Understanding the clinical reasoning processes involved in the management of multimorbidity in an ambulatory setting: study protocol of a stimulated recall research.

BACKGROUND: Primary care physicians are at the very heart of managing patients suffering from multimorbidity. However, several studies have highlighted that some physicians feel ill-equipped to manage these kinds of complex clinical situations. Few studies are available on the clinical reasoning processes at play during the long-term management and follow-up of patients suffering from multimorbidity. This study aims to contribute to a better understanding on how the clinical reasoning of primary care physicians is affected during follow-up consultations with these patients. METHODS: A qualitative research project based on semi-structured interviews with primary care physicians in an ambulatory setting will be carried out, using the video stimulated recall interview method. Participants will be filmed in their work environment during a standard consultation with a patient suffering from multimorbidity using a "button camera" (small camera) which will be pinned to their white coat. The recording will be used in a following semi-structured interview with physicians and the research team to instigate a stimulated recall. Stimulated recall is a research method that allows the investigation of cognitive processes by inviting participants to recall their concurrent thinking during an event when prompted by a video sequence recall. During this interview, participants will be prompted by different video sequence and asked to discuss them; the aim will be to encourage them to make their clinical reasoning processes explicit. Fifteen to twenty interviews are planned to reach data saturation. The interviews will be transcribed verbatim and data will be analysed according to a standard content analysis, using deductive and inductive approaches. CONCLUSION: Study results will contribute to the scientific community's overall understanding of clinical reasoning. This will subsequently allow future generation of primary care physicians to have access to more adequate trainings to manage patients suffering from multimorbidity in their practice. As a result, this will improve the quality of the patient's care and treatments.

Comparative field study evaluating the activity of recombinant factor VIII Fc fusion protein in plasma samples at clinical haemostasis laboratories.

Discrepancies exist for some of the modified coagulation factors when assayed with different one-stage clotting and chromogenic substrate assay reagents. The aim of this study was to evaluate the performance of a recombinant factor VIII Fc fusion protein (rFVIIIFc), currently in clinical development for the treatment of severe haemophilia A, in a variety of one-stage clotting and chromogenic substrate assays in clinical haemostasis laboratories. Haemophilic plasma samples spiked with rFVIIIFc or Advate(®) at 0.05, 0.20 or 0.80 IU mL(-1) were tested by 30 laboratories using their routine procedures and plasma standards. Data were evaluated for intra- and inter-laboratory variation, accuracy and possible rFVIIIFc-specific assay discrepancies. For the one-stage assay, mean recovery was 95% to 100% of expected for both Advate(®) and rFVIIIFc at 0.8 IU mL(-1). Intra-laboratory percent coefficient of variance (CV) ranged from 6.3% to 7.8% for Advate(®), and 6.0% to 10.3% for rFVIIIFc. Inter-laboratory CV ranged from 10% for Advate(®) and 16% for rFVIIIFc at 0.8 IU mL(-1), to over 30% at 0.05 IU mL(-1) for both products. For the chromogenic substrate assay, the average FVIII recovery was 107% ± 5% and 124% ± 8% of label potency across the three concentrations of Advate(®) and rFVIIIFc, respectively. Plasma rFVIIIFc levels can be monitored by either the one-stage or the chromogenic substrate assay routinely performed in clinical laboratories without the need for a product-specific rFVIIIFc laboratory standard. Accuracy by the one-stage assay was comparable to that of Advate(®), while marginally higher results may be observed for rFVIIIFc when using the chromogenic assay.

Evolutionary conservation of a microbody targeting signal that targets proteins to peroxisomes, glyoxysomes, and glycosomes.

Peroxisomes, glyoxysomes, glycosomes, and hydrogenosomes have each been classified as microbodies, i.e., subcellular organelles with an electron-dense matrix that is bound by a single membrane. We investigated whether these organelles might share a common evolutionary origin by asking if targeting signals used for translocation of proteins into these microbodies are related. A peroxisomal targeting signal (PTS) consisting of the COOH-terminal tripeptide serine-lysine-leucine-COOH has been identified in a number of peroxisomal proteins (Gould, S.J., G.-A. Keller, N. Hosken, J. Wilkinson, and S. Subramani. 1989. J. Cell Biol. 108:1657-1664). Antibodies raised to a peptide ending in this sequence (SKL-COOH) recognize a number of peroxisomal proteins. Immunocryoelectron microscopy experiments using this anti-SKL antibody revealed the presence of proteins containing the PTS within glyoxysomes of cells from Pichia pastoris, germinating castor bean seeds, and Neurospora crassa, as well as within the glycosomes of Trypanosoma brucei. Western blot analysis of purified organelle fractions revealed the presence of many proteins containing this PTS in both glyoxysomes and glycosomes. These results indicate that at least one of the signals, and therefore the mechanism, for protein translocation into peroxisomes, glyoxysomes, and glycosomes has been conserved, lending support to a common evolutionary origin for these microbodies. Hydrogenosomes, the fourth type of microbody, did not contain proteins that cross-reacted with the anti-PTS antibody, suggesting that this organelle is unrelated to microbodies.

Characterization of an in vitro assay for import of 3-phosphoglycerate kinase into the glycosomes of Trypanosoma brucei.

Glycosomes are microbody organelles found in kinetoplastida, where they serve to compartmentalize the enzymes of the glycolytic pathway. In order to identify the mechanism by which these enzymes are targeted to the glycosome, we have modified the in vitro import assay developed by Dovey et al. (Proc. Natl. Acad. Sci. USA 85:2598-2602, 1988). This assay measures the uptake of in vitro-translated Trypanosoma brucei glycosomal 3-phosphoglycerate kinase (gPGK) by purified glycosomes. Up to 50% of the total 35S-gPGK in the glycosomal fraction was resistant to extraction by 3 M urea or treatment with proteinase K (500 micrograms/ml). The glycosome-associated 35S-gPGK could be chemically cross-linked to the endogenous glycosomal proteins to form a sodium dodecyl sulfate-resistant complex, suggesting that it is close to the intraglycosomal protein matrix. Deoxycholate solubilized the glycosome and thereby rendered the glycosome-associated 35S-gPGK fully susceptible to proteinase K. However, the glycosome-associated 35S-gPGK was not digested by proteinase K in the presence of Triton X-100, which cannot dissolve the glycosomal protein core. The 35S-gPGK synthesized in vitro was able to bind directly to protein cores, where it became resistant to urea extraction and proteinase K digestion. However, the 35S-gPGK-protein core complex exhibited a much higher density than the 35S-gPGK-glycosome complex and was readily separable in sucrose gradients. Thus, in our in vitro import assay, the 35S-gPGK appeared to associate with intact glycosomes, possibly reflecting import of protein into the organelle. Complete denaturation of the 35S-gPGK in 8 M urea prior to the assay enhanced the efficiency of its association with glycosomes. Native gPGK did not compete with the association of in vitro-translated gPGK unless it was denatured. The assay exhibited time and temperature dependence, but it did not require externally added ATP and was not inhibited by the nonhydrolyzable analogs adenosine-5'-(beta,gamma-imido)-triphosphate and gamma-S-ATP. However, the presence of 20 to 30 microM ATP inside the glycosome may fulfill the requirement for protein import.

In vivo import of firefly luciferase into the glycosomes of Trypanosoma brucei and mutational analysis of the C-terminal targeting signal.

The compartmentalization of glycolytic enzymes into specialized organelles, the glycosomes, allows the bloodstream form of Trypanosoma brucei to rely solely on glycolysis for its energy production. The biogenesis of glycosomes in these parasites has been studied intensively as a potential target for chemotherapy. We have adapted the recently developed methods for stable transformation of T. brucei to the in vivo analysis of glycosomal protein import. Firefly luciferase, a peroxisomal protein in the lantern of the insect, was expressed in stable transformants of the procyclic form of T. brucei, where it was found to accumulate inside the glycosomes. Mutational analysis of the peroxisomal targeting signal serine-lysine-leucine (SKL) located at the C-terminus of luciferase showed that replacement of the serine residue (Serine548) with a small neutral amino acid (A, C, G, H, N, P, T) still resulted in an import efficiency of 50-100% of the wild-type luciferase. Lysine549 could be substituted with an amino acid capable of hydrogen bonding (H, M, N, Q, R, S), whereas the C-terminal leucine550 could be replaced with a subset of hydrophobic amino acids (I, M, Y). Thus, a peroxisome-like C-terminal SKL-dependent targeting mechanism may function in T. brucei to import luciferase into the glycosomes. However, a few significant differences exist between the glycosomal targeting signals identified here and the tripeptide sequences that direct proteins to mammalian or yeast peroxisomes.

Pseudoreversion analysis indicates a direct role of cell division genes in polar morphogenesis and differentiation in Caulobacter crescentus.

A pseudoreversion analysis was used to examine the role of cell division genes in polar morphogenesis in Caulobacter crescentus. Extragenic suppressors of temperature sensitive mutations in pleC, a pleiotropic gene required for cell motility, formation of polar phi CbK bacteriophage receptors, and stalk formation, were isolated. These suppressors, which restored motility at 37 degrees C, simultaneously conferred a cold sensitive cell division phenotype and they were mapped to the three new cell division genes divJ, divL and divK. The cold-sensitive mutations in divL, and to a lesser extent divJ, exhibited a relatively narrow range of suppression. The cold-sensitive cell division mutation in divK, by contrast, suppressed all pleC mutations examined and behaved as a classical bypass suppressor. The direct role of this cell division gene in the regulation of motility is suggested by the observation that divK341 mapped to the same locus as pleD301, a pleiotropic mutation that prevents loss of motility and stalk formation. These results provide strong evidence that the cell division and developmental pathways are interconnected and they support our earlier conclusion that cell division is required for the regulation of polar morphogenesis and differentiation in C. crescentus.

Phosphoenolpyruvate carboxykinase of Trypanosoma brucei is targeted to the glycosomes by a C-terminal sequence.

Import of proteins into the glycosomes of T. brucei resembles the peroxisomal protein import in that C-terminal SKL-like tripeptide sequences can function as targeting signals. Many of the glycosomal proteins do not, however, possess such C-terminal tripeptide signals. Among these, phosphoenolpyruvate carboxykinase (PEPCK (ATP)) was thought to be targeted to the glycosomes by an N-terminal or an internal targeting signal. A limited similarity to the N-terminal targeting signal of rat peroxisomal thiolase exists at the N-terminus of T. brucei PEPCK. However, we found that this peroxisomal targeting signal does not function for glycosomal protein import in T. brucei. Further studies of the PEPCK gene revealed that the C-terminus of the predicted protein does not correspond to the previously deduced protein sequence of 472 amino acids due to a -1 frame shift error in the original DNA sequence. Readjusting the reading frame of the sequence results in a predicted protein of 525 amino acids in length ending in a tripeptide serine-arginine-leucine (SRL), which is a potential targeting signal for import into the glycosomes. A fusion protein of firefly luciferase, without its own C-terminal SKL targeting signal, and T. brucei PEPCK is efficiently imported into the glycosomes when expressed in procyclic trypanosomes. Deletion of the C-terminal SRL tripeptide or the last 29 amino acids of PEPCK reduced the import only by about 50%, while a deletion of the last 47 amino acids completely abolished the import. These results suggest that T. brucei PEPCK may contain a second, internal glycosomal targeting signal upstream of the C-terminal SRL sequence.

The C-terminal tripeptide of glycosomal phosphoglycerate kinase is both necessary and sufficient for import into the glycosomes of Trypanosoma brucei.

Glycosomal phosphoglycerate kinase (gPGK) of Trypanosoma brucei differs from the cytoplasmic isozyme (cPGK) in its higher isoelectric point characterized by clusters of positive charges along the polypeptide chain, and a 20 amino acid C-terminal extension ending in serine-serine-leucine (SSL). While a C-terminal SSL tripeptide is apparently not capable of directing luciferase to the peroxisomes in mammalian cells [J. Cell Biol. 108 (1989), 1657-1664], we show here that it is sufficient for the import of luciferase as well as an unrelated protein, beta-glucuronidase, into the glycosomes of T. brucei, as determined by immunoelectron microscopy. The analysis of luciferase-gPGK fusion proteins indicates that the only targeting signal for import of gPGK into the glycosome resides in this C-terminal SSL sequence.

Cloning, expression and characterization of an unusual guanine phosphoribosyltransferase from Giardia lamblia.

Giardia lamblia is one of the most ancient eukaryotes identified to date. It lacks de novo purine biosynthesis and is thought to rely solely on the functions of two salvage enzymes, adenine and guanine phosphoribosyltransferases (APRTase and GPRTase). We have cloned the gene encoding the G. lamblia GPRTase by complementation of the E. coli strain Sø609 (delta gpt-pro-lac, thi, hpt, pup, purH,J, strA) with a genomic library consisting of Sau3AI-digested G. lamblia DNA inserted into the Bluescript vector. Transformed Sø609 colonies grew on minimal medium supplemented with guanine at a frequency of 3.3 x 10(-5) ampicillin-resistant colonies, but were unable to salvage hypoxanthine or xanthine, as predicted from previous studies of the native G. lamblia GPRTase. The sequence analysis of cloned DNA fragments reveals an open reading frame of 690 bp, encoding a protein of 26.3 kDa with an estimated pI of 6.83, in agreement with the reported subunit molecular weight of the native G. lamblia GPRTase. The deduced protein has less than 20% sequence identity to the human and other known HGPRTases, and features several significant changes in the primary sequence of the putative active sites of the enzyme, which may reflect the stringent substrate specificity of GPRTase. The recombinant GPRTase was expressed in E. coli and purified to > 95% homogeneity. Kinetic studies of the recombinant enzyme showed an apparent K(m) of 74 microM for guanine. Hypoxanthine as an alternate purine substrate was used only when present in millimolar amounts, and xanthine was not utilized at all. This Giardia enzyme is thus a highly unique purine PRTase without a known parallel in any other living organisms.

Cloning by functional complementation in Trypanosoma brucei.

A procyclic Trypanosoma brucei double-knockout mutant lacking the ornithine decarboxylase (ODC) gene was transfected with a T. brucei genomic library in the expression vector pTSO-HYG4, which utilizes the PARP promoter and replicates extrachromosomally by virtue of a minicircle origin of replication. Transfectants which grew in the absence of exogenous putrescine, the product of the ODC-catalyzed reaction, were obtained at a frequency of 1.6 x 10(-7) and shown to restore ODC protein synthesis and enzymatic activity. Restriction enzyme patterns and Southern blot analysis of plasmids recovered from these cells and propagated in E. coli showed that the inserts contained a single copy of the T. brucei ODC gene. These results demonstrate for the first time the feasibility of identifying novel T. brucei genes by direct complementation of mutant T. brucei cell lines.

Turning off flagellum rotation requires the pleiotropic gene pleD: pleA, pleC, and pleD define two morphogenic pathways in Caulobacter crescentus.

We have identified mutations in three pleiotropic genes, pleA, pleC, and pleD, that are required for differentiation in Caulobacter crescentus. pleA and pleC mutants were isolated in an extensive screen for strains defective in both motility and adsorption of polar bacteriophage phi CbK; using temperature-sensitive alleles, we determined the time at which the two genes act. pleA was required for a short period at 0.7 of the swarmer cell cycle for flagellum biosynthesis, whereas pleC was required during an overlapping period from 0.6 to 0.95 of the cell cycle to activate flagellum rotation as well as to enable loss of the flagellum and stalk formation by swarmer cells after division. The third pleiotropic gene, pleD, is described here for the first time. A pleD mutation was identified as a bypass suppressor of a temperature-sensitive pleC allele. Strains containing this mutation were highly motile, did not shed the flagellum or form stalks, and retained motility throughout the cell cycle. Since pleD was required to turn off motility and was a bypass suppressor of pleC, we conclude that it acts after the pleA and pleC gene functions in the cell cycle. No mutants defective in both flagellum biosynthesis and stalk formation were identified. Consequently, we propose that the steps required for formation of swarmer cells and subsequent development into stalked cells are organized into at least two developmental pathways: a pleA-dependent sequence of events, responsible for flagellum biosynthesis in predivisional cells, and a pleC-pleD-dependent sequence, responsible for flagellum activation in predivisional cells and loss of motility and stalk formation in progeny swarmer cells.

Sequential regulation of developmental events during polar morphogenesis in Caulobacter crescentus: assembly of pili on swarmer cells requires cell separation.

Pili, along with the flagellum and DNA bacteriophage receptors, are structural markers for polar morphogenesis in Caulobacter crescentus. Pili act as primary receptors for a number of small, C. crescentus-specific DNA and RNA bacteriophages, and the timing of pilus-dependent adsorption of bacteriophage phiCb5 in synchronized cell populations has led to the general conclusion that pili are formed coordinately with the flagellum and other polar surface structures in the predivisional cell. The use of rotary platinum shadow casting and electron microscopy as a direct assay for formation of flagella and pili in synchronous cell cultures now shows, however, that when expressed as fractions of the swarmer cell cycle, flagella are assembled on the predivisional cells at approximately 0.8 and that pili are assembled on the new swarmer cells at approximately 0.1 of the next cell cycle. Adsorption of pilus-specific bacteriophage phiCb5 prevented the loss of pili from swarmer cells during development, which suggests that these structures are retracted at the time of stalk formation. Examination of temperature-sensitive cell division mutants showed that the assembly of pili depends on completion of cell separation. These results indicate that the stage-specific events required for polar morphogenesis in C. crescentus occur sequentially, rather than coordinately in the cell cycle, and that the timing of these events reflects the order of underlying cell cycle steps.

Targeting proteins to the glycosomes of African trypanosomes.

Glycosomes are microbodies found in protozoa belonging to the order Kinetoplastida. These highly specialized organelles compartmentalize most of the glycolytic enzymes normally located in the cytosol of other eukaryotic cells. The recent success in expressing foreign proteins in Trypanosoma brucei has permitted a detailed analysis of glycosomal protein targeting signals in these organisms. These studies have revealed that the previously identified C-terminal tripeptide peroxisomal targeting signal also functions in the import of proteins into the glycosomes of T. brucei. However, the glycosomal and peroxisomal targeting signals differ in a few important ways. The C-terminal tripeptide sequence requirements for glycosomal protein targeting are comparatively relaxed. Of the three C-terminal amino acids, the first can be any small, neutral amino acid; the second should be capable of forming hydrogen bondings, whereas the third is a hydrophobic amino acid. This degenerate tripeptide sequence differs significantly from the more stringent requirements observed for the import of proteins into mammalian peroxisomes and thus represents an opportunity for designing peptide analogues that specifically block the glycosomal protein import for a possible antitrypanosomal chemotherapy. A recently described N-terminal signal that targets thiolase to the mammalian peroxisomes does not appear to function in import into the glycosomes. However, a novel internal targeting signal has tentatively been identified in at least one of the glycosomal proteins that can target a reporter protein to the glycosomes of T. brucei. Glycosome-deficient mutants have been isolated recently, which will aid in the identification of genes involved in the biogenesis of the glycosome.

A histidine protein kinase homologue required for regulation of bacterial cell division and differentiation.

Differentiation in the dimorphic bacterium Caulobacter crescentus results from a sequence of discontinuous, stage-specific events that leads to the production of a stalked cell and a new motile swarmer cell after each asymmetric cell division. As reported previously, pseudoreversion analysis of mutations in the pleiotropic developmental gene pleC identified three cell division genes: divJ, divK, and divL. We show here that one of these genes, divJ, encodes a predicted protein of 596 residues with an extensive hydrophobic N-terminal region and a C-terminal domain containing all of the invariant residues found in the family of bacterial histidine protein kinases. Our results also show that divJ is discontinuously transcribed early in the swarmer cell cycle during a period that coincides with the G1 to S transition. We propose that the DivJ protein is one member of a signal transduction pathway regulating the cell cycle and differentiation in Caulobacter and that protein modification by phosphorylation may play a central role in coupling developmental events to progress through the cell division cycle.

An essential single domain response regulator required for normal cell division and differentiation in Caulobacter crescentus.

Signal transduction pathways mediated by sensor histidine kinases and cognate response regulators control a variety of physiological processes in response to environmental conditions. Here we show that in Caulobacter crescentus these systems also play essential roles in the regulation of polar morphogenesis and cell division. Previous studies have implicated histidine kinase genes pleC and divJ in the regulation of these developmental events. We now report that divK encodes an essential, cell cycle-regulated homolog of the CheY/Spo0F subfamily and present evidence that this protein is a cognate response regulator of the histidine kinase PleC. The purified kinase domain of PleC, like that of DivJ, can serve as an efficient phosphodonor to DivK and as a phospho-DivK phosphatase. Based on these and earlier genetic results we propose that PleC and DivK are members of a signal transduction pathway that couples motility and stalk formation to completion of a late cell division cycle event. Gene disruption experiments and the filamentous phenotype of the conditional divK341 mutant reveal that DivK also functions in an essential signal transduction pathway required for cell division, apparently in response to another histidine kinase. We suggest that phosphotransfer mediated by these two-component signal transduction systems may represent a general mechanism regulating cell differentiation and cell division in response to successive cell cycle checkpoints.

Mouse ornithine decarboxylase is stable in Trypanosoma brucei.

The cDNA encoding mouse ornithine decarboxylase (ODC) was incorporated into a transforming vector pTSA-NEO2 carrying a procyclic acidic repetitive protein promoter and a neomycin phosphotransferase gene. The plasmid thus constructed, pMOD300, was introduced into the procyclic forms of Trypanosoma brucei via electroporation, and the transformants, selected under G418, expressed an ODC activity 100 times above the background level. Contrary to the commonly observed short half-life of mouse ODC in mammalian cells, however, the mouse ODC activity expressed in T. brucei remained stable for at least 6 h when protein synthesis was inhibited by cycloheximide. Pulse labelings and chase experiments with the irreversible ODC inhibitor [3,4-3H]difluoromethylornithine followed by gel electrophoresis, or with L-[35S] methionine followed by immunoprecipitation and gel electrophoresis indicated that the stable mouse ODC expressed in T. brucei has the same subunit molecular weight as the native enzyme. By an in vitro assay of protein stability in rabbit reticulocyte lysates (Loetscher, P., Pratt, G., and Rechsteiner, M. (1991) J. Biol. Chem. 266, 11213-11220), the native mouse ODC and the enzyme expressed in T. brucei had the same degree of instability. Thus, the mouse ODC expressed in T. brucei is probably identical to the native mouse ODC. Its remarkable stability in T. brucei must be due to the absence in trypanosomes of the proteolytic machinery present in mammalian cells responsible for rapid degradation of mouse ODC.

Trypanosoma brucei: identification of an internal region of phosphoglycerate kinase required for targeting to glycosomal microbodies.

Among the microbodies found in eukaryotes are the glycosomes of Trypanosoma brucei, thought to be closely related to peroxisomes. Two types of targeting signals for glycosomes have been identified thus far: type 1 at the C-terminus and type 2 at the N-terminus. In this report, we use an epitope-tagging system to characterize the targeting signal found on the minor glycosomal isozyme of phosphoglycerate kinase, 56PGK. No type 1 or 2 signal was found; rather, the topogenic information was found to be internal. Chimeric molecules formed with the cytoplasmic phosphoglycerate kinase isozyme indicate that a region between amino acids 24 and 91 of 56PGK is essential for glycosomal targeting. No homology was found between this region and peroxisomal proteins containing internal targeting signals.