Differential prolyl hydroxylation by six Physcomitrella prolyl-4 hydroxylases.

Hydroxylation of prolines to 4-trans-hydroxyproline (Hyp) is mediated by prolyl-4 hydroxylases (P4Hs). In plants, Hyps occur in Hydroxyproline-rich glycoproteins (HRGPs), and are frequently O-glycosylated. While both modifications are important, e.g. for cell wall stability, they are undesired in plant-made pharmaceuticals. Sequence motifs for prolyl-hydroxylation were proposed but did not include data from mosses, such as Physcomitrella. We identified six moss P4Hs by phylogenetic reconstruction. Our analysis of 73 Hyps in 24 secretory proteins from multiple mass spectrometry datasets revealed that prolines near other prolines, alanine, serine, threonine and valine were preferentially hydroxylated. About 95 % of Hyps were predictable with combined established methods. In our data, AOV was the most frequent pattern. A combination of 443 AlphaFold models and MS data with 3000 prolines found Hyps mainly on protein surfaces in disordered regions. Moss-produced human erythropoietin (EPO) exhibited O-glycosylation with arabinose chains on two Hyps. This modification was significantly reduced in a p4h1 knock-out (KO) Physcomitrella mutant. Quantitative proteomics with different p4h mutants revealed specific changes in protein amounts, and a modified prolyl-hydroxylation pattern, suggesting a differential function of the Physcomitrella P4Hs. Quantitative RT-PCR revealed a differential effect of single p4h KOs on the expression of the other five p4h genes, suggesting a partial compensation of the mutation. AlphaFold-Multimer models for Physcomitrella P4H1 and its target EPO peptide superposed with the crystal structure of Chlamydomonas P4H1 suggested significant amino acids in the active centre of the enzyme and revealed differences between P4H1 and the other Physcomitrella P4Hs.

The Impact of the SARS-CoV-2 Pandemic on Stress and Anxiety of Dental Students.

OBJECTIVES: To investigate the impact of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) pandemic on stress and anxiety of preclinical and clinical dental students. MATERIALS AND METHODS: Dental students (participants) in their clinical course (CC; n = 64) or preclinical course (PCC; n = 53) were included in the study. The subjective perceived levels of stress and anxiety were evaluated using the Dental Environment Stress (DES) questionnaire and the Depression, Anxiety and Stress Scales (DASS) questionnaire. Cortisol levels were measured in saliva samples collected from participants. Knowledge of the pandemic was evaluated using a 100-mm visual analog scale. All data were collected twice: once during the university holidays and once during term time. STATISTICAL ANALYSIS: Results from DES, DASS, and salivary cortisol tests were compared between baseline and follow-up using descriptive and bivariate statistics. Multivariate linear regression models were computed with DES, DASS, and cortisol values as dependent variables to analyze possible influencing factors. RESULTS: Participants showed medium levels of stress and anxiety at baseline and follow-up. The DASS score in the "anxiety" subdomain was significantly higher in the PCC group than in the CC group at baseline (p < 0.001) and increased during term time. DASS scores in the "stress" subdomain also increased during term time. However, both subdomain scores were lower than the cutoff value for a psychological disorder. The mean total DES scores were 615.9 ± 97.7 in the CC group and 580.40 ± 98.9 in the PCC group. These scores indicated medium stress levels and were not significantly different between the groups, nor did they change during the study period. Mean saliva cortisol levels were higher in the CC group (9.2 ± 5.2) than in the PCC group (4.9 ± 2.2) at baseline (p < 0.001) but converged by follow-up. Multivariate regression models showed that intraindividual perception of stress at baseline was consistently the most important aspect for changes in stress and anxiety levels during term time. The SARS-CoV-2 pandemic affected stress and anxiety levels in some participants, but this was not ubiquitous. CONCLUSION: Intraindividual differences in stress perception seem to be more relevant than course affiliation (preclinical or clinical) or the SARS-CoV-2 pandemic to stress and anxiety levels in dental students.

Adapting the German version of the dental environment stress questionnaire.

AIMS: To develop a German version of the Dental Environment Stress (DES) questionnaire, and to evaluate its reliability and validity. METHODS: The original English DES questionnaire was translated into German using a forward-backward translation process. To evaluate construct stability, a subgroup of dental students (n = 43) completed the DES twice (interval: 1 week). To evaluate how the DES responds to anticipated changes in stress, all dental students' (n = 64) DES scores and saliva cortisol levels administered from a clinical study were compared between holiday and term time. Furthermore, the Depression, Anxiety and Stress Scale (DASS) and a stress self-assessment were used to determine the validity of the DES. Reliability analyses were calculated using Kendall's tau correlations. To estimate reliability strength, correlation coefficients and intraclass correlations (ICCs) were used. RESULTS: Regarding construct stability, 24 of 25 DES items had at least moderate correlations, and most items showed strong correlations. Correlations for the seven subdomains were good (range: 0.778-1.000). The same was true for the total DES score (ICC: 0.944). Correlations for response to term-time stress were weaker and more varied. Validity analyses revealed fair correlations between the DES and students' self-assessment (Pearson's r = .592) and DASS score (Pearson's r = .392), suggesting satisfactory validity. Stress levels were quite similar between baseline and follow-up. CONCLUSION: The German DES is a reliable tool for evaluating stress in dental students. Because it can be used to identify individual stressors in various categories, it might enable the detection of specific stress situations in educational situations and facilitate solutions (adjustment of curricula, tailored consulting services).

Trafficking of PfExp1 to the parasitophorous vacuolar membrane of Plasmodium falciparum is independent of protein folding and the PTEX translocon.

Having entered the mature human erythrocyte, the malaria parasite survives and propagates within a parasitophorous vacuole, a membrane-bound compartment separating the parasite from the host cell cytosol. The bounding membrane of this vacuole, referred to as the parasitophorous vacuolar membrane (PVM), contains parasite-encoded proteins, but how these membrane proteins are trafficked to the PVM remains unknown. Here, we have studied the trafficking of PfExp1 to the PVM. We find that trafficking of PfExp1 to the PVM is independent of the folding state of the protein and also continues unabated upon inactivation of the PVM translocon Plasmodium Translocon of Exported proteins (PTEX). Our data strongly suggest that the trafficking of membrane proteins to the PVM occurs by as yet unknown mechanism, potentially unique to Plasmodium.

Acute pulmonary edema following liposuction due to heart failure and atypical pneumonia.

Microcannular liposuction in tumescent anesthesia is the most effective treatment for painful lipedema. Tumescent anesthesia is an established and safe procedure in local analgesia when performed according to guidelines. Major adverse effects are rare. In patients with advanced lipedema, however, the commonly presented comorbidities bear additional risks.We report on post-surgical acute pulmonary edema after tumescent liposuction according to guidelines in a 52-year-old female patient with lipedema of the legs. We discuss in detail possible scenarios that might be involved in such emergency. In the present case the most likely was a retarded community acquired atypical pneumonia with aggravation of pre-existent comorbidities.A combined treatment with intravenous b-lactam antibiosis, positive pressure ventilation, and continuous venovenous hemodialysis and filtration resulted in complete remission in a couple of days. In conclusion, tumescent liposuction of advanced lipedema patients should only be performed in well-trained centers with sufficient infrastructure.

Desmoteplase in acute massive pulmonary thromboembolism.

Alteplase is standard therapy for patients with acute, massive pulmonary embolism. The novel plasminogen activator desmoteplase displays high fibrin specificity and selectivity for fibrinbound plasminogen. In a preclinical model desmoteplase was twice as potent with a shorter lysis time and lower reocclusion rate. We conducted a phase II study comparing 125, 180, and 250 microg/kg bodyweight desmoteplase with 100 mg alteplase. Efficacy criteria were total pulmonary resistance (TPR), mean pulmonary artery pressure (mPAP), and Miller Index. Intention to treat analysis of 34 patients. The reduction of TPR after 24 hours was comparable between desmoteplase 180 microg/kg and alteplase (-48.0 +/- 22.4 vs. -50.4 +/- 16.3%; p = n.s. vs. alteplase; p = 0.0002 and p<0.0001 vs. baseline). The greatest effect was achieved with desmoteplase 250 microg/kg (-56.0 +/- 29.4%; p = n.s. vs. alteplase, p = 0.0055 vs. baseline). Two hours after treatment PAP was reduced by 27.9 (p = 0.0004 vs. baseline) and 30.4% (p = 0.015 vs. baseline) with the higher doses of desmoteplase and 29.6% with alteplase (p = 0.0006 vs. baseline). Further PAP reduction after 6 hours was most pronounced in the desmoteplase 250 microg/kg group (-40.1 +/- 18.0%; p = 0.0028 vs. baseline). The reduction of the Miller Index was greatest using desmoteplase 250 microg/kg (-35.0 +/- 21.7%; p = 0.011 vs. baseline), and alteplase (-41.6 +/- 27.2%; p = 0.0003 vs. baseline). Safety did not differ among the 4 groups. The study results suggest that desmoteplase at doses of 180 and 250 microg/kg had similar or greater efficacy compared to alteplase 100 mg. Onset of action was faster, safety was comparable.