Development of a project for interprofessional collaboration between medical and pharmacy students to improve medication safety in polypharmacy (PILLE).

Aim: Interprofessional collaboration is particularly relevant to patient safety in outpatient care with polypharmacy. The educational project "PILLE" is meant to give medical and pharmacy students an understanding of the roles and competencies needed for cooperation in the provision of healthcare and to enable interprofessional learning. Method: The curriculum is aimed at pharmacy and medical students and was developed in six steps according to the Kern cycle. It is comprised of an interprofessional seminar, a joint practical training in a simulated pharmacy, and a tandem job shadowing at a primary care practice. The project was implemented in three stages due to the pandemic: The interprofessional online seminar based on the ICAP model and the digital inverted classroom was held in the 2020 winter semester; the interprofessional practical training was added in the 2021 summer semester; and the interprofessional tandem job shadowing at a primary care practice in the 2021 winter semester. Attitudes toward interprofessional learning, among other things, was measured in the evaluation using the SPICE-2D questionnaire (Student Perceptions of Physician-Pharmacist Interprofessional Clinical Education). Results: In the first three semesters, a total of 105 students (46 pharmacy, 59 medicine) participated in the project, of which 78 participated in the evaluation (74% response rate). The students stated, in particular, that they had learned about the competencies and roles of the other profession and desired additional and more specific preparatory materials for the course sessions. The SPICE-2D questionnaire showed high values for both groups of students already in the pre-survey and these increased further as a result of the project. Conclusion: Joint case-based learning could be implemented under the conditions imposed by the pandemic. Online teaching is a low-threshold means to enable interprofessional exchange.

Does the degree of osteoarthritis influence the clinical outcome after anatomic total shoulder arthroplasty?

BACKGROUND: The degree of preoperative osteoarthritis has been shown to influence the postoperative outcome and the patients' satisfaction rate in hip and knee joint replacement surgery. However, no corresponding information is available for total shoulder arthroplasty (TSA). We therefore set out to evaluate the influence of preoperatively measured end-stage osteoarthritis on the postoperative clinical outcome of TSA. METHODS: A retrospective analysis of 103 anatomic total shoulder replacements (96 patients) was performed. Patients were evaluated radiologically with X-rays in two planes and clinically using the Constant and Murley score (CS) and the self-reported satisfaction with the result. The degree of osteoarthritis was radiographically analyzed with the aid of the classifications according to Kellgren/Lawrence, Gerber, Guyette, and Allain and according to whether complete narrowing of the glenohumeral joint was present or not [bone-on-bone contact (BOB) or no bone-on-bone contact (No BOB)]. RESULTS: The clinical results of TSA did not differ significantly among the various stages of osteoarthritis in any of the classifications (p > 0.05). The CS was significantly higher postoperatively for both the BOB and the No BOB group (p < 0.0001). Patients with BOB had a significantly lower CS preoperatively than patients with No BOB (p = 0.0172). In addition, the preoperative pain level was significantly higher in patients with BOB (p = 0.014). Postoperatively, no significant difference in CS (p = 0.6738) was found between the BOB group and the No BOB group. The mean improvement in CS was not statistically significant (p = 0.2218). CONCLUSION: In contrast to hip and knee joint replacement procedures, a milder grade of osteoarthritis does not adversely influence the functional result or subjective satisfaction rate after TSA. The degree of osteoarthritis on conventional X-rays has no bearing on the postoperative clinical outcome. Therefore, the decision on when to carry out anatomic total shoulder arthroplasty should depend on the patient's pain level and loss of quality of life.

The RNA-Protein Interactome of Differentiated Kidney Tubular Epithelial Cells.

BACKGROUND: RNA-binding proteins (RBPs) are fundamental regulators of cellular biology that affect all steps in the generation and processing of RNA molecules. Recent evidence suggests that regulation of RBPs that modulate both RNA stability and translation may have a profound effect on the proteome. However, regulation of RBPs in clinically relevant experimental conditions has not been studied systematically. METHODS: We used RNA interactome capture, a method for the global identification of RBPs to characterize the global RNA-binding proteome (RBPome) associated with polyA-tailed RNA species in murine ciliated epithelial cells of the inner medullary collecting duct. To study regulation of RBPs in a clinically relevant condition, we analyzed hypoxia-associated changes of the RBPome. RESULTS: We identified >1000 RBPs that had been previously found using other systems. In addition, we found a number of novel RBPs not identified by previous screens using mouse or human cells, suggesting that these proteins may be specific RBPs in differentiated kidney epithelial cells. We also found quantitative differences in RBP-binding to mRNA that were associated with hypoxia versus normoxia. CONCLUSIONS: These findings demonstrate the regulation of RBPs through environmental stimuli and provide insight into the biology of hypoxia-response signaling in epithelial cells in the kidney. A repository of the RBPome and proteome in kidney tubular epithelial cells, derived from our findings, is freely accessible online, and may contribute to a better understanding of the role of RNA-protein interactions in kidney tubular epithelial cells, including the response of these cells to hypoxia.

Structural analysis of coniferyl alcohol 9-O-methyltransferase from Linum nodiflorum reveals a novel active-site environment.

Coniferyl alcohol 9-O-methyltransferase from Linum nodiflorum (Linaceae) catalyzes the unusual methylation of the side-chain hydroxyl group of coniferyl alcohol. The protein was heterologously expressed in Escherichia coli as a hexahistidine derivative and purified for crystallization. Diffracting crystals were obtained of the pure protein and of its selenomethionine derivative, as well as of complexes with coniferyl alcohol and with S-adenosyl-L-homocysteine together with coniferyl alcohol 9-O-methyl ether (PDB entries 4ems, 4e70 and 4evi, respectively). The X-ray structures show that the phenylpropanoid binding mode differs from other phenylpropanoid O-methyltransferases such as caffeic acid O-methyltransferase. Moreover, the active site lacks the usually conserved and catalytic histidine residue and thus implies a different reaction mode for methylation. Site-directed mutagenesis was carried out to identify critical amino acids. The binding order of coniferyl alcohol and S-adenosyl-L-methionine was investigated by isothermal titration calorimetry experiments.

Methyl allyl ether formation in plants: novel S-adenosyl L-methionine:coniferyl alcohol 9-O-methyltransferase from suspension cultures of three Linum species.

A novel 41 kDa methyltransferase displaying high regiospecificity towards the allylic hydroxyl moiety of coniferyl alcohol was cloned from suspension cultures of Linum nodiflorum L. and expressed in E. coli. The apparent K (m) for coniferyl alcohol is 7.23 microM with a V (max) of 707.5 pkat mg(-1) protein at 30 degrees C, whereas the K (m) for the co-substrate S-adenosyl-L-methionine is 18.5 microM. Structure-function relationship studies revealed stringent structure requirements. Even minor substructure deviations as the side-chain saturation or changes in the phenyl ring substitution result in activities decreased by 75-90%. Crotyl and allyl alcohols are not substrates, confirming that the aromatic ring itself is indispensable, and solely the derivatives with a C(3) side-chain are accepted. The enzyme shares only similarities under 46% on amino acid level with other known methyltransferases. The designated reaction product, coniferyl alcohol 9-methyl ether, could be detected in suspension cells. The highest content of up to 0.02% of the dry mass is concurrent with an increase of the specific enzyme activity that reaches its maximum of 3.94 pkat mg(-1) on day 6 of the culture period. Transcript levels estimated by semi-quantitative RT-PCR remain constant until day 6 and recede thereafter. The corresponding methyltransferase from Linum flavum L. differs mainly by one short variable fragment. Biochemical characterization revealed a higher catalytic efficiency and a slightly broader substrate plasticity together with a lower sensitivity to the presence of Zn(2+), Cu(2+) and Co(2+). This is to our knowledge the first report of a regiospecific allylic O-methylation of phenylpropanoids in plants.

Catalytic activity, duplication and evolution of the CYP98 cytochrome P450 family in wheat.

A burst of evolutionary duplication upon land colonization seems to have led to the large superfamily of cytochromes P450 in higher plants. Within this superfamily some clans and families are heavily duplicated. Others, such as genes involved in the phenylpropanoid pathway have led to fewer duplication events. Eight coding sequences belonging to the CYP98 family reported to catalyze the 3-hydroxylation step in this pathway were isolated from Triticum aestivum (wheat) and expressed in yeast. Comparison of the catalytic properties of the recombinant enzymes with those of CYP98s from other plant taxa was coupled to phylogenetic analyses. Our results indicate that the unusually high frequency of gene duplication in the wheat CYP98 family is a direct or indirect result from ploidization. While ancient duplication led to evolution of enzymes with different substrate preferences, most of recent duplicates underwent silencing via degenerative mutations. Three of the eight tested CYP98s from wheat have phenol meta-hydroxylase activity, with p-coumaroylshikimate being the primary substrate for all of these, as it is the case for CYP98s from sweet basil and Arabidopsis thaliana. However, CYP98s from divergent taxa have acquired different additional subsidiary activities. Some of them might be significant in the metabolism of various free or conjugated phenolics in different plant species. One of the most significant is meta-hydroxylation of p-coumaroyltyramine, predominantly by the wheat enzymes, for the synthesis of suberin phenolic monomers. Homology modeling, confirmed by directed mutagenesis, provides information on the protein regions and structural features important for some observed changes in substrate selectivity. They indicate that the metabolism of quinate ester and tyramine amide of p-coumaric acid rely on the same recognition site in the protein.

Beta-peltatin 6-O-methyltransferase from suspension cultures of Linum nodiflorum.

S-Adenosyl-L-methionine:beta-peltatin 6-O-methyltransferase was isolated and characterized from cell suspension cultures of Linum nodiflorum L. (Linaceae), a Linum species accumulating aryltetralin lignans such as 6-methoxypodophyllotoxin. The enzyme transfers a methyl group from S-adenosyl-L-methionine to the only free OH-group of beta-peltatin in position 6 thus forming beta-peltatin-A methylether. This reaction is a putative biosynthetic step in the biosynthesis of 6-methoxypodophyllotoxin from deoxypodophyllotoxin. The enzyme has a pH-optimum at pH 7.7 and a temperature optimum at 40 degrees C. The enzyme activity is strongly inhibited by MnSO(4), FeCl(3), FeSO(4) and ZnSO(4) as well as S-adenosyl-homocysteine. Mg(2+) and EDTA did not influence the methylation of beta-peltatin. Substrate saturation curves were obtained for S-adenosyl-methionine and beta-peltatin and apparent K(m)-values of 15 microM and 40 microM, respectively, were determined for these substrates. Substrate inhibition was observed for beta-peltatin. No other lignan substrate tested nor caffeic acid were accepted. The suspension cell line of Linum nodiflorum was characterized with respect to growth, medium alterations and lignan production as well as activity of SAM:beta-peltatin 6-O-methyltransferase. Highest specific activities of beta-peltatin 6-O-methyltransferase were determined on day 7 of the culture period corresponding to the highest levels of 6-methoxypodophyllotoxin on days 7 to 12.