Glycoprotein biosynthesis in Saccharomyces cerevisiae: ngd29, an N-glycosylation mutant allelic to och1 having a defect in the initiation of outer chain formation

AbstractOuter chain glycosylation in Saccharomyces cerevisiae leads to heterogeneous and immunogenic asparagine-linked saccharide chains containing more than 50 mannose residues on secreted glycoproteins. Using a [3H]mannose suicide selection procedure a collection of N-glycosylation defective mutants (designated ngd) was isolated. One mutant, ngd29, was found to have a defect in the initiation of the outer chain and displayed a temperature growth sensitivity at 37°C allowing the isolation of the corresponding gene by complementation. Cloning, sequencing and disruption of NGD29 showed that it is a non lethal gene and identical to OCH1. It complemented both the glycosylation and growth defect. Membranes isolated from an ngd29 disruptant or an ngd29mnn1 double mutant were no longer able, in contrast to membranes from wild type cells, to transfer mannose from GDPmannose to Man8GlcNAc2, the in vivo acceptor for building up the outer chain. Heterologous expression of glucose oxidase from Aspergillus niger in an ngd29mnn1 double mutant produced a secreted uniform glycoprotein with exclusively Man8GlcNAc2 structure that in wild type yeast is heavily hyperglycosylated. The data indicate that this mutant strain is a suitable host for the expression of recombinant glycoproteins from different origin in S. cerevisiae to obtain mammalian oligomannosidic type N-linked carbohydrate chains

Final results of a phase I/II pilot study of capecitabine with or without vinorelbine after sequential dose-dense epirubicin and paclitaxel in high-risk early breast cancer

Background: The integration of the non-cross-resistant chemotherapeutic agents capecitabine and vinorelbine into an intensified dose-dense sequential anthracycline- and taxane-containing regimen in high-risk early breast cancer (EBC) could improve efficacy, but this combination was not examined in this context so far. Methods: Patients with stage II/IIIA EBC (four or more positive lymph nodes) received post-operative intensified dose-dense sequential epirubicin (150mg/m2 every 2 weeks) and paclitaxel (225mg/m2 every 2 weeks) with filgrastim and darbepoetin alfa, followed by capecitabine alone (dose levels 1 and 3) or with vinorelbine (dose levels 2 and 4). Capecitabine was given on days 1-14 every 21 days at 1000 or 1250 mg/m2 twice daily (dose levels 1/2 and 3/4, respectively). Vinorelbine 25 mg/m2 was given on days 1 and 8 of each 21-day course (dose levels 2 and 4). Results: Fifty-one patients were treated. There was one dose-limiting toxicity (DLT) at dose level 1. At dose level 2 (capecitabine and vinorelbine), five of 10 patients experienced DLTs. Therefore evaluation of vinorelbine was abandoned and dose level 3 (capecitabine monotherapy) was expanded. Hand-foot syndrome and diarrhoea were dose limiting with capecitabine 1250 mg/m2 twice daily. At 35.2 months' median follow-up, the estimated 3-year relapse-free and overall survival rates were 82% and 91%, respectively. Administration of capecitabine monotherapy after sequential dose-dense epirubicin and paclitaxel is feasible in node-positive EBC, while the combination of capecitabine and vinorelbine as used here caused more DLTs. Trial registration: Current Controlled Trials ISRCTN38983527

Carboxypeptidase E Modulates Intestinal Immune Homeostasis and Protects against Experimental Colitis in Mice

<div><p>Enteroendocrine cells (EEC) produce neuropeptides, which are crucially involved in the maintenance of the intestinal barrier. Hence, EEC dysfunction is suggested to be involved in the complex pathophysiology of inflammatory bowel disease (IBD), which is characterized by decreased intestinal barrier function. However, the underlying mechanisms for EEC dysfunction are not clear and suitable models for a better understanding are lacking. Here, we demonstrate that Carboxypeptidase E (CPE) is specifically expressed in EEC of the murine colon and ileum and that its deficiency is associated with reduced intestinal levels of Neuropeptide Y (NPY) and Peptide YY (PYY), which are both produced by EEC. Moreover, <i>cpe^−/−</i> mice exhibit an aggravated course of DSS-induced chronic colitis compared to wildtype littermates. In addition, we observed elevated mucosal IL-6 and KC transcript levels already at baseline conditions in <i>cpe^−/−</i> mice. Moreover, supernatants obtained from isolated intestinal crypts of <i>cpe^−/−</i> mice lead to increased IL-6 and KC expression in MODE-K cells in the presence of LPS. This effect was reversible by co-administration of recombinant NPY, suggesting a CPE mediated immunosuppressive effect in the intestines by influencing the processing of specific neuropeptides. In this context, the chemotaxis of bone marrow derived macrophages towards respective supernatants was enhanced. In conclusion, our data point to an anti-inflammatory role of CPE in the intestine by influencing local cytokine levels and thus regulating the migration of myeloid immune cells into the mucosa. These findings highlight the importance of EEC for intestinal homeostasis and propose EEC as potential therapeutic targets in IBD.</p></div

The Greifswald Post COVID Rehabilitation Study and Research (PoCoRe)–Study Design, Characteristics and Evaluation Tools

(1) Background: COVID-19 is often associated with significant long-term symptoms and disability, i.e., the long/post-COVID syndrome (PCS). Even after presumably mild COVID-19 infections, an increasing number of patients seek medical help for these long-term sequelae, which can affect various organ systems. The pathogenesis of PCS is not yet understood. Therapy has so far been limited to symptomatic treatment. The Greifswald Post COVID Rehabilitation Study (PoCoRe) aims to follow and deeply phenotype outpatients with PCS in the long term, taking a holistic and comprehensive approach to the analysis of their symptoms, signs and biomarkers. (2) Methods: Post-COVID outpatients are screened for symptoms in different organ systems with a standardized medical history, clinical examination, various questionnaires as well as physical and cardiopulmonary function tests. In addition, biomaterials are collected for the analysis of immunomodulators, cytokines, chemokines, proteome patterns as well as specific (auto)antibodies. Patients are treated according to their individual needs, adhering to the current standard of care. PoCoRe’s overall aim is to optimize diagnostics and therapy in PCS patients

CPE deficiency aggravates experimental chronic colitis.

<p>(A) Calculation of the disease activity index (DAI) by determining clinical parameters of inflammation (body weight development, stool consistency, rectal bleeding) through 30 days of experimental colitis. n = 9 (<i>cpe</i>^+/+), n = 8 (<i>cpe</i>^−/−). (B-C) Determination of macroscopic colitis severity via mouse endoscopy. Representative endoluminal pictures of the distal colon on day 30 of experimental colitis (B) and calculation of the murine endoscopic index of colitis severity (MEICS) by analyzing mucosal morphology, stool consistency and shape of the vascular pattern via mouse endoscopy (C). n = 9 (<i>cpe</i>^+/+), n = 8 (<i>cpe</i>^−/−). (D–E) Determination of microscopic colitis severity via histology. Representative histological pictures of the distal colon on day 30 of experimental colitis (D) and calculation of the histology score by analyzing mucosal architecture and infiltration of immune cells (E). n = 8 per genotype. (F) Determination of expression level of TNF-α, IL-6 and KC in colonic punch biopsies by real time RT-PCR after 30 days of experimental colitis and at baseline. n = 8 per genotype. *p<0.05, **p<0.01, ***p<0.001 by t-test.</p

Proinflammatory properties of colonic crypt supernatants of CPE-deficient mice.

<p>(A) Experimental set-up for the acquirement and utilization of forskolin-stimulated supernatants of isolated colonic crypts. (B–C) Determination of KC (B) and IL-6 (C) transcript levels produced in MODE-K cells after incubation with LPS (50 ng/ml) and forskolin-stimulated supernatants of <i>cpe</i>^+/+ and <i>cpe</i>^−/− colonic crypts via RT-PCR. (D). KC transcript levels produced in MODE-K cells after incubation with forskolin-stimulated supernatants of of <i>cpe</i>^+/+ and <i>cpe</i>^−/− mice and LPS together with recombinant NPY +/− PYY (1 µM/ml). KC expression levels are expressed in percent of the Mean of <i>cpe^+/+</i>. (E–F) BMDM migration via Boyden chamber assay. Representative pictures of migrated BMDM (E) and quantification (F) of BMDM migration towards supernatants of forskolin-stimulated colonic crypts of <i>cpe</i>^+/+ and <i>cpe</i>^−/− mice. *p<0.05, **p<0.01, ***p<0.001 by t-test.</p

Table_1_Effects of intermittent fasting on quality of life tolerance of chemotherapy in patients with gynecological cancers: study protocol of a randomized-controlled multi-center trial.docx

Fatigue is a very common side effect during intravenous chemotherapy. Unfortunately, only few effective therapeutic options are available, mostly based on daily activity. In our pilot trial we were able to demonstrate that intermittent fasting can reduce fatigue in healthy people, thus we aimed to assess the effects of the fasting dietary on quality of life during chemotherapy in patients with gynecological cancer, especially on the domain of fatigue. The IFAST trial is designed as a prospective, randomized-controlled, multi-center trial. Participation will be offered to women with gynecological cancers (breast cancer, ovarian cancer including peritoneal and fallopian tube cancers, endometrial cancer and cervical cancer) who are planned to receive intravenous chemotherapy for at least three months. Eligible patients will be randomized 1:1, stratified by tumor type and study center. Primary endpoint is the difference in mean change in fatigue, assessed with the Functional Assessment of Chronic Illness Therapy-Fatigue Scale (FACIT- FS©). Exploratory secondary endpoints will include general Quality of Life impairment, tolerance of chemotherapy, immunological changes, peripheral cell damage in blood cells, as well as tumor response to chemotherapy. There is new evidence that prolonged fasting periods of 46-96 hours during chemotherapy can positively influence the quality of life during chemotherapy. However, these fasting regiments are not feasible for many patients. Intermittent fasting could be a feasible (manageable) option for many patients to actively improve their quality of life and tolerance to chemotherapy and possibly even enhance the effectiveness of chemotherapy.Trial Registrationhttps://drks.de, identifier DRKS00031429.</p