Promoting Patient Portals: An Application of Social Cognitive Theory to Post-Adoption Patient Portal Use.

Many healthcare clinics encourage the use of online patient portals so that patients can have easier access to their health information, yet some patients are hesitant to interact with these portals. We used social cognitive theory to develop and test a theoretically grounded model that incorporates several (1) technological factors, (2) individual factors, and (3) social factors that influence individuals' post-adoption, active use of patient portals. Based on cross-sectional survey data from a sample of healthcare clinic patients (N = 431), we found that individuals' severity of illness predicted active use of patient portals and that trust in doctors predicted attitudes toward patient portals. Moreover, attitudes toward patient portals mediated the relationship between technology factors (i.e., perceived usefulness, ease of use, customization, and interactivity), and active use of patient portals. The paper concludes with a discussion of key findings, implications, and directions for future research.

Developing and validating the Unhurried Conversations Assessment Tool (UCAT).

OBJECTIVE: Given the importance of unhurried conversations for providing careful and kind care, we sought to create, test, and validate the Unhurried Conversations Assessment Tool (UCAT) for assessing the unhurriedness of patient-clinician consultations. METHODS: In the first two phases, the unhurried conversation dimensions were identified and transformed into an assessment tool. In the third phase, two independent raters used UCAT to evaluate the unhurriedness of 100 randomly selected consultations from 184 videos recorded for a large research trial. UCAT's psychometric properties were evaluated using this data. RESULTS: UCAT demonstrates content validity based on the literature and expert review. EFA and reliability analyses confirm its construct validity and internal consistency. The seven formative dimensions account for 89.93% of the variance in unhurriedness, each displaying excellent internal consistency (α > 0.90). Inter-rater agreement for the overall assessment item was fair (ICC = 0.59), with individual dimension ICCs ranging from 0.26 (poor) to 0.95 (excellent). CONCLUSION: UCAT components comprehensively assess the unhurriedness of consultations. The tool exhibits content and construct validity and can be used reliably. PRACTICE IMPLICATIONS: UCAT's design and psychometric properties make it a practical and efficient tool. Clinicians can use it for self-evaluations and training to foster unhurried conversations.

Mechanisms of AMPA Receptor Inhibition by Diminazene.

Diminazene is an anti-infection agent for animals and is a member of the diarylamidine group. This study reports the first detection of its inhibitory effect on AMPA-type ionotropic glutamate receptors. Experiments were carried out on isolated Wistar rat neurons: striatal giant cholinergic interneurons were used to study calcium-permeable AMPA receptors and hippocampal field CA1 pyramidal neurons were used to study calcium-impermeable AMPA receptors. Cells were isolated by vibrodissociation and currents were recorded by voltage clamping in the whole cell configuration. Diminazene produced concentration-dependent inhibition of currents evoked by application of kainate in both neuron types. IC50 values for calcium-permeable and calcium-impermeable AMPA receptors were 60 ± 11 and 160 ± 30 µM, respectively. Of note is that the inhibitory action of diminazene increased with increases in agonist concentration. The plot of the voltage dependence of inhibition at a fixed diminazene concentration for calcium-permeable AMPA receptors was biphasic: minimal inhibition was seen at positive potentials and maximum at -40 to -60 mV, while further hyperpolarization produced a gradual decrease in blockade efficacy. All these properties provide evidence that diminazene blocks AMPA receptor channels, perhaps with penetration through channels into cells.

Screening for Activity Against AMPA Receptors Among Anticonvulsants-Focus on Phenytoin.

The interest in AMPA receptors as a target for epilepsy treatment increased substantially after the approval of perampanel, a negative AMPA receptor allosteric antagonist, for the treatment of partial-onset seizures and generalized tonic-clonic seizures. Here we performed a screening for activity against native calcium-permeable AMPA receptors (CP-AMPARs) and calcium-impermeable AMPA receptors (CI-AMPARs) among different anticonvulsants using the whole-cell patch-clamp method on isolated Wistar rat brain neurons. Lamotrigine, topiramate, levetiracetam, felbamate, carbamazepine, tiagabin, vigabatrin, zonisamide, and gabapentin in 100-µM concentration were practically inactive against both major subtypes of AMPARs, while phenytoin reversibly inhibited them with IC50 of 30 ± 4 µM and 250 ± 60 µM for CI-AMPARs and CP-AMPARs, respectively. The action of phenytoin on CI-AMPARs was attenuated in experiments with high agonist concentrations, in the presence of cyclothiazide and at pH 9.0. Features of phenytoin action matched those of the CI-AMPARs pore blocker pentobarbital, being different from classical competitive inhibitors, negative allosteric inhibitors, and CP-AMPARs selective channel blockers. Close 3D similarity between phenytoin and pentobarbital also suggests a common binding site in the pore and mechanism of inhibition. The main target for phenytoin in the brain, which is believed to underlie its anticonvulsant properties, are voltage-gated sodium channels. Here we have shown for the first time that phenytoin inhibits CI-AMPARs with similar potency. Thus, AMPAR inhibition by phenytoin may contribute to its anticonvulsant properties as well as its side effects.

Priming affects the activity of a specific region of the promoter of the human beta interferon gene.

Treatment of Daudi or HeLa cells with human interferon (IFN) alpha 8 before induction with either poly(I)-poly(C) or Sendai virus resulted in an 8- to 100-fold increase in IFN production. The extent of priming in Daudi cells paralleled the increase in the intracellular content of IFN-beta mRNA. IFN-alpha mRNA remained undetectable in poly(I)-poly(C)-treated Daudi cells either before or after priming. An IFN-resistant clone of Daudi cells was found to produce 4- to 20-fold more IFN after priming, indicating that priming was unrelated to the phenotype of IFN sensitivity. IFN treatment of either Daudi or HeLa cells transfected with the human IFN-beta promoter (-282 to -37) linked to the chloramphenicol acetyltransferase (CAT) gene resulted in an increase in CAT activity after induction with poly(I)-poly(C) or Sendai virus. A synthetic double-stranded oligonucleotide corresponding to an authentic 30-base-pair (bp) region of the human IFN-beta promoter between positions -91 and -62 was found to confer virus inducibility upon the reporter CAT gene in HeLa cells. IFN treatment of HeLa cells transfected with this 30-bp region of the IFN-beta promoter in either the correct or reversed orientation also increased CAT activity upon subsequent induction. IFN treatment alone had no detectable effect on the activity of either the 30-bp region or the complete human IFN promoter.

Interferon modulation of c-myc expression in cloned Daudi cells: relationship to the phenotype of interferon resistance.

Treatment of interferon-sensitive Daudi cell with electrophoretically pure human interferon alpha markedly reduced the level of c-myc mRNA, increased the level of class I histocompatibility antigen (HLA) mRNA, and did not affect the level of actin mRNA within the same cells. In contrast, the level of c-myc mRNA or HLA mRNA did not change significantly following interferon treatment in different clones of Daudi cells selected for resistance to the antiproliferative action of interferon. These cells possessed interferon receptors, however, and responded to interferon modulation of other genes, including 2',5' oligoisoadenylate synthetase (M. G. Tovey, M. Dron, K. E. Mogensen, B. Lebleu, N. Metchi, and J. Begon-Lours, Guymarho, J. Gen. Virol., 64:2649-2653, 1983; M. Dron, M. G. Tovey, and P. Eid, J. Gen. Virol., 66:787-795, 1985). A clone of interferon-resistant Daudi cells which had reverted to almost complete sensitivity to both the antiproliferative action of interferon and the interferon-enhanced expression of HLA mRNA remained refractory, however, to interferon modulation of c-myc expression, suggesting that a reduced level of c-myc mRNA may not be a prerequisite for inhibition of cell proliferation in interferon-treated cells. Our results do not exclude the possibility, however, that posttranscriptional modification(s) of c-myc expression may precede an inhibition of cell proliferation in interferon-treated cells.

clk1, a serine/threonine protein kinase-encoding gene, is involved in pathogenicity of Colletotrichum lindemuthianum on common bean.

A random insertional mutagenesis in Colletotrichum lindemuthianum, the causal agent of common bean anthracnose, generated four mutants that showed altered pathogenicity when tested on intact seedlings, excised leaves, and/or excised hypocotyls. One of these mutants, H290, produced very few lesions on bean leaves and appeared affected in its ability to penetrate the leaf cuticle. Molecular analyses showed that the border sequences of the unique integration site of the disrupting pAN7-1 plasmid in the mutant exhibited homology with conserved domains of serine/threonine protein kinases. The corresponding wild-type sequences were cloned and a gene replacement vector with a mutated copy harboring a selection marker constructed. Transformation of the wild-type pathogen produced a strain with a phenotype identical to the original mutant. Genomic and cDNA sequences indicated that the disrupted gene is a member of the serine/threonine protein kinase family. The gene, called clk1 (Colletotrichum lindemuthianum kinase 1), was weakly expressed in the mycelium of the wild-type strain grown on rich and minimal synthetic media but was undetectable during the infection even when a sensitive reverse transcriptase-polymerase chain reaction methodology was used. This study represents the first characterization of altered pathogenicity mutants in C. lindemuthianum produced by random mutagenesis and demonstrates the involvement of a member of the serine/threonine kinase gene family in the early steps of the infection process.

Inheritance of partial resistance against Colletotrichum lindemuthianum in Phaseolus vulgaris and co-localization of quantitative trait loci with genes involved in specific resistance.

Anthracnose, one of the most important diseases of common bean (Phaseolus vulgaris), is caused by the fungus Colletotrichum lindemuthianum. A "candidate gene" approach was used to map anthracnose resistance quantitative trait loci (QTL). Candidate genes included genes for both pathogen recognition (resistance genes and resistance gene analogs [RGAs]) and general plant defense (defense response genes). Two strains of C. lindemuthianum, identified in a world collection of 177 strains, displayed a reproducible and differential aggressiveness toward BAT93 and JaloEEP558, two parental lines of P. vulgaris representing the two major gene pools of this crop. A reliable test was developed to score partial resistance in aerial organs of the plant (stem, leaf, petiole) under controlled growth chamber conditions. BAT93 was more resistant than JaloEEP558 regardless of the organ or strain tested. With a recombinant inbred line (RIL) population derived from a cross between these two parental lines, 10 QTL were located on a genetic map harboring 143 markers, including known defense response genes, anthracnose-specific resistance genes, and RGAs. Eight of the QTL displayed isolate specificity. Two were co-localized with known defense genes (phenylalanine ammonia-lyase and hydroxyproline-rich glycoprotein) and three with anthracnose-specific resistance genes and/or RGAs. Interestingly, two QTL, with different allelic contribution, mapped on linkage group B4 in a 5.0 cM interval containing Andean and Mesoamerican specific resistance genes against C. lindemuthianum and 11 polymorphic fragments revealed with a RGA probe. The possible relationship between genes underlying specific and partial resistance is discussed.

Identification of an ancestral resistance gene cluster involved in the coevolution process between Phaseolus vulgaris and its fungal pathogen Colletotrichum lindemuthianum.

The recent cloning of plant resistance (R) genes and the sequencing of resistance gene clusters have shed light on the molecular evolution of R genes. However, up to now, no attempt has been made to correlate this molecular evolution with the host-pathogen coevolution process at the population level. Cross-inoculations were carried out between 26 strains of the fungal pathogen Colletotrichum lindemuthianum and 48 Phaseolus vulgaris plants collected in the three centers of diversity of the host species. A high level of diversity for resistance against the pathogen was revealed. Most of the resistance specificities were overcome in sympatric situations, indicating an adaptation of the pathogen to the local host. In contrast, plants were generally resistant to allopatric strains, suggesting that R genes that were efficient against exotic strains but had been overcome locally were maintained in the plant genome. These results indicated that coevolution processes between the two protagonists led to a differentiation for resistance in the three centers of diversity of the host. To improve our understanding of the molecular evolution of these different specificities, a recombinant inbred (RI) population derived from two representative genotypes of the Andean (JaloEEP558) and Mesoamerican (BAT93) gene pools was used to map anthracnose specificities. A gene cluster comprising both Andean (Co-y; Co-z) and Mesoamerican (Co-9) host resistance specificities was identified, suggesting that this locus existed prior to the separation of the two major gene pools of P. vulgaris. Molecular analysis revealed a high level of complexity at this locus. It harbors 11 restriction fragment length polymorphisms when R gene analog (RGA) clones are used. The relationship between the coevolution process and diversification of resistance specificities at resistance gene clusters is discussed.

The Arabidopsis SHAGGY-related protein kinase (ASK) gene family: structure, organization and evolution.

Higher plants contain a multigene family encoding proteins that share a highly conserved catalytic protein kinase domain about 70% identical to SHAGGY protein kinase (SGG) and glycogen synthase kinase-3 (GSK-3), respectively, from Drosophila and mammals. In this study we have characterized the structure and evolution of the Arabidopsis SHAGGY-related protein kinase (ASK) gene family. At least ten ASK genes are present per haploid genome of Arabidopsis. The genomic sequences of five ASK genes show a strikingly high conservation of intron positions and exon lengths. Phylogenetic analyses suggested that the Arabidopsis gene family contains at least three ancient classes of genes that diverged early in land plant evolution. The different classes may reflect specificity of substrates and/or biological functions. Eight out of the ten predicted ASK genes were mapped and shown to be dispersed over the five Arabidopsis chromosomes. A tentative model for the organization and evolution of the Arabidopsis ASK genes is presented.

Enhanced levels of scrapie responsive gene mRNA in BSE-infected mouse brain.

The expression of the mRNA of nine scrapie responsive genes was analyzed in the brains of FVB/N mice infected with bovine spongiform encephalopathy (BSE). The RNA transcripts of eight genes were overexpressed to a comparable extent in both BSE-infected and scrapie-infected mice, indicating a common series of pathogenic events in the two transmissible spongiform encephalopathies (TSEs). In contrast, the serine proteinase inhibitor spi 2, an analogue of the human alpha-1 antichymotrypsin gene, was overexpressed to a greater extent in the brains of scrapie-infected animals than in animals infected with BSE, reflecting either an agent specific or a mouse strain specific response. The levels of spi 2 mRNA were increased during the course of scrapie prior to the onset of clinical signs of the disease and the increase reached 11 to 45 fold relative to uninfected controls in terminally ill mice. Spi 2, in common with four of the other scrapie responsive genes studied, is known to be associated with pro-inflammatory processes. These observations underline the importance of cell reactivity in TSE. In addition, scrg2 mRNA the level of which is enhanced in TSE-infected mouse brain, was identified as a previously unrecognized long transcript of the murine aldolase C gene. However, the level of the principal aldolase C mRNA is unaffected in TSE. The increased representation of the longer transcript in the late stage of the disease may reflect changes in mRNA processing and/or stability in reactive astrocytes or in damaged Purkinje cells.

Production and in vivo biologic actions of recombinant mouse interferon alpha 2.

We have expressed a recombinant mouse interferon alpha (r.Mu-IFN alpha 2) in Escherichia coli under the control of a tryptophan promoter using a synthetic adaptor formed by annealing two partially complementary oligonucleotides which introduced an ATG start codon and re-established the complete coding sequence of the mature IFN alpha 2 protein in the expression vector. Levels of up to 10(7) reference units of Mu-IFN alpha 2 per liter of culture were obtained using this construction. This recombinant mouse interferon alpha 2 exhibited antiviral activity in mice infected with EMC virus and antitumor activity in mice inoculated with Friend leukemia cells in a manner similar to that of natural mouse interferon alpha/beta.

Genetic Diversity and Pathogenic Variation of Colletotrichum lindemuthianum in the Three Centers of Diversity of Its Host, Phaseolus vulgaris.

ABSTRACT Population subdivision of Colletotrichum lindemuthianum, the causal agent of anthracnose, was studied in three regions located in three centers of diversity of its host, Phaseolus vulgaris. Random amplified polymorphic DNA (RAPD) markers, restriction endonuclease analysis of the amplified ribosomal internal transcribed spacer region, and virulence on a set of 12 cultivars were used to assess the genetic diversity of C. lindemuthianum strains isolated in Mexican, Ecuadorian, and Argentinean wild common bean populations. The three regions were significantly differentiated for molecular markers. For these markers, Mexico was the most polymorphic and the most distant from Ecuador and Argentina. The majority of the RAPD alleles present in Ecuador and Argentina were found in Mexico, suggesting that Andean populations have been derived from the Mesoamerican center. Pathogenicity tests on a set of 12 cultivars showed that all but one of the Mexican strains were virulent exclusively on Mesoamerican cultivars. Argentinean strains were virulent preferentially on southern Andes cultivars, and the Ecuadorian strains, except for one strain, were avirulent on all cultivars. These results suggest an adaptation of strains on cultivars of the same geographic origin. Thus, based on molecular and virulence markers, C. lindemuthianum strains isolated from wild common bean populations were divided into three groups corresponding to host gene pools.

Visualization of viral candidate cDNAs in infectious brain fractions from Creutzfeldt-Jakob disease by representational difference analysis.

Creutzfeldt-Jakob Disease (CJD), a neurodegenerative and dementing disease of later life, is caused by a viruslike entity that is incompletely characterized. As in scrapie, all more purified infectious brain preparations contain nucleic acids. However, it has not been possible to visualize unique bands that may derive from a viral genome. We here used a subtractive strategy known as representational difference analysis (RDA) to uncover such sequences. To reduce the complexity of starting target nucleic acids, sucrose gradients were first used to select nuclease resistant particles with a defined 120S size. In CJD this single 120S gradient peak is highly enriched for infectivity, and contains reduced amounts of PrP (Proc. Natl. Acad. Sci. 92, 5124-8, 1995). Parallel 120S fractions from uninfected brain were made to generate subtractor sequences. 120S particles were lysed in GdnSCN, and ng amounts of released RNA were purified for random-primed cDNA synthesis. To capture representative fragments of 100-500 bp, cDNAs were cleaved with Mbo I for adaptor ligation and amplification. In the first experiment with moderate RDA selection, it was possible to visualize clones from CJD cDNA that did not hybridize to control cDNA. In the second experiment, more exhaustive subtractions yielded a discrete set of CJD derived gel bands. Competitive hybridization showed a subset of these bands were not present in either the control 120S cDNA or in the hamster genome. This represents the first demonstration of apparently CJD-specific nucleic acid bands in more purified infectious preparations. Although exhaustive cloning, sequencing and correlative titration studies need to be done, it is encouraging that most of the viral candidates selected thus far have no significant homology with any previously described sequence in the database.

Interferon-resistant Daudi cells are deficient in interferon-alpha-induced ISGF3 alpha activation, but remain sensitive to the interferon-alpha-induced increase in ISGF3 gamma content.

Low levels of the transcription factor ISGF3 alpha were detected in the cytoplasm and nucleus of untreated Daudi cells, which increased markedly following interferon (IFN) treatment. In contrast no ISGF3 alpha was detected in an IFN-resistant clone of Daudi cells, DIF8, and only low levels were detected in these cells after IFN-alpha treatment. High levels of ISGF3 were produced in vitro, however, by the addition of ISGF3 alpha to extracts of IFN-treated DIF8 cells, indicating that IFN is unable to produce substantial amounts of functional ISGF3 alpha in DIF8 cells. A second clone of IFN-resistant Daudi cells, DIF3, also exhibited defective ISGF3 alpha production, which was restored to normal in the subclone DIF3REV5 that had reverted to high IFN sensitivity. Thus, the antiproliferative effect of IFN on Daudi cells and derived clones is closely related to the level of ISGF3 present in the nucleus of these cells. IFN-alpha, however, also enhances the content of ISGF3 gamma in IFN-resistant cells as well as certain proteins of unknown function, raising the possibility that a second pathway of IFN-alpha signal transduction, distinct from the ISGF3 pathway, remains functional in both DIF8 and DIF3 cells.

Light and developmental regulation of the Anp-controlled anthocyanin phenotype of bean pods.

In the presence of the dominant allele of the Anp gene, bean pods present a purple-mottled phenotype. The purple pigmentation is variable from cell to cell in the pod epidermal layer and develops as a random mosaic. Three anthocyanidins, delphinidin, petunidin and malvidin, are involved in this purple pigmentation. Anthocyanins accumulated in vacuoles; anthocyanoplasts and cristal bodies were also observed occasionally. A developmental switch is a prerequisite for anthocyanin accumulation in the pods. This does not occur before day 4 after pollination and is controlled by light in competent pods. mRNAs for PAL, CHS, CHI, DFR and UFGT are induced in the pods, indicating that the general anthocyanin biosynthetic pathway is well conserved at both the biochemical and molecular levels in this species. mRNA steady-state level studies of PAL and CHS suggest that the light regulation occurs at the transcriptional level.

Isolation of Daudi cells with reduced sensitivity to interferon. I. Characterization.

Treatment of Daudi cells with successively increasing concentrations of interferon-alpha resulted in the selection of a cell population which multiplied in the continued presence of 10(4) units/ml of interferon-alpha. A number of clones of interferon-resistant Daudi cells were isolated from this population. Two clones, DIF2 and DIF3, were found to exhibit moderate and pronounced resistance, respectively, to both the antiviral and antiproliferative actions of human interferons-alpha and -beta. These clones were also less responsive to the enhancement by interferon of Epstein-Barr virus early antigen expression. Both the surface antigens and karyotype of the interferon-resistant clones were similar to those of parental Daudi cells. After prolonged cultivation in the absence of interferon, DIF3 cells were found to 'revert' to an intermediate interferon sensitivity. The interferon sensitivity of clone DIF2 remained unchanged even after more than 1 year in culture.

Small tandemly repeated DNA sequences of higher plants likely originate from a tRNA gene ancestor.

Several monomers (177 bp) of a tandemly arranged repetitive nuclear DNA sequence of Brassica oleracea have been cloned and sequenced. They share up to 95% homology between one another and up to 80% with other satellite DNA sequences of Cruciferae, suggesting a common ancestor. Both strands of these monomers show more than 50% homology with many tRNA genes; the best homologies have been obtained with Lys and His yeast mitochondrial tRNA genes (respectively 64% and 60%). These results suggest that small tandemly repeated DNA sequences of plants may have evolved from a tRNA gene ancestor. These tandem repeats have probably arisen via a process involving reverse transcription of polymerase III RNA intermediates, as is the case for interspersed DNA sequences of mammalians. A model is proposed to explain the formation of such small tandemly repeated DNA sequences.

Isolation of Daudi cells with reduced sensitivity to interferon. III. Interferon-induced proteins in relation to the phenotype of interferon resistance.

The pattern of both constitutive and interferon-induced proteins was determined by two-dimensional gel electrophoresis in parental and interferon-resistant clones of Daudi cells in relation to the phenotype of interferon resistance. The complement of constitutive proteins present in clones DIF3, DIF8, DIF9 and DIF10 appeared to be identical to that of parental Daudi cells even though these cells were resistant to both the antiviral and anti-proliferative actions of interferon. Treatment of Daudi cells for 20 h with 10(3) reference units/ml of electrophoretically pure human interferon-alpha resulted in the induction of 15 proteins of molecular weights ranging from 15000 to 62000 detected after a 4 h labelling period with L-[35S]methionine. A number of these proteins were also induced in interferon-resistant clones of Daudi cells although some of these proteins appeared later and in smaller amounts than in the interferon-treated parental cells. However, seven proteins with molecular weights ranging from 18000 to 58000 which were induced by interferon in parental Daudi cells were not induced in any of the four interferon-resistant clones, suggesting that the phenotype of interferon resistance of these cells may be related to a reduction or absence of certain interferon-induced protein(s).