Significant improvement of bone marrow-derived MSC expansion from MDS patients by defined xeno-free medium.

BACKGROUND: Robust and reliable in vitro and in vivo models of primary cells are necessary to study the pathomechanisms of Myelodysplastic Neoplasms (MDS) and identify novel therapeutic strategies. MDS-derived hematopoietic stem and progenitor cells (HSPCs) are reliant on the support of bone marrow (BM) derived mesenchymal stroma cells (MSCs). Therefore, isolation and expansion of MCSs are essential for successfully modeling this disease. For the clinical use of healthy MSCs isolated from human BM, umbilical cord blood or adipose tissue, several studies showed that xeno-free (XF) culture conditions resulted in superior growth kinetics compared to MSCs cultured in the presence of fetal bovine serum (FBS). In this present study, we investigate, whether the replacement of a commercially available MSC expansion medium containing FBS with a XF medium is beneficial for the expansion of MSCs derived from BM of MDS patients which are often difficult to cultivate. METHODS: MSCs isolated from BM of MDS patients were cultured and expanded in MSC expansion medium with FBS or XF supplement. Subsequently, the impact of culture media on growth kinetics, morphology, immunophenotype, clonogenic potential, differentiation capacity, gene expression profiles and ability to engraft in immunodeficient mouse models was evaluated. RESULTS: Significant higher cell numbers with an increase in clonogenic potential were observed during culture of MDS MSCs with XF medium compared to medium containing FBS. Differential gene expression showed an increase in transcripts associated with MSC stemness after expansion with XF. Furthermore, immunophenotypes of the MSCs and their ability to differentiate into osteoblasts, adipocytes or chondroblasts remained stable. MSCs expanded with XF media were similarly supportive for creating MDS xenografts in vivo as MSCs expanded with FBS. CONCLUSION: Our data indicate that with XF media, higher cell numbers of MDS MSCs can be obtained with overall improved characteristics in in vitro and in vivo experimental models.

Replication stress signaling is a therapeutic target in myelodysplastic syndromes with splicing factor mutations.

Somatic mutations in genes coding for splicing factors, e.g. SF3B1, U2AF1, SRSF2, and others are found in approximately 50% of patients with Myelodysplastic Syndromes (MDS). These mutations have been predicted to frequently occur early in the mutational hierarchy of the disease therefore making them particularly attractive potential therapeutic targets. Recent studies in cell lines engineered to carry splicing factor mutations have revealed a strong association with elevated levels of DNA:RNA intermediates (R-loops) and a dependency on proper ATR function. However, data confirming this hypothesis in a representative cohort of primary MDS patient samples have so far been missing. Using CD34+ cells isolated from MDS patients with and without splicing factor mutations as well as healthy controls we show that splicing factor mutation-associated R-loops lead to elevated levels of replication stress and ATR pathway activation. Moreover, splicing factor mutated CD34+ cells are more susceptible to pharmacological inhibition of ATR resulting in elevated levels of DNA damage, cell cycle blockade, and cell death. This can be enhanced by combination treatment with low-dose splicing modulatory compound Pladienolide B. We further confirm the direct association of R-loops and ATR sensitivity with the presence of a splicing factor mutation using lentiviral overexpression of wild-type and mutant SRSF2 P95H in cord blood CD34+ cells. Collectively, our results from n=53 MDS patients identify replication stress and associated ATR signaling to be critical pathophysiological mechanisms in primary MDS CD34+ cells carrying splicing factor mutations, and provide a preclinical rationale for targeting ATR signaling in these patients.

Anamnestic risk factor evaluation of patients carrying carbapenem-resistant Enterobacterales and/or Acinetobacter baumannii - impact on infection control management at a German University Hospital.

Background: Carbapenem-resistant Enterobacterales and Acinetobacter baumannii are of major concern in terms of infection prevention and control. This study evaluated factors that may increase the frequency of Enterobacterales and A. baumannii with carbapenem resistance (CR) in patients admitted to a German University Hospital for implementation of optimized infection control management. Methods: A five-year-retrospective epidemiological cohort analysis was conducted on anamnestic risk factors for carrying Enterobacterales and/or A. baumannii with CR in patients who were first tested positive for these species at University Hospital Frankfurt (UHF) between January 2013 and June 2018. Results: 364 patients were tested positive for Enterobacterales and/or A. baumannii with CR, resulting in n=400 bacterial isolates in total, with Klebsiella pneumoniae being the most frequently detected species (n=146/400; 36.5%; 95% confidence interval: 31.8-41.4). In patients who were tested positive for Enterobacterales and/or A. baumannii with CR, any hospital stay within the previous 12 months was the most frequently reported common factor (n=275/364; 75.5%; 70.8-79.9). Conclusion: A hospital stay within the previous 12 months, including hospitals in Germany and abroad, is a frequent characteristic of patients who tested positive for Enterobacterales and/or A. baumannii with CR. Upon admission, any previous hospital stay of the given patient within the previous 12 months should be determined. Infection control strategies such as screening measures need to be adapted to these patient groups in hospital settings. In order to reflect the varying determinants in "nosocomial" cases in greater detail, the existing criteria used to characterize "nosocomial detection" of gram-negative bacteria with CR should be reviewed.

Inner-Shell Water Rearrangement Following Photoexcitation of Tris(2,2'-bipyridine)iron(II).

The solvent dynamics in Fe-tris-bipyridine [Fe(bpy)3](2+) upon electronic excitation (oxidation) and subsequent relaxation is followed on the picosecond time scale by using atomistic simulations. Starting from the low spin (LS) Fe(II)LS state the transition to the excited Fe(III) (1,3)MLCT (metal-to-ligand charge transfer) state decreases the water coordination in immediate proximity of the central iron atom. This readjustment of the solvent shell occurs on the subpicosecond time scale. Full relaxation of the water environment would occur on the 10 ps time scale which is, however, never reached as the lifetime of the (1,3)MLCT state is only 200 fs. Further relaxation toward the long-lived (665 ps) [Fe(II)HS(bpy)3] high spin (HS) state does not change the degree of solvation. The results support a model in which the change in the degree of solvation is driven by electronic effects (charge redistribution) and not by structural changes (change in bond lengths). Furthermore, the results are consistent with recent combined X-ray emission (XES) and X-ray diffusion (XDS) scattering experiments which provided evidence for a reduced solvent density upon excitation of the [Fe(II)LS(bpy)3] initial state. However, the time scale for water exchange dynamics is faster than that found in the experiments.

Toward force fields for atomistic simulations of iridium-containing complexes.

The structural and energetic characterization of metal complexes is important in catalysis and photochemical applications. Unraveling their modes-of-action can be greatly assisted by computation, which typically is restricted to computationally demanding methods including electronic structure calculations with density functional theory. Here, we present an empirical force field based on valence bond theory applicable to a range of octahedral Ir(III) complexes with different coordinating ligands, including iridium complexes with a chiral P,N ligand. Using an approach applicable to metal-containing complexes in general, it is shown that with one common parametrization 85% of the 116 diastereomers--all within 21 kcal/mol of the lowest energy conformation of each series--can be correctly ranked. For neutral complexes, all diastereomers are ranked correctly. This helps to identify the most relevant diastereomers which, if necessary, can be further investigated by more demanding computational methods. Furthermore, if one specific complex is considered, the root mean square deviation between reference data from electronic structure calculations and the force field is ≈1 kcal/mol. This, together with the possibility to carry out explicit simulations in solution paves the way for an atomistic understanding of iridium-containing complexes in catalysis.

Structure and dynamics of solvent shells around photoexcited metal complexes.

Understanding the geometry, energetics and dynamics of solvated transition metal (TM) compounds is decisive in characterizing and optimizing their function. Here, we demonstrate that it is possible to quantify the structural dynamics of solvated [Ru(II)(bpy)3], an important TM-complex for solar-energy harvesting research, by using state-of-the art force fields together with molecular simulations. Electronic excitation to [Ru(III)(bpy)3] leads to a nonequilibrium system in which excess energy is redistributed to the surrounding solvent following a cascade of dynamical effects that can be characterized by the simulations. The study reveals that the structure of the surrounding solvent relaxes towards the equilibrium on a sub-picosecond to a few-picosecond time scale. Analysis of solvent residence and rotational reorientation times during relaxation demonstrates increased dynamics in the inner solvation sphere on the picosecond time scale. Energy transfer to the solvent occurs on different time scales for the different degrees of freedom which range from a few hundred fs to several picoseconds.

[Evaluation of nursing and leadership competencies - a baseline for practice development]. / Evaluation der Pflege- und Führungskompetenz - eine Ausgangslage für die weitere Praxisentwicklung.

Competent nursing and effective leadership are essential attributes for the provision of high-quality care which is patient-centred, evidence-based and outcome-oriented. The Department of Clinical Nursing science (DCN) of the University Hospital Basel (USB) initiated and implemented programmes of targeted practice development to promote competence in nursing and leadership. With the aim of generating data on nursing and leadership competencies, as well as on the quality of nursing care and the nursing work environment, an evaluation study with a mixed-method design was implemented in 2007. Within the quantitative portion of the study 679 nurses and 27 nurse managers participated. The descriptive results showed that nurses rated their overall level of competence on the Nurse Competence Scale high with a mean score of 75.1 (VAS 0 - 100). The rating of leadership competencies of nurse managers on the Leadership Practice Inventory was in the upper third of the 10-point Likert scale with mean scores of 40 to 50 (6 - 60). In order to permit ongoing monitoring of practice development, follow-up evaluations at regularly scheduled intervals are planned. With the introduction of a reimbursement system on the basis of Diagnosis Related Groups into Swiss health care, effective monitoring of nursing service contextual factors is crucial, now and in the future.