Non-specific Components Research Articles

A randomized group intervention trial to enhance mood and self‐efficacy in people with multiple sclerosis

To document mood, self-efficacy, and resiliency in people with multiple sclerosis (MS) following a brief group psychological intervention, and to examine whether benefits were greater than those derived from provision of education or group social interaction. A randomized controlled intervention trial assessing outcomes at five time points over a 1-year follow-up. Participants with MS were assigned to one of three groups: one receiving brief group psychological intervention (PG) comprising three 90 minute cognitive behavioural sessions supported by an Information Booklet dealing with mental and emotional issues relating to MS; a group provided only with educational material - the information booklet group (IBG); and a group who not only received the booklet but also participated in non-structured social discussion (SDG) sessions similar in length and number to PG participants. Outcomes were documented using questionnaires. Outcomes were assessed using area under the curve (AUC) analysis: a summary measure that considers individual changes serially over time to provide a more meaningful picture than the one based on single time points. Ninety participants were followed up over the 12-month post-intervention, and their data are included in the analysis. Analyses indicated benefits in all outcome dimensions for the psychotherapeutic (PG) and social discussion groups (SDG) relative to the IBG group, but no differences between PG and SDG. The study indicates benefits from psychosocial intervention compared with bibliotherapy, with some additional benefit from psychological intervention compared with a social discussion group. Results suggest that much of the benefit may derive from non-specific therapeutic components. Without psychosocial intervention, the psychological status of people with MS worsened over time.

Role of P63 (CKAP4) in binding of surfactant protein-A to type II pneumocytes

We have recently described a putative receptor for lung surfactant protein-A (SP-A) on rat type II pneumocytes. The receptor, P63, is a 63-kDa type II transmembrane protein. Coincubation of type II cells with P63 antibody (Ab) reversed the inhibitory effect of SP-A on secretagogue-stimulated surfactant secretion from type II cells. To further characterize SP-A interactions with P63, we expressed recombinant P63 protein in Escherichia coli and generated antibodies to P63. Immunogold electron microscopy confirmed endoplasmic reticulum and plasma membrane localization of P63 in type II cells with prominent labeling of microvilli. Binding characteristics of iodinated SP-A to type II cells in the presence of P63 Ab were determined. Binding (4 degrees C, 1 h) of (125)I-SP-A to type II cells demonstrated both specific (calcium-dependent) and nonspecific (calcium-independent) components. Ab to P63 protein blocked the specific binding of (125)I-SP-A to type II cells and did not change the nonspecific SP-A association. A549 cells, a pneumocyte model cell line, expressed substantial levels of P63 and demonstrated specific binding of (125)I-SP-A that was inhibited by the P63 Ab. The secretagogue (cAMP)-stimulated increase in calcium-dependent binding of SP-A to type II cells was blocked by the presence of P63 Ab. Transfection of type II cells with small interfering RNA to P63 reduced P63 protein expression, attenuated P63-specific SP-A binding, and reversed the ability of SP-A to prevent surfactant secretion from the cells. Our results further substantiate the role of P63 as an SP-A receptor protein localized on the surface of lung type II cells.

An improved approach to detection of amplitude of low-frequency fluctuation (ALFF) for resting-state fMRI: Fractional ALFF

Most of the resting-state functional magnetic resonance imaging (fMRI) studies demonstrated the correlations between spatially distinct brain areas from the perspective of functional connectivity or functional integration. The functional connectivity approaches do not directly provide information of the amplitude of brain activity of each brain region within a network. Alternatively, an index named amplitude of low-frequency fluctuation (ALFF) of the resting-state fMRI signal has been suggested to reflect the intensity of regional spontaneous brain activity. However, it has been indicated that the ALFF is also sensitive to the physiological noise. The current study proposed a fractional ALFF (fALFF) approach, i.e., the ratio of power spectrum of low-frequency (0.01–0.08 Hz) to that of the entire frequency range and this approach was tested in two groups of resting-state fMRI data. The results showed that the brain areas within the default mode network including posterior cingulate cortex, precuneus, medial prefrontal cortex and bilateral inferior parietal lobule had significantly higher fALFF than the other brain areas. This pattern was consistent with previous neuroimaging results. The non-specific signal components in the cistern areas in resting-state fMRI were significantly suppressed, indicating that the fALFF approach improved the sensitivity and specificity in detecting spontaneous brain activities. Its mechanism and sensitivity to abnormal brain activity should be evaluated in the future studies.

Structural and thermodynamic parameters and intermolecular interactions in aqueous formic acid amides

The structural and thermodynamic characteristics of aqueous solutions of formamide, N-methylformamide, N,N-dimethylformamide, and N,N-diethylformamide were calculated. The specific and nonspecific components of the total energy of intermolecular interactions were determined. The limits of the concentration regions with differently arranged structures of solutions were established, and the preferable solvation parameters of the solution components were evaluated.

Evaluation of non-specific immune components from the skin mucus of olive flounder ( Paralichthys olivaceus)

The innate immune system, particularly the external body surface, plays a frontier role in protecting fish under intensive aquaculture and at prolonged low temperatures from relevant infections due to inadequate adaptive immune responses. In the present study we aimed to understand the mucosal immunity of an economically important mariculture fish, olive flounder ( Paralichthys olivaceus) by evaluating the immune components from its skin mucus. The activities of lysozyme (233.33 ± 171.82 units mg −1), trypsin-like protease (42.84 ± 1.249 units mg −1), alkaline phosphatase (0.376 ± 0.005 units mg −1) and esterase (0.170 ± 0.006 units mg −1) were detected in the skin mucus. Transferrin was identified by MALDI-TOF/MS analysis. ELISA and immunoblot assays using anti-flounder IgM monoclonal antibody showed the presence of a significant level (1.80 ± 0.001, n = 3) of monomer immunoglobulin M (IgM) with approximate molecular weight of 160 and 25 kDa under non-denaturing and denaturing states, respectively. Skin mucus showed strong antibacterial activity against tested fish pathogenic bacteria. In addition, skin mucus successfully agglutinated (HA titre 2 8), but completely failed to haemolyse, rabbit erythrocytes. In conclusion, the major immune components of the skin mucus, identified in the present study, are possibly involved in the broad spectrum non-specific immunity of olive flounder.

Structral and thermodynamic characteristics and intermolecular interactions in aqueous solutions of diols

The structural and thermodynamic characteristics of aqueous solutions of ethanediol, 1,2-and 1,3-propanediols, and 1,2-and 1,4-butanediols were calculated over the whole range of the compositions of the mixtures. The specific and nonspecific components of the total energy of intermolecular interactions were determined. The boundaries of the concentration regions with different structural organizations of solutions were established, and the parameters of preferable solvation of the solution components were evaluated.

Explaining differences in saturation levels for Affymetrix GeneChip® arrays

The experimental spike-in studies of microarray hybridization conducted by Affymetrix demonstrate a nonlinear response of fluorescence intensity signal to target concentration. Several theoretical models have been put forward to explain these data. It was shown that the Langmuir adsorption isotherm recapitulates a general trend of signal response to concentration. However, this model fails to explain some key properties of the observed signal. In particular, according to the simple Langmuir isotherm, all probes should saturate at the same intensity level. However, this effect was not observed in the publicly available Affymetrix spike-in data sets. On the contrary, it was found that the saturation intensities vary greatly and can be predicted based on the probe sequence composition. In our experimental study, we attempt to account for the unexplained variation in the observed probe intensities using customized fluidics scripts. We explore experimentally the effect of the stringent wash, target concentration and hybridization time on the final microarray signal. The washing effect is assessed by scanning chips both prior to and after the stringent wash. Selective labeling of both specific and non-specific targets allows the visualization and investigation of the washing effect for both specific and non-specific signal components. We propose a new qualitative model of the probe-target hybridization mechanism that is in agreement with observed hybridization and washing properties of short oligonucleotide microarrays. This study demonstrates that desorption of incompletely bound targets during the washing cycle contributes to the observed difference in saturation levels.

Dissecting the Non-specific and Specific Components of the Initial Folding Reaction of Barstar by Multi-site FRET Measurements

Initial polypeptide chain collapse plays a major role in the development of subsequent structure during protein folding, but it has been difficult to elucidate the coupling between its cooperativity and specificity. To better understand this important aspect of protein folding, nine different intramolecular distances in the protein have been measured by fluorescence resonance energy transfer (FRET) in the product(s) of the initial, sub-millisecond collapse reaction during the folding of barstar, under different folding conditions. All nine distances contract in these initial folding products, when the denaturant concentration is reduced. Two of these distances were also measured in peptides corresponding to sequence segments 38–55 and 51–69 of the protein. Surprisingly, both distances do not contract in the peptides which remain fully unfolded when the denaturant concentration is reduced. This suggests that the contraction of at least some segments of the polypeptide chain may be facilitated only by contraction of other segments. In the case of the initial product of folding of the protein, the dependence on denaturant concentration of the relative change in each distance suggests that there are two components to the initial folding reaction. One is a nonspecific component, which appears to be driven by the change in denaturant concentration that is used to initiate refolding. This component corresponds to the collapse of completely unfolded protein ( U) to unfolded protein in refolding conditions ( U C). The extent of nonspecific collapse can be predicted by the response of completely unfolded protein to a change in denaturant concentration. All distances undergo such solvent-induced contraction, but each distance contracts to a different extent. There is also a specific component to initial sub-millisecond folding, in which some distances (but not all) contract more than that predicted by solvent-induced contraction. The observation that only some of the distances undergo contraction over and above solvent-induced contraction, suggest that this specific component is associated with the formation of a specific intermediate ( I E). FRET efficiency and distance change differently for the different donor–acceptor pairs, with a change in denaturant concentration, indicating that the formation or dissolution of structure in U C and I E does not happen in a synchronized manner across different regions of the protein molecule. Also, all nine FRET efficiencies and intramolecular distances in the product(s) of sub-ms folding, change continuously with a change in denaturant concentration. Hence, it appears that the transitions from U to U C and to I E are gradual transformations, and not all-or-none structural transitions. Nevertheless, the product of these gradual transitions, I E, possesses specific structure.

Spontaneous tumor rejection by cbl-b–deficient CD8+ T cells

The concept of tumor surveillance implies that specific and nonspecific components of the immune system eliminate tumors in the early phase of malignancy. Understanding the biochemical mechanisms of tumor immunosurveillance is of paramount significance because it might allow one to specifically modulate spontaneous antitumor activity. We report that inactivation of the E3 ligase Casitas B cell lymphoma-b (Cbl-b) confers spontaneous in vivo rejection of tumor cells that express human papilloma virus antigens. Moreover, cbl-b−/− mice develop significantly fewer ultraviolet B (UVB)–induced skin malignancies and reject UVB-induced skin tumors. CD8+ T cells were identified as key players in the spontaneous tumor rejection response. Loss of Cbl-b not only enhances antitumor reactivity of CD8+ T cells but also occurs in the absence of CD4+ T cells. Mechanistically, cbl-b−/− CD8+ T cells are resistant to T regulatory cell–mediated suppression and exhibit enhanced activation and rapid tumor infiltration. Importantly, therapeutic transfer of naive cbl-b−/− CD8+ T cells is sufficient to mediate rejection of established tumors. Even up to 1 yr after the first encounter with the tumor cells, cbl-b−/− mice carry an “anticancer memory.” These data identify Cbl-b as a key signaling molecule that controls spontaneous antitumor activity of cytotoxic T cells in different cancer models. Inhibition of Cbl-b is a novel approach to stimulate long-lasting immunity against cancer.

DEFENSE MECHANISMS OF THE SURFACE EPITHELIUM OF HUMAN ESOPHAGEALMUCOSA

This review, which is based on the literature data and the results of personal research, contains an analysis of the current concepts on the tissue, cellular and molecular mechanisms, protecting human esophageal epithelium (EE) from gastric juice, bile, hot and rough food, microorganisms, alcohol, carcinogens, drugs and oxidizing agents. The response of EE to concrete environmental factors includes both specific and non-specific components, which depend on the nature of injurious agent. EE is damaged structurally and functionally only when it is exposed to the injurious factors of high intensity and /or long duration, which result in the exhaustion of resources of defense mechanisms. The insufficiency of EE defense mechanisms may be based on various genetic defects.

Enhancing Macroautophagy Protects against Ischemia/Reperfusion Injury in Cardiac Myocytes

Cardiac myocytes undergo programmed cell death as a result of ischemia/reperfusion (I/R). One feature of I/R injury is the increased presence of autophagosomes. However, to date it is not known whether macroautophagy functions as a protective pathway, contributes to programmed cell death, or is an irrelevant event during cardiac I/R injury. We employed simulated I/R of cardiac HL-1 cells as an in vitro model of I/R injury to the heart. To assess macroautophagy, we quantified autophagosome generation and degradation (autophagic flux), as determined by steady-state levels of autophagosomes in relation to lysosomal inhibitor-mediated accumulation of autophagosomes. We found that I/R impaired both formation and downstream lysosomal degradation of autophagosomes. Overexpression of Beclin1 enhanced autophagic flux following I/R and significantly reduced activation of pro-apoptotic Bax, whereas RNA interference knockdown of Beclin1 increased Bax activation. Bcl-2 and Bcl-x(L) were protective against I/R injury, and expression of a Beclin1 Bcl-2/-x(L) binding domain mutant resulted in decreased autophagic flux and did not protect against I/R injury. Overexpression of Atg5, a component of the autophagosomal machinery downstream of Beclin1, did not affect cellular injury, whereas expression of a dominant negative mutant of Atg5 increased cellular injury. These results demonstrate that autophagic flux is impaired at the level of both induction and degradation and that enhancing autophagy constitutes a powerful and previously uncharacterized protective mechanism against I/R injury to the heart cell.

Non-linear analysis of GeneChip arrays

The application of microarray hybridization theory to Affymetrix GeneChip data has been a recent focus for data analysts. It has been shown that the hyperbolic Langmuir isotherm captures the shape of the signal response to concentration of Affymetrix GeneChips. We demonstrate that existing linear fit methods for extracting gene expression measures are not well adapted for the effect of saturation resulting from surface adsorption processes. In contrast to the most popular methods, we fit background and concentration parameters within a single global fitting routine instead of estimating the background before obtaining gene expression measures. We describe a non-linear multi-chip model of the perfect match signal that effectively allows for the separation of specific and non-specific components of the microarray signal and avoids saturation bias in the high-intensity range. Multimodel inference, incorporated within the fitting routine, allows a quantitative selection of the model that best describes the observed data. The performance of this method is evaluated on publicly available datasets, and comparisons to popular algorithms are presented.

On the parameterization of surface structures

The procedure for the parameterization of complex surface structures is proposed on the basis of the analysis of spatial “series” formed by the projections on the coordinate axes of roughness profiles recorded during the surface scanning with the sensitive element of atomic force microscope. To extract parameters of thus obtained “spatial series,” flicker-noise spectroscopy is used that allows retrieval of information relevant to specific (“resonance”) and nonspecific (chaotic) components of analyzed complex signals.

Demonstration of a common artifact in immunosorbent assays of brain extracts: Development of a solid-phase extraction protocol to enable measurement of amyloid-β from wild-type rodent brain

In the process of developing species-specific, immunosorbent assays for brain amyloid-β (Aβ) in non-transgenic animals, we have demonstrated an artifact that impedes accurate quantitation of Aβ in this assay format. Using synthetic peptides, cerebrospinal fluid (CSF), or plasma samples, no nonspecific binding or cross-species immunoreactivity was detected in human or rodent Aβ assays. However, extracts of guinea pig brain (human Aβ sequence) or rat brain (rodent Aβ sequence) demonstrated immunoreactivity regardless of which capture antibody, detection antibody, or reporter method (colorimetric or fluorescent) was used. This immunoreactivity remained even in the absence of a capture antibody. Various blocking conditions failed to resolve the nonspecific binding of detection antibodies in the presence of brain extracts. Fractionation of DEA-extracted guinea pig brain over Sephadex G-50 demonstrated the feasibility of separating specific from nonspecific binding components in the brain extracts. Thus, a solid phase extraction method, compatible with multiple extraction buffers, has been developed to isolate and concentrate Aβ from brain extracts. This isolation method eliminates non-specific binding components from brain extracts and allows for accurate quantitation and robust detection of multiple Aβ peptides in extracts from wild-type animals.

Effect of biomaterial surface properties on fibronectin– α5β1 integrin interaction and cellular attachment

The ability of fibronectin (Fn) to mediate cell adhesion through binding to α 5 β 1 integrins is dependent on the conditions of its adsorption to the surface. Using a model system of alkylsilane SAMs with different functional groups ( X=OH, COOH, NH 2 and CH 3) and an erythroleukemia cell line expressing a single integrin ( α 5 β 1), the effect of surface properties on the cellular adhesion with adsorbed Fn layers was investigated. 125I-labeled Fn, a modified biochemical cross-linking/extraction technique and a spinning disc apparatus were combined to quantify the Fn adsorption, integrin binding and adhesion strength, respectively. This methodology allows for a binding equilibrium analysis that more closely reflects cellular adhesion found in stable tissue constructs in vivo. Differences in detachment strength and integrin binding were explained in terms of changes in the adhesion constant ( ψ, related to affinity) and binding efficiency of the adsorbed Fn for the α 5 β 1 integrins (CH 3≈NH 2<COOH≈OH) and the resulting average bond strength. Fn interacted more strongly with α 5 β 1 integrins when adsorbed on COOH vs. OH surfaces suggesting that negative charge may be a critical component of inducing efficient cellular adhesion. As evident by the low ψ values, Fn adsorbed on NH 2 and CH 3 surfaces interacted inefficiently with α 5 β 1 integrins and also possessed significant non-specific components to adhesion. Lastly, comparison of cellular adhesion to Fn adsorbed onto smooth and rough surfaces showed that nano-scale roughness altered cellular adhesion by increasing the surface density of adsorbed Fn.

Biogenesis and speciation of nascent apoA-I-containing particles in various cell lines

It is generally thought that the large heterogeneity of human HDL confers antiatherogenic properties; however, the mechanisms governing HDL biogenesis and speciation are complex and poorly understood. Here, we show that incubation of exogenous apolipoprotein A-I (apoA-I) with fibroblasts, CaCo-2, or CHO-overexpressing ABCA1 cells generates only alpha-nascent apolipoprotein A-I-containing particles (alpha-LpA-I) with diameters of 8-20 nm, whereas human umbilical vein endothelial cells and ABCA1 mutant (Q597R) cells were unable to form such particles. Interestingly, incubation of exogenous apoA-I with either HepG2 or macrophages generates both alpha-LpA-I and prebeta1-LpA-I. Furthermore, glyburide inhibits almost completely the formation of alpha-LpA-I but not prebeta1-LpA-I. Similarly, endogenously secreted HepG2 apoA-I was found to be associated with both prebeta1-LpA-I and alpha-LpA-I; by contrast, CaCo-2 cells secreted only alpha-LpA-I. To determine whether alpha-LpA-I generated by fibroblasts is a good substrate for LCAT, isolated alpha-LpA-I as well as reconstituted HDL [r(HDL)] was reacted with LCAT. Although both particles had similar V(max) (8.4 vs. 8.2 nmol cholesteryl ester/h/microg LCAT, respectively), the K(m) value was increased 2-fold for alpha-LpA-I compared with r(HDL) (1.2 vs. 0.7 microM apoA-I). These results demonstrate that 1) ABCA1 is required for the formation of alpha-LpA-I but not prebeta1-LpA-I; and 2) alpha-LpA-I interacts efficiently with LCAT. Thus, our study provides direct evidence for a new link between specific cell lines and the speciation of nascent HDL that occurs by both ABCA1-dependent and -independent pathways.

Atomic force microscopy study on specificity and non-specificity of interaction forces between Enterococcus faecalis cells with and without aggregation substance

Enterococcus faecalis is one of the leading causes of hospital-acquired infections, and indwelling medical devices are especially prone to infection. E. faecalis expressing aggregation substance (Agg) adheres to biomaterial surfaces by means of positive cooperativity, i.e. the ability of one adhering organism to stimulate adhesion of other organisms in its immediate vicinity. In this study, atomic force microscopy (AFM) was used to measure the specificity and non-specificity of interaction forces between E. faecalis cells with and without Agg. Bacteria were attached to a substratum surface and a tip-less cantilever. Two E. faecalis strains expressing different forms of Agg showed nearly twofold higher interaction forces between bacterial cells than a strain lacking Agg [adhesive force (F(adh)), -1.3 nN]. The strong interaction forces between the strains with Agg were reduced after adsorption of antibodies against Agg from -2.6 and -2.3 nN to -1.2 and -1.3 nN, respectively. This suggests that the non-specific interaction force between the enterococci amounts to approximately 1.2 nN, while the specific force component is only twofold stronger. Comparison of the results of the AFM interaction forces with the positive cooperativity after adhesion to a biomaterial in a parallel-plate flow chamber showed that in the absence of strong interaction forces between the cells, positive cooperativity was also absent. In conclusion, this is believed to be the first time that the influence of specific antibodies on interaction forces between E. faecalis cells has been demonstrated by AFM, thereby experimentally distinguishing between specific and non-specific force components.

Structural modification of plasma HDL by phospholipids promotes efficient ABCA1-mediated cholesterol release

It has been suggested that ABCA1 interacts preferentially with lipid-poor apolipoprotein A-I (apoA-I). Here, we show that treatment of plasma with dimyristoyl phosphatidylcholine (DMPC) multilamellar vesicles generates prebeta(1)-apoA-I-containing lipoproteins (LpA-I)-like particles similar to those of native plasma. Isolated prebeta(1)-LpA-I-like particles inhibited the binding of (125)I-apoA-I to ABCA1 more efficiently than HDL(3) (IC(50) = 2.20 +/- 0.35 vs. 37.60 +/- 4.78 microg/ml). We next investigated the ability of DMPC-treated plasma to promote phospholipid and unesterified (free) cholesterol efflux from J774 macrophages stimulated or not with cAMP. At 2 mg DMPC/ml plasma, both phospholipid and free cholesterol efflux were increased ( approximately 50% and 40%, respectively) in cAMP-stimulated cells compared with unstimulated cells. Similarly, both phospholipid and free cholesterol efflux to either isolated native prebeta(1)-LpA-I and prebeta(1)-LpA-I-like particles were increased significantly in stimulated cells. Furthermore, glyburide significantly inhibited phospholipid and free cholesterol efflux to DMPC-treated plasma. Removal of apoA-I-containing lipoproteins from normolipidemic plasma drastically reduced free cholesterol efflux mediated by DMPC-treated plasma. Finally, treatment of Tangier disease plasma with DMPC affected the amount of neither prebeta(1)-LpA-I nor free cholesterol efflux. These results indicate that DMPC enrichment of normal plasma resulted in the redistribution of apoA-I from alpha-HDL to prebeta-HDL, allowing for more efficient ABCA1-mediated cellular lipid release. Increasing the plasma prebeta(1)-LpA-I level by either pharmacological agents or direct infusions might prevent foam cell formation and reduce atherosclerotic vascular disease.

Mindfulness and Healing Intention: Concepts, Practice, and Research Evaluation

This paper deals with the role of the health care professional in creating an optimal healing environment (OHE), with a special focus on which inner state and way of being in the world can create a healing intention. A core thesis is that every healing effort and every healing intention starts within the health care professional. An accepting, mindful, and warm-hearted relationship with self is primary to any healing intention. Important concepts to develop such an attitude are mindfulness, love, compassion, and awareness. The concept of a healing attitude toward the self is described, as is the mirror principle that states there is symmetry between someone's relationship with the outer world and his or her inner world. The mindfulness concept is outlined through a set of mental and heart qualities. Mindfulness is strongly related to compassion, and it is compassion that serves as a source for all healing intentionality. Compassion connects the suffering of the patient with the healer's own suffering, and this emotional connection instantiates a healing relationship. The power and importance of mindfulness and compassion for healing are explored along the Frank model of nonspecific therapeutic components. This is the approach whereby a health care professional can elicit self-healing powers in patients through his or her inner attitudes. Finally, a research program and some hypotheses on how to implement and research this specific approach toward the creation of an OHE are outlined.

A MAP kinase cascade composed of cell type specific and non-specific elements controls mating and differentiation of the fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle in which the alpha allele of the mating type locus is linked to virulence and haploid differentiation. Here we analysed a conserved MAP kinase cascade composed of mating-type specific (Ste11alpha, Ste12alpha) and non-specific (Ste7, Cpk1) elements. Gene disruption experiments demonstrate that this specialized MAP kinase pathway is required for both mating and cell type-specific differentiation but not for virulence. The Ste11alpha, Ste7 and Cpk1 kinases were found to act as a co-ordinate signalling module, whereas the Ste12alpha transcription factor functions with a redundant partner or in a branched or parallel signalling pathway. Our studies illustrate how MAP kinase cascades can be constructed from cell type-specific and non-specific components, yielding pathways that contribute to cell type-specific patterns of signalling and differentiation.