). Thus, the polarized distribution of transgenic TfR-GFP recapitulated that of endogenous TfR (Cameron et al., 1991). TfR has a cytosolic tail of 67 amino MTMR9 acids comprising an endocytic YXXØ signal (YTRF, residues 20–23) (Figure 1A) (Collawn et al., 1990). Previous deletion analyses showed that several segments of the TfR tail are required for somatodendritic sorting (West et al., 1997), but the exact signals involved were not defined. We performed a mutational analysis of the TfR tail and found that single substitution of alanine for Y20 resulted in loss of polarized distribution of TfR-GFP, with the mutant protein being evenly distributed among the dendrites, soma, and axon (Figures 1B and 1C) (polarity index: 1.3 ± 0.2; Table 1).

Paired-associative learning of this form has long been recognized, both within the animal learning community in studies of intentional actions (Dickinson, 1980) and in neuroscience

starting with the seminal studies of Miyashita on the electrophysiological signature of fractal pairings (Miyashita et al., 1993). Research on “systems consolidation” at the memory circuits level, which is distinct from research on “cellular consolidation” at VE-821 chemical structure the single-cell level (Dudai and Morris, 2000), has led to the idea that the distributed circuitry of the hippocampus performs a variety of encoding-related operations to stimuli such as pattern separation and pattern completion before subsequently creating event-event or event-context associations that may then be subject to consolidation in neocortex (McClelland and Goddard, 1996). The hippocampus and neocortex are hence considered as complementary learning systems (CLSs; McClelland and Goddard,

1996). Whereas the hippocampus is good at putting anything together with anything, and particularly with spatial information in the case of rodents, the neocortex readily forms representations of individual stimuli but is more restricted functionally in its capacity to link disparate information (e.g., information in distinct sensory processing systems). The neuroanatomical connectivity required may be present, but the strength of connections is initially weak, with experience being the guide as to what gets functionally http://www.selleckchem.com/products/Gemcitabine-Hydrochloride(Gemzar).html connected to what. The combined forces of flexible hippocampal-dependent learning, systems consolidation, and the vast storage capacities of through the neocortex collectively realize the “binding” task of understanding and representing the world around us and not just changing behavior adaptively to deal with specific types of association. However, this systems consolidation process is now revealed as one that is influenced by what has gone before. One recent example that combines thinking about prior knowledge with representational

associations is the idea of forming “schemas” around related paired associates that then alter the rate at which new paired associates can be learned and consolidated (Tse et al., 2007). Specifically, animals are trained to enable one of several flavors of food to be associated with and thus predict the location where more of that foodstuff is available. In this case, neither the different flavors of food nor the locations change “value” in the manner that a context does in context fear conditioning; what changes is the ability of one set of cues (flavors) to evoke a memory of the other (places). The use of places also enables the animals to gradually build up a representation of the testing space, over several weeks of training, such that they may be thought to have a mental schema that connects these otherwise independent associations into some kind of framework.