NFAT and NFκB Activation in T Lymphocytes: A Model of Differential Activation of Gene Expression

Mathematical models for the regulation of the Ca2+-dependent transcription factors NFAT and NFκB that are involved in the activation of the immune and inflammatory responses in T lymphocytes have been developed. These pathways are important targets for drugs, which act as powerful immunosuppressants by suppressing activation of NFAT and NFκB in T cells. The models simulate activation and deactivation over physiological concentrations of Ca2+, diacyl glycerol (DAG), and PKCθ using single and periodic step increases. The model suggests the following: (1) the activation NFAT does not occur at low frequencies as NFAT requires calcineurin activated by Ca2+ to remain dephosphorylated and in the nucleus; (2) NFκB is activated at lower Ca2+ oscillation frequencies than NFAT as IκB is degraded in response to elevations in Ca2+ allowing free NFκB to translocate into the nucleus; and (3) the degradation of IκB is essential for efficient translocation of NFκB to the nucleus. Through sensitivity analysis, the model also suggests that the largest controlling factor for NFAT activation is the dissociation/reassociation rate of the NFAT:calcineurin complex and the translocation rate of the complex into the nucleus and for NFκB is the degradation/resynthesis rate of IκB and the import rate of IκB into the nucleus.
Introduction
Nuclear factor of activated T cells (NFAT) and nuclear factor κB (NFκB) are Ca2+-dependent and PKCθ-dependent transcription factors that mediate the immune response in T lymphocytes (T cells).8,16,25,44,49 In resting cells both NFAT and NFκB reside primarily in the cytoplasm, NFAT in its phosphorylated form and NFκB bound to the inhibitor IκB. T cell receptor stimulation initiates a cascade of intracellular reactions causing an increase in the cytosolic calcium concentration and a resulting translocation of both NFAT and NFκB to the nucleus.19,48 Both NFAT and NFκB may bind to promoter regions of DNA and, in concert with other transcription factors, allow expression of genes responsible for the production of the interleukins IL-2 by NFAT50 and IL-8 by NFκB.58
In the NFAT pathway (Fig. 1a), Ca2+ activates the phosphatase calcineurin, which binds to and dephosphorylates NFAT.34,45,59 Dephosphorylation exposes nuclear localization signals,10 resulting in rapid translocation of the NFAT-calcineurin complex to the nucleus where it can participate in promoting gene transcription.51 The cytoplasm-to-nucleus translocation is opposed by kinases such as GSK-3 and possibly protein kinase A that block the Ca2+-calcineurin dephosphorylation of NFAT in the cytoplasm, or that rephosphorylate free NFAT in the nucleus.9,43 Association of NFAT with activated calcineurin inhibits the kinase activity; in the absence of activated calcineurin, dephosphorylated NFAT in the nucleus is rephosphorylated and exported. This futile shuttling of NFAT into and out of the nucleus results in little gene transcription, thus gene expression activated by NFAT requires continuously active calcineurin.54,62
Figure 1.
(a) Schematic diagram of the mechanism of action of NFAT. At rest, NFAT is in its phosphorylated form (NFAT:Pi) and located mainly in the cytosol. Upon activation, a rise in [Ca]i activates calcineurin (C) which binds NFAT:Pi and dephosphorylates it, allowing its import into the nucleus where it can act on the DNA. (b) Schematic diagram of the mechanism of action of NFκB. At rest, NFκB is bound to its inhibitor IκB mainly in the cytosol (NFκB:IκB). Activation of calcineurin (C) synergizes with PKCα activating an early phase of the kinase IKK and activation of PKCθ activates a later phase of IKK which phosphorylates IκB. It then dissociates from NFκB allowing NFκB to be imported into the nucleus where it can act on the DNA.
In contrast to NFAT, NFκB is retained in the cytoplasm (Fig. 1b) not by phosphorylation but by association with the inhibitory IκB proteins.7 In resting cells, NFκB and IκB can dissociate in the cytoplasm and translocate to the nucleus.14 Reassociation of NFκB and IκB masks nuclear localization signals on both species resulting in nuclear export. NFκB has little opportunity to bind to DNA during this shuttling and so little transcription occurs. Stimulation of the T cell receptor, however, initiates a cascade of reactions proceeding through Ca2+48 and PKCθ33,56 that results in activation of the IκB kinase IKK. IKK phosphorylates IκB associated with NFκB, allowing the IκB to dissociate from NFκB and marking the IκB for subsequent polyubiquitination and degradation by the proteasome.20,36 NFκB is freed by this degradation, nuclear localization signals on NFκB are exposed, and it is translocated to the nucleus where it can bind to DNA and promote gene transcription. IκB is resynthesized when the calcium and PKCθ concentration decrease. Newly formed IκB translocates to the nucleus where it binds to NFκB and masks its nuclear localization signals. The NFκB:IκB complex is then exported to the cytoplasm thus completing the activation-deactivation cycle. Recently, Hoffman and co-workers showed that oscillations in the IκB concentration are observed experimentally and modeled its mechanism.26
In many cells, interplay of the sources and sinks of Ca2+ results in oscillations in the intracellular calcium concentration when surface receptors are stimulated by agonist.11 In particular, T cells display calcium oscillations in response to T cell receptor activation.21 The transcription factors NFAT and NFκB respond in different ways to the frequency of these oscillations, thus differentiating the activation of NFAT and NFκB. In this way differential transcription factor activation and consequent gene expression is conferred through a ubiquitous second messenger, namely Ca2+.22 Calcium oscillations have also been shown to increase the level of NFAT activation at low levels of stimulation over a steady rise in Ca2+, increasing the efficiency of low-level signal detection.22 Furthermore, overexpression of calcineurin partially replaces the calcium requirement for NFAT activation, as does the expression of constitutively active calcineurin.39,15
Defects in the activation of NFAT and NFκB and expression of IL-2 and IL-8, respectively, have been implicated in a number of human diseases and conditions. In several patients, failure to activate NFAT was shown to cause severe combined immunodeficiency disease, SCID.23 Overexpression of IL-8 can cause psoriasis and rheumatoid arthritis.58 Other conditions related to defects in NFAT and NFκB activation include asthma, allergy, inflammation, and septic shock.
These pathways also contain an important target for drugs such as cyclosporin A and FK506, which act as powerful immunosuppressants by blocking the activation of calcineurin’s phosphatase activity,15,18,39 thereby suppressing activation of NFAT and NFκB in T cells. Furthermore, the PKCθ inhibitor rottlerin can block PKCθ translocation to the immunological synapse (IS),57 which suppresses the activation of NFκB in T cells. These properties make these drugs useful not only in combating immune system diseases but also in preventing the rejection of transplanted tissues. But the general immune system suppression that results with cyclosporin A and FK506 leads to severe side effects, such as secondary tumors and opportunistic infections,30 and progressive loss of renal function and neurotoxicity.5 Additionally, inhibition of transcription factor activation in other types of cells can have unintended and undesirable consequences.1
To understand better how signal transduction pathways function, to help find therapies and cures for immune system diseases, and to find better ways to control the immune system in transplant patients it would be useful to understand the NFAT and NFκB pathways in a quantitative fashion.
Models and Mechanisms
We have developed mathematical models for the action of NFAT and NFκB that simulate the activation and deactivation of each factor by changes in intracellular calcium concentration. Two separate models have been developed, one for NFAT and one for NFκB. We required both models to incorporate experimentally determined reaction pathways and to simulate activation and deactivation of the transcription factors through experimentally determined intermediates and over physiological calcium concentrations. Both models use parameters derived, as much as possible, from experimental observations (Tables 1–4). We required the models to be capable of simulating the system response to calcium concentration oscillations and, in line with experimental observations,22 to show a different response in the activation of NFAT and NFκB to changes in the frequency of the oscillations. We expect the NFAT model to show that oscillations in the concentration of calcium enhance the activity of NFAT at low calcium concentrations and that overexpression of calcineurin replaces the requirement for calcium in the activation of NFAT. For both the NFAT and NFκB models we expect the models to require sustained calcium signaling for gene expression.TABLE 1.NFAT rate constants.Rate constantValueSourcek10.0000256 s−1Estimatek20.00256 s−1Loh et al.34k30.005 s−1Estimatek40.5 s−1Estimatek50.0019 s−1Shibasaki et al.52k60.00092 s−1Shibasaki et al.52k70.005 s−1Estimatek80.5 s−1Estimatek90.5 s−1Estimatek100.005 s−1Estimatek116.63 μM−1 s−1Adjusted to control rate of activation observed by Shaw et al.51k120.00168 s−1Adjusted to control rate of deactivation observed by Beals et al. 10k130.5 s−1to agree with similar reactions given by Klee et al.32k140.00256 s−1Loh et al.34k150.00168 s−1Adjusted to control rate of deactivation observed by Beals et al.10k166.63μM−1s−1Adjusted to control rate of activation observed by Shaw et al.51k170.0015 s−1Zhu and McKeon62k180.00096 s−1Zhu and McKeon62k191.0μM−3s−1Klee et al.32; Kakalis et al.29k201.0 s−1Klee et al.32, Kakalis et al.29k210.21 s−1Estimate and adjusted relative to k22 to give equal calcium ion concentrations in nucleus and cytoplasmk220.5 s−1EstimateTABLE 2.Concentration and volume parameters.ParameterValueSourceCell diameter9000 nMEstimate (Alberts et al.2)Nucleus diameter6000 nMEstimate (Alberts et al.2)Nucleus volume (vn)113 μm3CalculatedCytosol volume (vc)269 μm3CalculatedResting cell [Ca2+]100 nMAlberts et al.2Activated cell [Ca2+]1000 nMAlberts et al.2Total calcineurin concentration60.0 nMCrabtree17Total NFκB concentration52.9 nMCarlotti et al.13Total NFAT concentration7.23 nMEstimateTotal IκB concentration55.0 nMCarlotti et al.13Number calcium ions required to activate each calcineurin molecule3Kakalis et al.29Total PKCθ concentration2000 nMEstimateTotal PKCα concentration1000 nMEstimateTABLE 3.PKCα membrane-binding parameters.ParameterDefinitionValueSourceKCaDissociation constant22.0 μMNalefski et al.38hCalcium binding cooperativity1.5Nalefski et al.38nStoichiometric coefficient2 mol/molNalefski et al.38KDAGDissociation constant10.2 nMAnanthanarayanan et al.3[DAG]Diacyl glycerol concentration2000.0 nMMogami et al.37tr2aIκB constitutive mRNA synthesis1.54 × 10−6 μM s−1Hoffmann et al.25tr2IκB inducible mRNA synthesis1.65 × 10−2μ M−1 s−1Hoffmann et al.25tr3IκB mRNA degradation2.8 × 10−4 s−1Hoffmann et al.26TABLE 4.NFκB rate constants.Rate constantValueSourcek50.0019 s−1Shibasaki et al.52k60.00092 s−1Shibasaki et al.52k191.0μM−3s−1Klee et al.32; Kakalis et al.29k201.0 s−1Klee et al.32, Kakalis et al.29k210.21 s−1Estimate and adjusted relative to k22 to give equal calcium ion concentrations in nucleus and cytoplasmk220.5 s−1Estimatek230.614 μM−1 s−1Carlotti et al.14, Huang et al.27k240.00184 s−1Carlotti et al.14k250.002 s−1Adjusted to control rate of activation/deactivation observed by Dolmetsch et al.22k261.0 μM−1 s−1Adjusted to control rate of activation/deactivation observed by Dolmetsch et al.22k270.00026 s−1k28/k27 = 50; Carlotti et al.14k280. 0134 s−1Estimated from Birbach et al.12k290.010 s−1k30/k29 = 2, Carlotti et al.14k300.02 s−1Estimatek310.000034 s−1Estimatek320.000034 s−1Estimatek330.02 s−1Estimatek340.000034 s−1Estimatek350.000000036 s−1Adjusted to agree with Trushin et al.56k360.00008 s−1Adjusted to agree with Trushin et al.56k370.0000016 s−1Adjusted to agree with Trushin et al.56k380.0008 s−1Adjusted to agree with Trushin et al.56k390.0016 s−1Adjusted to agree with Trushin et al.56k400.0006 s−1Adjusted to agree with Trushin et al.56k410.02 s−1Adjusted to agree with Yang et al.60k420.000177 s−1Adjusted to agree with Yang et al.60k430.02Estimated from Trushin et al.56k440.009Estimated from Trushin et al.56
NFAT Model
A schematic diagram of the mechanism for the action of NFAT is shown in Fig. 1b. A rise in the cytosolic calcium concentration leads to an increased calcium–calmodulin concentration which activates calcineurin (denoted by C*). Calcineurin binds to and dephosphorylates the cytosolic species NFAT:Pi, the phosphorylated form of NFAT.40,46 Without calmodulin, the phosphatase activity of calcineurin is much reduced.31 The dephosphorylated NFAT-calcineurin complex (NFAT:C*) translocates to the nucleus, where it may bind to promoter regions of DNA and assist in initiation of gene transcription. A lowering of the calcium concentration results in deactivation of calcineurin and its dissociation from the NFAT-calcineurin complex. Free nuclear NFAT may then be phosphorylated by ubiquitous kinases, masking nuclear localization signals and resulting in translocation from the nucleus to the cytoplasm. Thus rising and falling calcium ion concentrations result in activation and deactivation of the transcription factor NFAT.
There are several simplifying assumptions made in the model for NFAT activation: (1) the total cellular concentration of NFAT remains constant, that is, the degradation and synthesis rates are balanced; (2) dephosphorylated NFAT bound to calcineurin in the nucleus is the transcriptionally active form of NFAT; (3) calcineurin dephosphorylates NFAT in one composite dephosphorylation site (i.e. all sites are treated as one); (4) The cytosolic and nuclear free calcium concentrations are equal and are controlled by a calcium clamp protocol simulating the procedure used by Dolmetsch et al.;22 (5) calcineurin binds three calcium ions to become active; and (6) NFAT and calcineurin clearly bind to other molecules in the cell, however, these interactions are ignored for simplicity.
A reaction scheme consistent with this mechanism is given in Fig. 2a. Here nuclear species are shown in the lower half of the figure and given the subscript n. Cytosolic species are shown in the upper half of the figure and given the subscript c. At the low calcium concentrations present in the resting cell the dominant NFAT-containing species is phosphorylated NFAT in the cytoplasm, designated (NFAT:Pi)c (Fig. 2a – dark gray box at left). At the high calcium concentrations present in the activated cell the dominant species is NFAT associated with activated calcineurin in the nucleus, (NFAT:C*)n (Fig. 2a – light gray box at right). The model treats cytosolic and nuclear entities as distinct, and thus includes 12 separate chemical species, and cytosolic and nuclear Ca.2+. The arrows between species represent forward and reverse chemical reactions, or nuclear import and export. The rates of reaction, and of import and export, are represented by the 22 rate constants, designated k1 to k22.
Figure 2.
(a) Reaction scheme for the NFAT mechanism. The activation path for NFAT is shown by the light gray arrows. An increasing calcium concentration activates calcineurin (C), which binds to and dephosphorylates NFAT:Pi (dark gray box). The NFAT-activated calcineurin complex NFAT:C* is imported to the nucleus where it may be transcriptionally active (light gray box). The deactivation path for NFAT is shown by the dark gray arrows. A decreasing calcium concentration allows dissociation of NFAT and activated calcineurin (NFAT:C*) in the nucleus (light gray box). Free NFAT is phosphorylated and exported to the cytoplasm (dark gray box). (b) Reaction scheme for the NFκB mechanism. The activation path for NFκB is shown by light gray arrows. An increase in the calcium ion concentration activates calcineurin in synergy with PKCα which, through a sequence of reactions, activates an early phase of the kinase IKK. Activation of PKCθ leads to activation of a later phase of IKK. IKK phosphorylates the NFκB:IκB complex (dark gray box) which then dissociates. The phosphorylated IκB is degraded and the free NFκB imported into the nucleus to be the transcriptionally active form (light gray). The deactivation path for NFκB is shown by dark gray arrows. A decrease in the calcium ion concentration allows resynthesis of IκB:Pi, which enters the nucleus and associates with NFκB. The resulting complex dephosphorylates and is exported from the nucleus.
Using the law of mass action, this reaction scheme yields a system of 12 coupled first order differential equations describing the change in concentration with time for each of the 12 distinct chemical species (Appendix A). The parameters in the equations include the 22 rate constants and the number of calcium ions required to activate the phosphatase activity of each calcineurin molecule, designated by m in the figure. The time course for the concentrations of each of the chemical species from a given initial condition is determined by numerical solution of the system using a standard fourth order Runge–Kutta technique.
Table 1 lists the rate constants used in the NFAT model. They may be loosely divided into three groups: (1) calculated from published data, (2) estimated from published data, and (3) adjusted to fall within expected physiological ranges in order to make the model conform to published experimental data.
NFkB Model
A schematic diagram of the mechanism of action of NFκB is shown in Fig. 1b. As previous studies have shown, PKCθ and calcineurin act as co-activators of the transcription factor NFκB.55,56 A high calcium concentration activates calcineurin and PKCα, which through a sequence of reactions, co-activates the IKK complex with PKCθ. TCR/CD3 activation leads to the translocation of PKCθ to the immunological synapse (IS), and CD28 activation leads to PKCθ migration to the central supramolecular activation cluster (cSMAC) in the IS.28 IKK phosphorylates IκB in the complex NFκB:IκB, leading to immediate recognition of IκB by the F-box/WD-domain protein E3RS,IκB61 polyubiquitination of the IκB20 and subsequent degradation of the IκB by the proteasome. Our model does not explicitly include the polyubiquitination but includes it in the degradation rate of IκB which is essential for NFκB translocation and hence for gene transcription. Once freed of IκB, NFκB translocates to the nucleus where it may bind to DNA and promote gene transcription. When the calcium concentration falls to resting levels, newly synthesized IκB rapidly translocates to the nucleus where it combines with NFκB, masking nuclear localization signals on both species.6 The NFκB:IκB complex is then exported from the nucleus to the cytoplasm.
There are several simplifying assumptions used to render the model computationally tractable: (1) the total cellular NFκB concentration is constant due to a balance between its synthesis and degradation processes; (2) IκB production is enhanced by activated NFκB as described by Hoffman et al.;26 (3) the cytosolic and nuclear free calcium concentrations are equal and are controlled by a calcium clamp protocol simulating the procedure used by Dolmetsch et al.;22 (4) the IKK kinases are lumped into one reaction; (5) in the model IκB represents IκBα; (6) the amount of free NFκB in the nucleus is used as a measure of the activation of the transcription factor; (7) calcineurin binds 3 calcium ions to become active; and (8) the binding of calcineurin, IκB, and NFκB to other molecules in the cell clearly occurs, but is not included in the model for simplicity.
A reaction scheme consistent with this schematic diagram is shown in Fig. 2b. As in the reaction scheme for NFAT, nuclear species are shown in the lower half of the figure and given the subscript n. Cytosolic species are shown in the upper half of the figure and given the subscript c. In the resting cell, the dominant NFκB containing species is NFκB:IκB in the cytoplasm (Fig. 2b – dark gray box at right). In the active cell, the dominant species is NFκBn (Fig. 2b – light gray box at left). The NFκB model has 17 different chemical species with the nuclear and cytosolic concentrations represented separately. As in the NFAT model, the arrows between species represent forward and reverse chemical reactions, or nuclear import and export. The rates of reaction, and of import and export, are represented by the 26 rate constants, designated k5 and k6 and k19 to k42. Note that k41 represents the rate of degradation of IκB and k42 the rate of its resynthesis.26 From the NFκB reaction scheme a system of 17 coupled first order differential equations may be written, one for the change in concentration with time of each of the 17 distinct chemical species (Appendix B). Given the initial concentration of each species, numerical integration of the system yields the time course of the concentration for each species.
Results and Discussion
A number of simulations were performed to duplicate experimental results to validate the model. In all simulations, the parameters listed in Tables 1–4 were used unless stated in the figure legends. The only difference in the simulated results was the calcium clamp protocol used.
NFAT Model Results
Using the parameter values in Tables 1 and 2, the model was allowed to come to steady state at rest ([Ca]i = 0.1μM). The resting state concentrations are shown in Table 5. These concentrations were used as initial values when simulating the evolution of the concentration of each species during a transition from the resting state ([Ca]i = 0.1 μM) to the active state ([Ca]i = 1.0 μM). The results are shown in Fig. 3a. The high calcium concentration activates calcineurin, which associates with and dephosphorylates NFAT:Pi in the cytoplasm; the concentration of NFAT:Pi in the cytoplasm falls rapidly. Only a small amount of NFAT:Pi:C* is formed in the cytoplasm when activated calcineurin associates with NFAT:Pi because it is rapidly dephosphorylated to NFAT:C*, which in turn is rapidly translocated to the nucleus where it is transcriptionally active. Note in this figure that the change in concentration of the nuclear species appears large relative to the concentrations of the cytosolic species because the volume of the nucleus is smaller than the volume of the cytoplasm by a factor of about 2.4. The light gray arrows in Fig. 2a show the NFAT activation pathway on the reaction scheme for NFAT. Calcineurin associates with calcium ion, activating calcineurin. The activated calcineurin associates with phosphorylated NFAT and dephosphorylates it. The NFAT:C* resulting from this dephosphorylation is rapidly imported to the nucleus where it is transcriptionally active.
Figure 3.
(a) Activation of NFAT from the resting state. (b) Deactivation of NFAT from the active state. The reaction paths (Fig. 2a) for the activation and deactivation of NFAT are determined by observing the sequence of the formation of the various species in the reaction scheme.
TABLE 5.
NFAT resting steady-state concentrations.
Species
Concentration (nM)
NFATn
0.5219
NFATc
0.1101
C*n
0.0505
C*c
0.0091
(NFAT:Pi)n
0.2272
(NFAT:Pi)c
9.4397
(NFAT:Pi:C*)n
0.0025
(NFAT:Pi:C*)c
0.0022
(NFAT:C*)n
0.9477
(NFAT:C*)c
0.0061
Cn
49.198
Cc
9.7108
Can2+
100
Cac2+
100
A simulation of the deactivation of the activated state is shown in Fig. 3b. Here the initial concentrations were those of the activated steady state, and the calcium ion concentration was set to 0.1 μM. Figure 3b shows that the concentration of the transcriptionally active species (NFAT:C*)n falls rapidly as NFAT and calcineurin dissociate. Consequently the concentration of NFATn rises as NFAT is freed by the dissociation, and then falls as it is phosphorylated. The concentration of the phosphorylated species (NFAT:Pi)n never rises very high because this species is rapidly exported from the nucleus. The dark gray arrows in Fig. 2a show the NFAT deactivation pathway on the reaction scheme for NFAT. After (NFAT:C*)n dissociates, free NFAT is phosphorylated and exported from the nucleus.
Dolmetsch et al.22 suggest that non-linearity in the response of NFAT to calcium ion concentration is important in determining how the activity of NFAT depends on oscillations in the calcium ion concentration. Their experiments show that oscillation frequency is the critical factor in differentiating the response of NFAT and NFκB. Our models for both NFAT and NFκB simulate the effect of calcium ion concentration oscillations on the activation of the transcription factors. One such simulation for NFAT is shown in Fig. 4a. The initial concentrations of the species were those of the resting state and the oscillation period of the calcium ion concentration was 100 s. The calcium ion concentration was fixed at 1.0 μM during the initial 10 s and 0.1 μM during the final 90 s of each oscillation. Figure 4a shows that the concentration of the transcriptionally active (NFAT:C*)n rises rapidly during the high calcium concentration pulse and then decays more slowly during the low calcium concentration portion of the period. The concentration of (NFAT:Pi)c follows the reverse trend, decreasing rapidly when the calcium ion concentration is high and increasing slowly when the concentration is low. A fraction of the NFAT exists as free NFAT in the nucleus, oscillating between the form NFATn and (NFAT:C*)n as calcineurin associates with and dissociates from NFAT. We can easily see that in the absence of constant signaling the concentration of transcriptionally active (NFAT:C*)n would soon fall to resting state levels. This is shown in Fig. 4a where at 1500 s the calcium ion concentration is set to 0.1 μM. The concentration of the transcriptionally active (NFAT:C*)n falls and that of (NFAT:Pi)c rises, both approaching their resting levels as time increases.
Figure 4.
(a) Activation of NFAT by oscillations in the calcium ion concentration. During each period the calcium concentration was high (1.0 μM) for 10 s and low (0.1μM) for 90 s. At 1500 s the calcium concentration was set to the resting state value (0.1 μM) and the system returned to the resting state. (b) The percent of NFAT in the active state is plotted as a function of calcium ion oscillation period. (c) The percent of NFAT in the active state is plotted as a function of average calcium ion concentration for steady state and oscillating calcium ion concentrations. The percent of NFAT in the active steady state decreases below 0.4 μM when the calcium concentration is constant. The percent of NFAT in the active state persists at a high level to less than 0.2 μM average concentration when the calcium concentration oscillates.
To study how the degree of NFAT activation varies with oscillation frequency, we simulated calcium concentration oscillations using 10 s intervals of high concentration and varying intervals of low concentration, with the total period ranging from 100 to 1800 s (Fig. 4b). Because the concentration of the transcriptionally active (NFAT:C*)n varies throughout the period, a sufficient number of periods was simulated to reach a stable state, then the lowest value of (NFAT:C*)n during the last period was chosen to calculate the percent of NFAT in the transcriptionally active form. We reasoned here that NFAT involved in shuttling in and out of the active form was not likely to bind to DNA and assist in initiation of transcription, thus the lowest value of the concentration of the active form was justified. In this way the fraction of NFAT in the transcriptionally active form was found as a function of the oscillation period. Figure 4b shows that the percent of NFAT that is transcriptionally active is almost 60% with a 100 s period, and that this fraction falls to less than 10% with a 900 s period. These results agree qualitatively with the results presented by Dolmetsch et al.,22 in their Fig. 3b for the percent of gene expression as a function of oscillation period. Their data shows 50% gene expression with a 100 s oscillation, with the percent expression falling to zero for periods of 400 s or more. The quantitative differences between our simulated results and the experimental observations by Dolmetsch et al.22 possibly occur because they have determined percent gene expression while we calculate percent of NFAT that is transcriptionally active. There might be additional non-linearity or cooperativity introduced in transcription factor binding to DNA and transcription. For example, as mentioned earlier, Fiering et al.24 observed that the binding of 3 NFAT molecules to a constructed promoter on a DNA strand produced cooperativity of transcription. They also suggested a similar requirement for NFκB activation of gene expression. While inclusion of these factors would result in a more quantitatively accurate reproduction of the reporter gene expression data, we chose to simplify the model to emphasize the mechanisms for activation of the transcription factors.
Dolmetsch et al.22 show that the percent of cells expressing a lacZ+ reporter gene, sensitive to transcriptionally active NFAT, decreased when the steady-state calcium concentration fell below about 0.35 μM. But if the calcium concentration exhibited oscillations, the gene expression persisted to an average calcium concentration at least as low as 0.2 μM. The model displays similar behavior (Fig. 4c). To simulate oscillations, 10 s periods of high calcium concentration and 90 s periods of low calcium concentration were used. The low calcium concentration was fixed at 0.1 μM, and the concentration during the high calcium intervals was fixed in each of several tests to produce a set of experiments each having a different average calcium concentration. Model data for the steady state was obtained following the protocol described previously. Figure 4c shows that simulating oscillations does indeed enhance the activity of NFAT at low calcium concentrations relative to the activity at a constant calcium concentration. The shape of the curves representing steady state and oscillating calcium concentration behavior are quite similar to those shown by Dolmetsch et al.,22 in their Fig. 2c. The two calculated curves meet at a calcium concentration of 0.39 μM, very close to the 0.35 μM shown in the experimental work. This corresponds to a period of approximately 30 s. If the period is longer than 30 s (the average calcium is lower than 0.39 μM), then the oscillations are more potent than steady-state calcium with the same average at activating NFAT. If higher, the opposite is true. The model data shows enhanced NFAT activity down to about 0.2 μM, below which the activity decreases rapidly.
Finally, the NFAT model was tested to determine if it correctly simulates the observation that overexpression of calcineurin partially replaces the requirement for calcium, as reported by Luo et al.35 These simulations were carried out by holding the calcium ion concentration fixed at the resting state value, 0.1 μM, and varying the calcineurin concentration, followed by calculating the degree of activation of the NFAT. Figure 5 shows the results obtained when the calcineurin concentration was increased as much as a factor of 25 times above the baseline concentration. The percent of NFAT in the transcriptionally active form does indeed increase when the calcineurin concentration increases, and high levels of activation are achieved with high calcineurin concentrations.
Figure 5.
Percent active NFAT as a function of calcineurin concentration at fixed low calcium ion concentration. The calcium concentration was fixed at a level characteristic of the resting state (0.1 μM) and the calcineurin concentration increased to simulate overexpression of calcineurin. Each data point shows the steady state concentration of transcriptionally active NFAT at a fixed calcineurin concentration. Overexpression of calcineurin replaces the requirement for calcium at fixed low calcium concentration.
NFκB Model Results
The NFκB model was characterized in a manner similar to that for the NFAT model. Using the parameters from Tables 2–4, the calcium concentration was fixed at 0.1 μM and the time evolution of the concentration of each species calculated until steady state values were observed. These concentrations were taken to be those of the steady state at rest (Table 6).TABLE 6.NFκB resting steady-state concentrations.SpeciesConcentration (nM)NFκBn7.1779NFκBc0.0731(IκB:Pi)n0.2035(IκB:Pi)c0.0525(NFκB:IκB:Pi)n0.2343(NFκB:IκB:Pi)c0.1076(NFκB:IκB)n0.2376(NFκB:IκB)c52.961C*n0.0505C*c0.0091Cn49.198Cc9.7108100100
Figure 6a shows the concentrations of species in the NFκB model during a transition from the resting to active states. Starting with the resting steady state concentrations, the calcium ion concentration was fixed at 1.0 μM and the time evolution of the concentration of each species calculated. The high calcium concentration activates calcineurin, which in synergy with PKCα and PKCθ, co-activates the kinase IKK. The activated kinase phosphorylates IκB associated with NFκB in the cytoplasm. Subsequent polyubiquitination and IκB degradation result in a decrease in the cytoplasmic NFκB:IκB:Pi concentration, a dissociation of NFκB and IκB, and rapid translocation of NFκB into the nucleus. The concentration of cytosolic NFκB never rises very high because of its rapid nuclear import, therefore the concentration of this species is not shown in Fig. 6a. The light gray arrows in Fig.2b show the NFκB activation pathway on the reaction scheme for NFκB. A rise in the calcium concentration activates calcineurin, which through a sequence of reactions, and in synergy with PKCα, activates an early phase of the kinase IKK. CD3/CD28 activate PKCθ, leading to activation of a later phase of IKK.56 Activated IKK phosphorylates the NFκB:IκB complex in the cytoplasm. After dissociation, the phosphorylated IκB is polyubiquitinated and degraded, and free NFκB is imported to the nucleus where it may be transcriptionally active.
Figure 6.
(a) Activation of NFκB from the resting state. (b) Deactivation of NFκB from the active state. The sequence of events depicted in Fig. 2b can be determined by observing the time evolution of the concentration of the different reaction species.
Deactivation of the activated state of NFκB is shown in Fig. 6b. The initial concentrations were those of the activated state at equilibrium, and the calcium ion concentration was fixed at 0.1 μM. The most important step in deactivation is synthesis of new IκB, which is rapidly translocated to the nucleus forming the phosphorylated complex with NFκB. This complex is dephosphorylated and rapidly translocated into the cytoplasm. The result is an overall increase in concentration of NFκB:IκB in the cytoplasm as the concentration of NFκB in the nucleus decreases. The dark gray arrows in Fig. 2b show the pathway for deactivation on the reaction scheme for NFκB. Newly synthesized IκB is imported to the nucleus where it associates with NFκB. The resulting complex dephosphorylates, and the NFκB:IκB complex is exported to the cytoplasm. As for NFAT, Dolmetsch et al.,22 have studied the degree of gene expression caused by NFκB as a function of steady-state calcium ion concentration. In their paper, Fig. 3a shows that NFAT and NFκB behave in a similar manner; they exhibit a highly non-linear sigmoidal dependence, with very little gene expression at calcium ion concentrations of 0.1 μM or less, a rapid rise in gene expression at calcium concentrations between 0.1 μM and 0.5 μM, and near 100% gene expression at calcium concentrations over 0.5 μM. Since we follow the calcium clamp protocols used by Dolmetsch and co-workers, which does not involve CD3 or CD28 activation and hence does not involve activation of PKCθ, we do not activate PKCθ in these simulations. Figure 7 shows our simulated results for NFκB without PKCθ activation, with the percent of NFκB that is transcriptionally active plotted as a function of the calcium ion concentration. These results were obtained by setting the calcium ion concentration to the desired value, then calculating the concentration of transcriptionally active NFκB as a function of time until a steady state was reached. There is good agreement between our simulated behavior and the experimental results of Dolmetsch et al.22 Both show activation of NFκB over the physiological range of calcium ion concentrations, with little activity at low concentrations and full activity at high concentrations. The sigmoidal shape of the calculated curve agrees with the experimental results.
Figure 7.
Dependence of the percent active NFκB on the steady-state calcium concentration. For each data point the calcium ion concentration was fixed at the indicated value.
As was done for the NFAT model, the NFκB model will simulate the system response to calcium oscillations. An example of NFκB activation by calcium concentration oscillations is shown in Fig. 8a. The initial concentrations were those of the resting steady state and the oscillation period was 100 s. The calcium ion concentration was fixed at 1.0 μM during the initial 10 s of each period and at 0.1 μM for the remaining 90 s of each period. When the calcium concentration is high, the concentration of NFκB:IκB in the cytoplasm falls rapidly, and when the calcium concentration is low the concentration of this species increases slowly. The concentration of NFκB in the nucleus follows the reverse trend, increasing rapidly during the periods of high calcium concentration and falling more slowly when calcium concentration is low.
Figure 8.
(a) Activation of NFκB by oscillations in the calcium ion concentration. During each period the calcium concentration was high (1.0 μM) for 10 s and low (0.1 μM) for 90 s. The concentration of the NFκB:IκB complex in the cytoplasm falls rapidly during periods of high calcium concentration. The concentration of the phosphorylated NFκB:IκB complex first rises, then falls as IκB is degraded. Free NFκB is imported to the nucleus, steadily increasing the concentration of the transcriptionally active nuclear NFκB. (b) Differentiation of the activity of NFAT and NFκB by calcium ion oscillation period. The activity of NFκB decreases as the oscillation period increases (upper curve) but not as much as for NFAT (lower curve); the percent active NFκB is greater than that of NFAT for a given calcium concentration oscillation period. This dissimilarity in behavior differentiates the response of NFAT and NFκB to calcium concentration oscillations.
During the calcium oscillations, the concentration of the transcriptionally active nuclear NFκB rises, showing little oscillation because of the relatively slow rate of the polyubiquitination and IκB degradation steps. In the absence of constant signaling the concentrations of the species return to their resting levels. In Fig. 8a the concentration of Ca2+ is set to 0.1μM at 2000 s, after which the concentration of nuclear NFκB falls, and that of (NFκB:IκB)c rises, to levels characteristic of the resting state.
We studied how the degree of NFκB activation varied with calcium concentration oscillation period as we did for the NFAT model. Figure 8b shows the results of simulations with periods varying from 100 to 2000 s. For the shortest period, the percent of active NFκB was 70%, and at the longest period the percent active NFκB was 20%. These results agree qualitatively with those shown by Dolmetsch et al.,22 in their Fig. 3b. Their figure shows over 90% gene expression with a 100 s period, falling to less than 20% gene expression for an 1800 s period. Note that they have determined percent gene expression while we calculate percent of transcriptionally active NFκB, a difference that may explain the quantitative difference in the results.
Figure 8b combines the model results for NFAT and NFκB to compare the effect of oscillation frequency on activation of NFκB to the activation of NFAT by including the curve from Fig. 4b Figure 8b shows that a higher fraction of the NFκB is transcriptionally active than that of NFAT for long oscillation periods. These results agree qualitatively with those shown in Dolmetsch et al.,22 Fig. 3b, where gene expression by NFAT declined rapidly with oscillation frequency but persisted to long periods for NFκB.
Figure 9a simulates the results of Yang et al.,60 who studied the degradation of IκBα with time in fibroblasts. To simulate this, we have used Ca2+ oscillations with a period of 100 s and assumed that degradation is the same for all members of the IκB family and then plotted the percent of IκB remaining with time. We have also assumed that the active PKCθ and PKCα concentrations drop after 30 min based on the experimental observations of Szamel et al.53 who report that PKCθ and PKCα translocated rapidly to the membrane within 1–10 min and start to decline after 30 min. This was simulated with the model by reducing the amounts of active PKCθ and PKCα concentrations to resting values at 40 min (Fig. 9a). The simulation shows that there is near complete degradation of IκB in about 30 min, which agrees with the experimental results shown in Yang et al.60 in their Fig. 2. They observed an increase in IκB concentration after about 40 min.
Figure 9.
(a) Degradation of IκB with time during Ca2+ oscillations with period 100 s. Activation of PKCθ and PKCα is terminated at 40 s. (b) Percent active NFκB as a function of the rate of degradation of NFκB. At steady state and at high calcium concentration the percent of active NFκB is sensitive to the rate of IκB degradation assumed in the model. As the degradation rate decreases the percent of active NFκB decreases. Thus the degradation rate must be sufficiently high to obtain significant activation of NFκB. The NFκB model uses a degradation rate of 0.02 s−1.
Figure 9b shows that the rate of degradation of IκB is an essential factor in the activation of NFκB. Here the dependence of the percent active NFκB is shown as a function of the IκB constitutive degradation rate, k41 in the NFκB model. Our NFκB model uses a degradation rate of 0.02 s−1. As the degradation rate decreases below this value the percent of active NFκB decreases, thus a sufficiently high IκB degradation rate is required for efficient NFκB activation.
Sensitivity Analysis
To further characterize the system, a sensitivity analysis was performed to determine which steps in the reaction schemes for NFAT and NFκB exerted the most influence on the activation of the transcription factors. To this end, each rate constant was increased and decreased by 10%. The percent change of steady-state active transcription factor at 1.0 μM calcium over the control was calculated (Fig. 10). The sensitivity was calculated by
Figure 10.
(a) Sensitivity analysis for NFAT activation. (b) Sensitivity analysis for NFκB activation. In both cases, the rate constants were increased and decreased by 10%. The sensitivity of the steady state concentration of active transcription factor at 1.0 μM calcium to changes in the rate constants is shown.
For NFAT (Fig. 10a), the rate constants that seem to exert the greatest effect on activation are k9, k10, k13, k14, k15, k16,k19, and k20. The rate constants k9 and k10 control the rate of nuclear import and export of NFAT and the rate constants k13 and k14 control the phosphorylation and dephosphorylation of NFAT by calcineurin. The sensitivities here suggest that increasing the binding affinity of NFAT and calcineurin (k15 and k16) will increase activation of NFAT. This is consistent with mutational studies that demonstrate that alteration of the calcineurin binding site on NFAT decreases activation of NFAT.4,41 It is interesting to note that even though the sensitivities of NFAT activation to changes in k11 and k12 are in the expected directions, the values of these sensitivities are relatively small. Also, increasing the rate of activation of calcineurin through calcium binding (k19 and k20) will also increase activation of NFAT. Other rate constants that exert a small but still significant effect are k5 and k6 suggesting that an increase in the amount of free cytosolic calcineurin will increase activation of NFAT. This is consistent with Fig. 5 and the experiments that demonstrate that increasing calcineurin increases the activation of NFAT.
For NFκB (Fig. 10b), the rate constants k30, k41, k42 and tr3 exert the greatest control on the activation of NFκB. The rate constants k23, k24, k25, k26, k27, k28, k43k44, tr2 and tr2a also exert a significant influence on activation of NFκB. The rate constants k41 and k42 control the degradation and synthesis of IκB. The rate constants tr2, tr2a and tr3 control the rate of IκB mRNA synthesis and degradation. The importance of these rate constants re-emphasizes the importance of the removal of free IκB from the cystosol for activation of NFκB as indicated by Fig. 9. The rate constants k43 and k44 control the shuttling of IKK between the cytoplasm and the nucleus. The rate constants k30 and k29 control the import of IκB into the nucleus. Preventing the import of IκB into the nucleus increases the activation of NFκB as it lowers the amount of IκB available to bind NFκB which leads to its export from the nucleus. The rate constants k23 and k24 control the dissociation of NFκB and IκB. Increasing the dissociation rate increases activation of NFκB. The effect of the rate constants k25 and k26 demonstrate that increasing the rate of phosphorylation of IκB by IKK increases the activation of NFκB. Finally, decreasing k27, the rate constant for export of NFκB out of the nucleus, or increasing k28, the rate of import NFκB to the nucleus, increases the amount of activation of NFκB. This effectively increases the amount of NFκB in the nucleus. It is interesting that although reducing the rate of phosphorylation of IKK by calcineurin does inhibit NFkB translocation to the nucleus, this is not one of the most sensitive parameters. The sensitivity analysis suggests that there are several other targets that will block NFAT and NFκB translocation. For several of the targets on NFAT, there is experimental data to support the model predictions about the sensitivities. On the other hand, the prediction that blocking of NFAT translocation decreases transcription has yet to be verified experimentally. This might be accomplished by developing a compound that either binds the nuclear import site of NFAT or that blocks the import mechanism. For NFκB the prediction of this analysis is that the most effective target would be to block IκB degradation or increase its re-synthesis. Another potent target would be to block NFκB translocation. Other possible interventions to block NFκB translocation would be to introduce compounds that increase the affinity of NFκB for IκB or that increase the activity of IKK. These are not the only possible sites as many accessory pathways regulate this system. Future studies that build on this model and explore these pathways will give further insight.
Conclusions
We have developed models for the calcium and PKCθ mediated activation and deactivation of the transcription factors NFAT and NFκB that incorporate experimentally determined reaction pathways and that simulate this action over physiological calcium concentrations. The model reproduces experimentally observed behaviors of both the NFAT and NFκB systems under a variety of conditions.
Several parameters values for T cells were not available and therefore were derived from experimental observations in cells other than the T cell. For the most part these choices are reasonable estimates, but should be examined further as new data on signaling during T cell activation becomes available. Variations in some estimates will have a greater influence on model outcomes than the others. For example, the total concentration of NFκB was taken from fibroblast data,13 but this mostly affects the amount activated rather than the percent activated yielding a small control coefficient (0.0127). On the other hand, sensitivity analysis indicates that the degradation rate of IκB that was estimated using data from fibroblasts60 would exert a greater influence on model results and thus its choice is more critical.
While the model is qualitatively accurate, simulation of the reporter gene levels observed by Dolmetsch and colleagues might be more closely approximated by the inclusion of additional features to the model. Several of these enhancements are obvious and consist of including elements both upstream and downstream of the system modeled. For example, binding of the transcription factors to the DNA might be added along with steps to describe the expression of the reporter genes observed in the experiment. Inclusion of these steps would require adding significant complexity to the model that might obstruct demonstration of the mechanisms governing activation of the calcium dependent transcription factors NFAT and NFκB. Thus, addition of these steps has been left for future work.
Although this model is able to simulate the calcium clamp in the in vitro experiments used by Dolmetsch and co-workers, in vivo the T cell has more complex calcium dynamics. One aspect of these dynamics is the calcium activation of PKC and its feedback on phospholipase C (PLC). Many studies have indicated that PKC acts as a co-activator of the transcription factors NFAT and NFκB through its effect on calcineurin.18,55 According to Pfeifhofer et al.,42 PKCθ is predominately expressed in T-lymphocytes. They found that PKCθ increases NFAT and NF-κB activation by reducing the intracellular calcium elevation through a reduction in IP3 production. They also found that PKCα does this to a lesser degree. The current model includes the activation of PKCθ and its effect on IKK complex. Future work might include any feedback of PKC on PLC and possibly the actions of other PKCs.47
Two computational models have been developed by other authors to model the dynamics of NFκB in T-lymphocytes. The model developed by Carlotti et al.,13 describes the association and dissociation of NFκB and IκB and their translocation into the nucleus both in the associated and dissociated forms. Their model demonstrates that NFκB is localized in the cytoplasm at rest due to its association with IκB and the export of NFκB from the nucleus. A second model, developed by Hoffman and co-workers, demonstrated that the temporal control was due to coordinated degradation and resynthesis of IκB and that IκB provides a strong negative feedback that can turn off the NFκB response.

t cell
immune response
calcium
computational model


How Complementary and Alternative Medicine Practitioners Use PubMed

Background PubMed is the largest bibliographic index in the life sciences. It is freely available online and is used by professionals and the public to learn more about medical research. While primarily intended to serve researchers, PubMed provides an array of tools and services that can help a wider readership in the location, comprehension, evaluation, and utilization of medical research.
Introduction
This study considers the use of PubMed (www.pubmed.gov) by practitioners of complementary and alternative medicine (CAM). In 2001, Americans invested US $50 billion in this form of health care, according to John Weeks, editor of The Integrator, a newsletter tracking “the business of alternative medicine,” with 40% of American adults turning at some point to alternative medicine [1]. While some in the medical research community have argued that “there is no alternative medicine” as “there is only scientifically proven, evidence-based medicine supported by solid data, or unproven medicine for which there is no scientific evidence” [2], the US National Center for Complementary and Alternative Medicine (one of the National Institutes of Health) sponsors research in this field and serves as a clearinghouse for clinical trials and research summaries.
Within the scope of its standing as the largest bibliographic index on the life sciences, PubMed (containing Medline, formerly known as Index Medicus, as a subset) indexes medical research articles and includes material useful for physicians and pharmacists, as well as traditional Chinese medicine practitioners [3,4]. A study of nurses, health care assistants, midwives, and health visitors (registered nurses or midwives who promote mental, physical, and social well-being in the community) has shown that, among those who had sufficient access to online sources of health information, the access to journal literature was felt to have led to improved patient care [5].
A prerequisite for the successful pursuit of health information is what Norman and Skinner identify as eHealth literacy, or the skills that enable people “to seek, find, understand, and appraise health information” [6]. This study focuses on how, among a group of CAM practitioners, those abilities are supported by an array of PubMed tools and services. Up to this point, investigators have recognized that users of online health services are not a monolithic group. Studies have been conducted on the reading levels demanded of cancer patients [7], the overwhelming quantity of information available for first-time mothers [8], the limited search skills of college students studying health [9], the growing use of electronic resources by nurses with limited search skills [10,11], and the growing confidence among doctors in their search strategies, although few use this skill on a real-time basis with patients [12].
The variety of approaches that these studies apply testifies to the idea that reading health literature online is situation specific and calls for context, and that investigations into eHealth literacy are fruitful when specific populations are delineated. Information retrieval studies have been done on citation retrieval [13] and literature searching [14] of PubMed, as well as on the use of the medical subject heading (MeSH) and Limits services [15]. Work has also been done in advising users how to best conduct CAM searches [16,17] with at least one study finding that PubMed indexing is not yet adequate for this field of medicine, especially with CAM clinical trials [17].
This study is intended to complement the more common focus among information science researchers on users’ information retrieval strategies with indexing services such as PubMed [18,19]. It addresses the value of PubMed’s specific tools and services for CAM practitioners, as these tools are introduced to them in the context of their own interests in the life sciences and their interest in learning about the use of PubMed. In focusing on the value and role of the tools and services, we are drawing on literacy and learning research in the subject domains, or “domain knowledge” as Alexander and others refer to it [20]. Alexander has established the degree to which a combination of personal interest and background knowledge contribute to the degree of learning in domain areas such as biology and physics, and she holds, in particular, that “one’s knowledge base is a scaffold that supports the construction of all future learning” [21].
In this sense, PubMed’s provision of tools and services (such as Related Articles and Bookshelf) can be seen to help users easily and immediately augment, in a limited way, shortcomings in their knowledge base. The tools and services compensate for shortcomings not only in searching for articles but also when comprehending, evaluating, and utilizing abstracts and full-text articles. The tools and services (such as Full-Text Access, Email Alert, Send To) also appear to be poised to support the user’s interest in and engagement with the indexed material (which is the other critical factor Alexander has identified for domain learning). By introducing CAM practitioners to the tools and services, one by one, and gaining from them a sense of the perceived value and contribution of each, this study seeks to determine the potential contribution of these tools and services to the knowledge-base scaffolding and the personal interest of health care workers who lie outside of PubMed’s originally intended audience of researchers and physicians. This work has obvious implications for the design of the tools, in PubMed’s effort to serve as broad an audience of health care practitioners as possible, and as a way, in turn, of extending the impact and benefits of life science research.
Methods
Sample
The participants in this study were drawn from among those practising CAM. They were recruited from three sources: (1) participants 1-5 were drawn from among students taking an online professional development course at a community college on research literacy for the health professions, (2) participants 6-9 were recruited from the Massage Therapy Association of British Columbia, and (3) participants 10-18 were drawn from the Canadian Memorial Chiropractic College. The eight women and 10 men who agreed to participate in the study ranged in age from their 20s to their 50s. Of the 18 participants, 10 were pursuing or possessed a chiropractic degree, seven were registered massage therapists, and one practised homeopathy. A total of 13 participants had a university degree, with one holding a master’s degree and another, a PhD. In terms of background with online research, participants 1-5 were taking a course on research literacy, while participants 13-18 had been introduced to the topic in a session with a librarian, with some one-on-one follow-up.
Design
Participants were offered an opportunity to learn how to use PubMed in one-on-one sessions with the researcher (MQ-R) in exchange for helping the researchers understand the value of PubMed’s various features to users such as themselves. The 10 PubMed tools and services considered in this study (Table 1) can be divided into three functions: information retrieval (Boolean Search, Limits, Related Articles, Author Links, MeSH), information access (Publisher Link, LinkOut, Bookshelf), and information management (History, Send To, Email Alert). The sessions with the participants took place between April 2004 and December 2006 and averaged 2 hours in length. Sessions held with participants 1-9 were conducted over the phone, with both participant and researcher accessing PubMed through the Internet, while sessions with participants 10-18 were conducted face-to-face with a single computer connected to the Internet, which the participant operated. Participants were introduced to between six and 10 PubMed tools. The participants in the phone sessions covered 7.1 items on average compared to 7.7 items covered by participants in the face-to-face sessions.
Prior to their PubMed training session, the researcher asked participants by email to identify a health science topic of interest to them. The researcher then found a suitable article based on the interest of the 14 participants who responded and sent it to them in advance of the PubMed session (Multimedia Appendix 1). The participants were asked to read the paper prior to the session. The starting point for each session, after introductions and preliminary questions about the participant’s background, was an initial search of PubMed on a topic of interest. Participants were then guided through a range of six to 10 PubMed tools and services depending on the pace of the 2-hour session and where the interests of the participants led (Multimedia Appendix 2). For each tool and service, they were introduced to how the feature worked, using concepts and papers of interest to them, and were asked to provide their thoughts on its potential value for their use of PubMed.
Table 1
PubMed tools and services introduced to participants
Tool/Service
Function
Boolean Search
Search terms can be entered using Boolean operators AND, OR, NOT.
Search Limits
Allows the user to limit the search by language; type of journal; gender; human or animal subjects; age; type of article; date; inclusion into PubMed.
Related Articles
This appears as a button/link with each citation and brings up a list of matching articles based on the original article.
Full-Text Access
This is provided through the publisher’s link on the abstract page and on the LinkOut page, with publisher, aggregator, PubMed Central, and/or subscribing libraries providing open access for roughly 15% of articles.
Send To
Send To allows users to email citations, convert citations to a text file, or save citations for editing and further action.
Search History
This tracks terms, time, and number of results in searches, while allowing Boolean searches combining different searches from the Search History.
Bookshelf
This provides links to relevant passages in a set of full-text life science books that range from basic science to clinical practices.
MeSH Vocab
MeSH is a controlled vocabulary database for ascertaining the most commonly used terms in PubMed.
Email Alert
The National Center for Biological Information (NCBI) of the National Library of Medicine (NLM) offers email notification of new work in specified topics.
Author Links
Authors’ names have links leading to a listing of all of their work in PubMed.
Analysis
The sessions were transcribed (Multimedia Appendix 3), and the two researchers created a summary table of the results by rating the responses for each tool as (+) positive (“that’s valuable”), (++) positive with emphasis (“I’d really use that”), or (−) negative or indifferent (“that makes sense”). Because of the session’s 2-hour time constraint and the semistructured handling of the sessions, not every tool was covered with every participant. As a follow-up to the PubMed session, the researcher emailed the participants 2 weeks later, asking them to describe their current use, if any, of PubMed and other Internet resources.
Results
What follows is a detailed qualitative analysis of the participants’ responses to each of the 10 PubMed tools and services covered in this study, with the aim of better understanding how such aspects of PubMed might be seen as contributing to its use by the participants. Differences in the responses among the participants (on the basis of training or university degree) was not the focus of the study, although, as might be expected, those with prior training and with university degrees tended to value PubMed tools and services more highly. Participants with prior training in the use of online research literature thought that, on average, 6.0 out of the 7.5 tools and services that were introduced to them would be valuable in their use of PubMed, while those without such training responded positively to 4.6 of the 7.1 items introduced to them (Table 2). Similarly, those with a university degree responded positively to 5.7 out of 7.4 items, compared to those without a university degree, who gave a positive evaluation to 4.8 out of 7.4 items.
Table 2
Value of PubMed tools and services for participants (N = 18)
Participants
PubMed Tools and Services‡
Further Use of PubMed
Code†
UniversityDegree
Prior Online ResearchTraining
BooleanSearch
SearchLimits
RelatedArticles
Full-TextAccess
SendTo
Search History
Bookshelf
MeSHVocab
EmailAlert
AuthorLinks
MF1
yes
yes
+*
+*
+
++
+
++*
−
−*
+*
yes
CM2
yes
yes
++
+
++
+
+*
−
yes
HF3
no
yes
++*
+*
++*
+
+
++
−
+
no
MF4
yes
yes
+*
+*
+*
+*
++
++
MF5
yes
yes
+*
+
+
++
++
+
MM6
no
no
+
++
++
++*
−
+
−
−
MF7
no
no
++
−
++
+
+
++
yes
MF8
no
no
−
−
+
++
−
−
+
−
yes
MM9
yes
no
+
−
−
+
−
++
−
no
CF10
no
no
++
++
−
+
−
+
−
yes
CM11
yes
no
++
−
−
++
−
++
+
++
+
yes
CF12
yes
no
+
+
−
++
++
++
yes
CM13
yes
yes
+
−
+
+
−
+
−
+
+
no
CM14
yes
yes
+
−
+*
+
+*
++*
−
+
CM15
yes
yes
−
++
++
++
++
+*
+
−
yes
CM16
yes
yes
+*
+
++
+*
++
+
−
CM17
yes
yes
+*
+*
+
++*
++
+
−
−*
+
CM18
yes
yes
−
++
−*
+*
+
−
+
*Prior familiarity with tool or service.
†First letter of code: M=registered massage therapist; C=chiropractor; H=homeopath. Second letter of code: M=male; F=female.
‡ +: positive evaluation of tool or service; ++: additional positive emphasis; −: negative or indifferent response to tool or service
Blank cells indicate that tool or service was not covered in the participant’s session or participant did not respond to email follow-up.
Boolean Search
The basic starting point with PubMed, as with any online index or database, is the search, which typically allows the combining of search terms with AND, OR, or NOT for more accurate searching. These Boolean operators were familiar to a number of participants, but were a little vague for others: “I might have heard of it” (CF12). Learning about them made a considerable impression on a number of participants:
CM2
I will use the Boolean search methods even when using the different features of the database. This is incredibly important to know, use, and understand. It’s like the building blocks of searches. I’ve got to practise that one afterwards.
At least one participant recognized that the search results were profoundly influenced by the terms of the search:
CM11
The difficulties is [sic] the prejudice you take to your search question. Maybe you missed something because you describe[d] it badly.
Search Limits
A further search strategy that the participants found useful was the ability to apply Limits to the searches, including dates, author, links to free full-text articles, language, gender, human subjects, type of article, and so on (Figure 1): “I have used Limits with dates mainly...and if I am searching for a particular author” (MF1). Limits seemed useful for participants for finding specific information, such as when they needed to answer a particular question: “I think I would use this one [Limits] to look for something very specific, but if I wanted to browse around I would use Related Articles” (MF5), and “I think I would use the Limits if I have a problem patient who comes at me with a Google paper, like if she was a 45-year-old female who found something and said, ‘Why don’t I try this?’” (CF10). The participants found Limits useful because it allowed them to be selective about the information they would find, such as limiting their searches specifically to include only core clinical journals. This is because “they [core clinical journals] are well recognized in the medical community” (MF7), and, as another participant explains, “Because sometimes I don’t want to know...what a really alternative journal says, I want to read the larger stuff” (CF12).
Figure 1Limits tab on PubMed
Related Articles
The ability to identify one very promising article and then find all of the related articles by clicking on a hyperlink (Figure 2) was highly valued by five participants: “I really like Related Articles; I saw it in the other database, and it is useful because it brings me to things that I would probably not have found otherwise, outside my own searching” (CM2). However, participants also intuited how Related Articles could be used in relation to Boolean searches to achieve a very efficient focus on the desired research: “These look really great.… I could spend hours just seeing what comes up from this feature after starting from one search” (HF3), and “This list [based on Related Articles] makes more sense to me than the original list that came up” (MF4). It did seem to a number of the participants that “Related Articles worked perfectly” (MF7). Yet, Related Articles did not work for everyone, as one chiropractor commented, “What [PubMed] thinks is similar is not what I think is similar” (CM18).
Full-Text Access
PubMed provides access to the complete articles that it indexes through a publisher’s link on the article’s abstract page (see Figure 2), as well as a link to PubMed Central if the journal’s contents have been placed in this open-access repository. In addition, LinkOut provides links to sites where the article can be accessed, including sites of the publisher, journal aggregators such as EBSCO and ProQuest, and university libraries that hold print and/or electronic editions of the article (which are accessible to members of those libraries). Many of the publishers and the aggregators provide nonsubscribers the ability to purchase access. For example, Elsevier’s Science Direct changes US $30 to purchase an article in any of its 2200 journals. In addition, PubMed clearly identifies that a small proportion (roughly 15%) of the literature has been made “open access” by its publisher, either immediately upon publication or after a certain period (“moving wall” model), typically from 6 to 24 months following publication.
Figure 2Partial results of a PubMed search on “complementary medicine,” showing icons (from top to bottom) for “no abstract,” “abstract only,” and “open access to full-text article” (although no abstract is available) and hyperlinks to “Related Articles” and “Links” (to the full text on publisher and/or library websites)
While the article’s abstract was typically included in PubMed’s indexing of an article, it was judged insufficient by participants as a way of both understanding and evaluating the research, whether they had had very little experience with the research or were well oriented to their field’s literature. As one first-time PubMed user stated as she looked at an abstract, “[I] definitely need to read the article. It [abstract] does not tell us enough” (MF8). This response is consistent with previous studies on the shortcomings of abstracts [22-24].
For most participants, the abstract was necessary as a starting point but was insufficient as source of information on its own. It served as a screen, as it would for a researcher:
CM2
So an abstract is really useful. If I felt the abstract was interesting, I would definitely need the full text to actually find the article useful to my work. An abstract only provides enough information to pass the first screening test.
This chiropractor initially felt, on first looking through the list of PubMed search results, that “This information is not what I am looking for; it is too technical, too specialized.” However, by the time he had had the chance to download and look at an open-access article, he was expressing regrets: “This is incredible; it is so easy; I wish I could just finish reading the article right now” (CM2). Frustration over not being able to view full-text articles was a common theme: “There was an article that I was interested in looking at, and it said that it would not be available for about a year or 6 months” (CM14). “The only problem,” as a registered massage therapist put it, “is that the ones that are free are not the ones I was wanting” (MF8).
For some, visiting a research library was not an option, and the cost of purchasing a single article posed a serious obstacle: “It deters me, big time, having to pay for articles” (MF8), and “Oh, you can order papers; I don’t have money to order papers” (CM14). For others, using a credit card online was an issue: “I don’t think that that’s a safe measure, with my credit card online” (MF5). Yet the interest in seeing an article was sufficient that a number of participants were prepared to give serious consideration to paying to download it: “It is incredibly important to have access to the full text of the article. I would even pay to access a full-text article” (CM2); “I think I’ll look for mostly free articles so I could actually read them. If I was desperate to find information and couldn’t find anything, then I might actually buy it” (HF3), and “depending on how valuable the article is I might be willing to do that” (MF7).
For one chiropractor, who administered a professional chiropractic organization, having access to the full-text article was a must. He felt that the full text offered a more accurate picture of the research and provided the language he needed to use for his own work, which was to lobby the Canadian government to further integrate chiropractic into standard public health policies. As he explained,
CM11
No one who has ever done research has ever done an abstract that exactly covers the detail of what’s done inside. So when you actually read the research paper you see more…. The abstract…never provides that robust meaty quote that I need.
He went on to give an example from the New England Journal of Medicine in which the results reported in the body of the article placed the chiropractor’s treatment of childhood asthma in a far better light than the abstract did.
Another chiropractor conveyed the frustration of coming across a very good source of research, only to find access to the resource restricted. In this case, it was a systematic review conducted by the Cochrane Collaboration, which are not freely available:
CM14
It says [reading from the abstract] “evidence which is based on the Cochrane review of the subject.” This review is like topnotch.... It’s called a Cochrane collaboration, and we don’t have free access to that.... It would be phenomenal if we had access to that.... It’s all based on a strict protocol, like how many people were in the study.... If you want to know the vast information about a subject, you should go to Cochrane reviews.
Even in the case of a chiropractor who felt that “often times I get most of the information I need from the abstract” (CM17) and thus did not need the full text, there was a recanting after a few moments:
CM17
Well, that’s not true. I do [need access to the full text]. Often times you are stuck because they tell you “in the article we describe” such and such. And then you do everything to get it. And we have different ways to get articles.
A registered massage therapist noted how much she appreciated it when the researcher sent, prior to her session, the full text of an article of interest:
MF7
I especially valued that it was not just a summary.... It is very relevant to my work as a therapist, and as an instructor. [I] also found the references very helpful.
And finally, a registered massage therapist who had recently obtained her MSc from a Canadian university, and thus had lost her ready access to the scholarly literature upon graduation, had learned about the issue of access in terms of how libraries were signing a new type of agreement with the journal publishers, which carefully controlled access:
MF1
The contracts that the academic institutions sign with the publishers, limiting access to students and faculty only, mean less access for the public. When hard copies were available, anyone could read, digest, copy relative sections, etc. Now, depending on the institutional policy, it may be difficult even to get access to view a full text. Needless to say, the cost of individual articles is exorbitant.
The library would allow the public access to its online journals at a small number of “public” terminals (for those designated as “walk-ins” in the contract), but as this participant noted,
MF1
[The university libraries] control [public access] very, very, very tightly. I went in and asked if I could use the computer.... I had 6 articles I wanted to see, and they let me do that.
She concluded that this represented “the publisher’s stranglehold on the universities” (MF1). Another participant had caught wind of a hopeful agreement that would greatly increase access to medical research, not only for him, but the public at large:
MM6
I was talking to a librarian here at the public library and she said in [British Columbia, Canada] they were...trying to link the public library with the university library so that anybody that has a library card [could view the journals].
It was PubMed’s clear identification of, as well as links to, the open-access articles that was the one aspect which participants consistently valued. The participants felt strongly about the importance of being able to read the full text, and while a few had access to research libraries (McGill and University of British Columbia cited), others were willing to consider paying for that access. But in all cases, the value of open access to this literature was judged as a critical aspect in becoming better informed about what the research had to offer their professional practice.
Send-To Tools
Users are able to select, organize, and save the citations they find by sending the selected citations to a file, text, printer, clipboard, email, or RSS feed. Five of the participants were very impressed by how this tool allowed one to build a personal set of research resources:
CM2
I’ve done this before [email search results to self] when I have found research on the Web. I think it’s great; it is very easy, and I like to keep things in my inbox. This is a great tool that I would use.
Another chiropractor commented on how it enabled him to integrate PubMed into his work:
CM11
Over the course of the day, I can sort it and then I have my list in order. Now I can take that display list and send it to text.
Search History
PubMed keeps a record of each search that a user conducts, under History, allowing users to not only review their previous searches but to combine them using Boolean operators. “Wow. That’s amazing. If I ever want to go back to what I did, this is how. It’s like you don’t have to write anything down because it is all recorded automatically. This is really, really great” (HF3). While this tool only made it into the sessions of 10 participants, only one of those participants proved relatively indifferent to its powers: “That makes sense” (CM18).
Bookshelf
PubMed has integrated a number of full-text medical and life science books that can be searched separately or in conjunction with an article abstract to gain background information about key terms in the abstract (which are highlighted with links to the relevant passages in the books). Only one participant saw how access to the books might be used for providing background: “I really like it; if I am writing a thesis or a paper, I would see this as very useful” (MF5). More frequently, the participants in the study made a number of observations about the currency and focus of the research articles as the reasons they preferred them over books: “Well that’s interesting [referring to Bookshelf], but I like articles because they are more current” (MF1), and “I like journal articles because they are more specific” (HF3).
A chiropractor reinforced this idea, to a degree that suggested how the background role of PubMed’s Bookshelf program was being missed by some:
CM14
That’s interesting [referring to Bookshelf]. But...once you get into reading research articles, they are much better than reading books. Books are always behind a few years.... There is a lot of bias behind the author of the book—reading an article, you get the actual truth.
Such responses suggest that the participants were picking up ideas about the importance of currency and the leading edge role of journals in the life sciences, ideas that need to be refined, certainly, but that are not far removed from common thinking within the research culture.
MeSH Vocabulary Database
The MeSH Database enables readers to look up the controlled vocabulary used to index the research literature. Among the 10 participants introduced to MeSH, there were challenges initially in comprehending it, to a degree not experienced with the other tools and services: “I don’t understand; what is this [MeSH Database]? Is this everything with the keywords?” (CF12), and “Well, I have taken the tutorial [on the MeSH Database], and I’ve tried to use it, and I know it provides you with headings” (MF1). However, with further explanation and a chance to work with it, at least one participant found it “totally awesome and very useful” (MF8), and another appreciated that “it helps to distinguish things, and so you can go in one direction or another depending on what you want” (MM9). However, that same participant pointed out that what he felt was an element of bias:
MM9
The word “subluxation”...means something quite different to a medical doctor than it does to a chiropractor when it comes to small tiny restriction in motion or small tiny misalignment. But the medical profession still doesn’t believe such things can occur, so I see here that there is no such thing here as subluxation in the sense that the chiropractor means it—very interesting.
PubMed’s sharing of its vocabulary used for indexing also spoke to the value of this service as a source of engagement and reflection.
Email Alert and Notification Services
The National Center for Biotechnology Information (NCBI) of the National Library of Medicine (NLM) is responsible for PubMed and offers users a personalized service that includes email alerts when materials of interest to them are indexed. While this tool came up in only five of the sessions, there was recognition of its value by all the participants: “I have an automatic referral for [from] NCBI every so often about updates of new research in this area” (MF1). A chiropractor had signed up with a similar service, PubCrawler, developed by a genetics research lab at Trinity College, Dublin, for alerting readers to new articles in areas of interest: “I’ve signed up to PubCrawler, which alerts me to new research in my area, and so [I] quite often scan these [almost daily]” (CM11).
Author Links
The ability to find other works by the same author, simply by clicking on the author’s name, was not generally valued by the participants with the exception of the homeopath and one of the chiropractors: “So I have heard of him before. I would think it would be interesting; it would be interesting to know what his perspective was on other things” (CF12). With the six participants who encountered this feature divided between interest in and indifference toward it, it may seem that this way of focusing on the work of an individual researcher is not as valued as the other means of tracking work.
Comprehending Research
In the course of reflecting on PubMed tools and services, some of the participants reflected on their understanding, as well as their critical reading and use, of the research they were searching. For example, a number of participants commented on the value of reading the abstract as a necessary first step in reading research: “I wouldn’t read a paper without an abstract” (HF3). By the same token, the abstract was not enough: “Definitely need to read the article. [Abstract] does not tell us enough here” (MF8). A chiropractor noted that the figures and illustrations available in the full text were needed to make sense of the orthopedic tests used in the study:
CM15
[The abstract is] a good summary, but...if the article had pictures and stuff, because maybe these are new tests that have come out that the school hasn’t taught us yet, and they might be better than the ones we’ve learned...[then access] would be important.
Another chiropractor spoke of the importance of seeing the research methodology described in detail:
CM14
The abstracts usually don’t show enough about the methods of an article.... It depends on the method, if the paper is actually good.
Similarly, a third chiropractor, who was satisfied with the abstract alone when it came to “advice or recommendations,” felt that when the “treatment” of a client was at issue, then more than the abstract was needed:
CM18
I can’t break down the methods they used [with the abstract alone], and if there’s any flaws in it...I won’t get it from the abstract. So I would like to see the whole thing.
The participants in this study were aware of their limits in understanding the research, which concerned specific domains of knowledge, as this chiropractor made clear: “I am trying to find one that is not as ‘biochemistry’ to read; those ones are terrible to read.” Yet, within a given search, the same participant was able to find research studies which, in dealing with “exercise for treating fibromyalgia,” were extremely helpful and comprehensible to the point where he “would want to prescribe” those exercises to his clients (CM16). More than one registered massage therapist was prepared to challenge the limits on her own understanding: “There’s a lot of qualitative research out there on complementary therapies, but not a lot of quantitative [work], so I am looking for quantitative-oriented stuff” (MF5). However, she realized that she was not clear on what “the p factor” (sic) was and stated that “I’m not really up with statistics.... I can understand some but not all” (MF5). A second registered massage therapist was willing to push her learning beyond the concerns of clinical practice: “Well, it’s not useful for me, for what I do, but it does tell me some information that I didn’t know before: the cell type that they are talking about, the engagement of it” (MF7).
If participants felt somewhat overwhelmed by the research at times—“It’s a little above my head” (CM13)—they also knew how to direct their reading in relation to their strengths. As a registered massage therapist put it: “This is a little too theoretical and general...[while I’m] looking for protocols for treating low back pain.... I’d be more on ‘protocol,’ ‘orthopedic,’ ‘massage therapy’” (MM9). Or, as a chiropractor explained: “It’s a bit too heavy; it is going to go beyond what I need clinically” (CF10). What was perhaps most notable about the participants’ responses to the research was their perception of the value of this public resource to their professional practice: “It’s pretty encouraging to find this [material in PubMed]; it’s not intimidating” (MF7), and “I use the database and feel quite comfortable with it” (CF10).
A Growing Research Culture
This research on CAM practitioners’ use of a life sciences research index reflects a growing influence of a research culture among the health professions and the public. Two participants made reference to their clients bringing in online health information:
MF5
My problem is telling [my clients] that what they are finding in Google is not that sound scientific information because any practitioner out there, anybody can put something out onto Google or their office website, but it’s not necessarily valid. I always have clients who are asking me about information that has come out on certain treatments, so that is primarily what I would tend to use this [PubMed] for.
That same increased interest in recent research had been expressed within the CAM community as well, as the administrator of a chiropractic organization stated:
CM11
Our membership expects us to know things instantly if they are in the public domain. They say, "Why don’t you know? Everyone else knows”.
The increasing prominence of research in the health services sector was also reflected in the training received by graduates of the Canadian Memorial Chiropractic College:
CM17
This school is very evidence-based. So a lot of the assignments are pretty much looking up research: sensitivity, specificity, tests, prognosis.... If you are efficient with any of these search engines, you can find the information quite quickly. I have used it in university as well, but not to a great extent. When I got here I had to use it much more. It depends on the clinician that you get; some are really, really, really, evidence-based, meaning they follow everything the research says.
Another participant noted:
CM16
They [at the Canadian Memorial Chiropractic College] talk so much about evidence-based care in medicine and chiropractic, and you have to have research articles in order to attempt to that. You need a way to do it and PubMed is the only way I know.
He went on to add that “it is free.” This was further confirmed by a classmate: “They [the school] thrust that down our throats: ‘Make sure you are up to date on that’” (CM15).
In turn, CAM practitioners are participating in the debates on the growing prominence of certain forms of medical research but in the following example also demonstrate limited understanding of the methodology:
CM11
I wrote an article about [how]...it is more or less impossible to do a [randomized clinical trial] of chiropractic. You can’t blind a practitioner; it’s more or less impossible to blind the patient, so any effect of randomization is nonsense.
Overall, there is a growing global awareness of the important role played by research in the life sciences, and these CAM practitioners are learning that this body of knowledge has much to offer, with PubMed providing one means of obtaining access to it: “There’s a lot going on in Europe and Asia that I know nothing about unless they publish in Lymphology or unless they present at the ISL [International Society of Lymphology]” (MF7).
Continuing Use of PubMed
In response to follow-up emails that were sent out some weeks after the session with the researcher, seven participants affirmed that they were using or were planning to use PubMed:
CM2
Yes, I have used PubMed since our last conversation. I use it on a weekly basis. I use it to find information for patients and for myself. I am in the process of developing an acupuncture-related website and writing a book on health-related topics.
Issues of access to research articles persisted in the use of PubMed for this chiropractor:
CM2
I have not purchased any articles, although I have been tempted. I tend to look at the full-text articles, and I have used the links LinkOut and Bookshelf with success.
As well, he had begun to see how he could best use PubMed in his practice:
CM2
I think I’ll use it to get research, to get on-the-fly research to answer questions. But I wouldn’t send my patients to use the database or conduct searches on this database after a consult with them because I think it would be too complicated. I would not refer them to PubMed to find their own research.
There were also indications from the participants of PubMed raising the quality of knowledge that informs their practice: “I’m using [PubMed] now to look something up instead of Google” (CF12).
Other participants spoke in the follow-up emails of their plans for continuing use of PubMed:
CM15
Now that I know more about it, I think I’ll use it more.... Now that we have had this session, I’ll be able to understand it a little bit better, especially this "Related Articles".
This same participant had explained in his session with the researcher how the Limits tool would serve his practice: “[This] would be great if I had a patient come in with a problem, and I had to really specify my search according to his needs” (CM15).
Another chiropractic student spoke of PubMed use in terms of its public access as a source of professional development:
CM17
I am not going to be in academia much longer. It would be great to know as much as I could before I leave. I plan on using this when I leave as well.
Of course, not everyone who participated in this study went on to use PubMed afterward:
HF3
I often learn about research studies from [online] medical news sources [eg, Medical Post] and access them through links provided in the article versus searching PubMed. There is so much information out there, and I find it hard to find time to access it all!.
A similar theme was sounded by a massage therapist:
MM9
I have no urgent reason to [use PubMed], and my time is too occupied to browse.... Currently, I do not [need PubMed].
And a chiropractor wrote to say that while he is sometimes “directed to PubMed as a resource” at his school, the problem is that he “could never really get the entire PDF files” that he wanted (CM13).
Discussion
This study confirmed our hypothesis that CAM practitioners would find that certain PubMed tools and services, on being introduced to them, had the potential to contribute to their engagement with and understanding of the research that interested them. This study did not seek to measure the actual contribution of these tools and services to the users’ learning. It was designed to establish whether these users perceived the tools and services as having value, as a first step to subsequent studies that will assess the differences that individual tools might make to the participants’ learning.
Among the PubMed tools and services to which the participants were introduced, only the service of full-text access proved a positive asset for engagement, comprehension, evaluation, and utilization. As well, the Boolean Search and Related Articles were also strong contributors to the participants’ work with PubMed, and all tools and services impressed more than a few participants as to their value and contribution.
In terms of the specific design of PubMed, it is clear that the NLM is continuing to improve its design and functionality [25]. For example, the participants found MeSH to be a particularly difficult tool to grasp; however, the NLM has since provided three animated tutorials (eg, ”Searching with MeSH”) with voice-over, which make its operation much clearer, with at least one recent study attesting to MeSH as the preferred search strategy [26]. Still, one of this paper’s reviewers wisely advised that training in MeSH would greatly improve user searches, and the ways in which a user’s health vocabulary contributes to recognition and comprehension has been the subject of a recent study [27].
There have been design improvements, as well, with Related Articles, Limits, and other tools. With Bookshelf, the only feature that left over half of the participants who tried it seeing little value in it, PubMed has withdrawn the book links from an article’s abstract, while still offering users the ability to search its book holdings. Yet, these medical and basic science books could well serve as background resources when a reader is stymied by concepts in biochemistry and statistics, to name two areas that this study’s participants identified as weak for them [28]. There is reason, then, for the NLM to continue exploring ways of integrating the books into the literature searches in a way that encourages professionals in the health services to seek background clarification and context, as a way of improving their engagement with the research.
The study also has bearing on what has developed, with the rise of the Internet, into the “access issue.” This goes back to at least 1999, when Harold Varmus, then director of the National Institutes of Health, proposed that all medical research journals make their work freely available in PubMed Central 6 months after publication, an initiative that met with the successful resistance of commercial and society publishers [29]. More recent measures include the National Institutes of Health Public Access Policy, which asks funded researchers to voluntarily deposit copies of their published work in PubMed Central, but which has managed to achieve a less than 4% compliance rate to date [30]. As well, the proposed Federal Research Public Access Act of 2006 is currently before the Senate, which would mandate open access within 6 months of publication for a large proportion of US government–funded research [31]. The results of this study would seem to support efforts to increase access to research by making it clear how greater access could contribute to the practice of CAM, as well as to more traditional forms of clinical practice.
The participants’ consistent interest in having access to the full text of the articles indexed in PubMed leads to a strong and immediate recommendation to improve that access: The NLM should make every effort to capture an otherwise missing and substantial source of open access to research and scholarship, namely, the published health sciences research that has been posted by authors in institutional repositories and on websites. While PubMed has an excellent system for identifying and connecting to open-access articles made available by publishers, it needs to develop similarly effective systems for tapping the published work that authors have posted, with the publisher’s permission, in archives and on websites. With numerous archiving mandates, both in place and pending, for this form of open access to research that has been funded by governments and foundations, PubMed needs to ensure that it is able to take advantage of this movement to greater openness. For example, PubMed could include a means of searching the more than 800 repositories worldwide.
For those who study the use of health information, we see a need to push beyond the obvious limitations of this study. There are sufficient grounds for further studies carefully designed to assess the contribution that particular aspects of these tools provide for different types of users, with a focus on how the design of the tools and services augments the users’ comprehension, evaluation, and utilization of the materials they encounter.
The prospect of the ongoing development of resources such as PubMed, along with an increasing degree of public and professional expectation of access to research and scholarship, holds much promise for the continuing educational quality of the Internet and society at large. While only a fraction of CAM practitioners and a proportion of their clients will pursue these forms of knowledge, there is a larger point to this. Through the combined efforts and commitment of the NLM, a good number of life science journals, and the researchers themselves (who post open-access copies of their work), the quality of knowledge that is publicly and universally available is increasing, adding to people’s understanding as well as to the health care practices that affect their lives. This study is one small demonstration of how the benefits of this greater access extend to a larger community than has been commonly considered when it comes to public resources such as PubMed.

complementary and alternative medicine
pubmed
literacy
information retrieval
professional development
information management
open access
research dissemination