Wnt9 directs zebrafish heart tube assembly via a combination of canonical and non-canonical pathway signaling.

During zebrafish heart formation, cardiac progenitor cells converge at the embryonic midline where they form the cardiac cone. Subsequently, this structure transforms into a heart tube. Little is known about the molecular mechanisms that control these morphogenetic processes. Here, we use light-sheet microscopy and combine genetic, molecular biological and pharmacological tools to show that the paralogous genes wnt9a/b are required for the assembly of the nascent heart tube. In wnt9a/b double mutants, cardiomyocyte progenitor cells are delayed in their convergence towards the embryonic midline, the formation of the heart cone is impaired and the transformation into an elongated heart tube fails. The same cardiac phenotype occurs when both canonical and non-canonical Wnt signaling pathways are simultaneously blocked by pharmacological inhibition. This demonstrates that Wnt9a/b and canonical and non-canonical Wnt signaling regulate the migration of cardiomyocyte progenitor cells and control the formation of the cardiac tube. This can be partly attributed to their regulation of the timing of cardiac progenitor cell differentiation. Our study demonstrates how these morphogens activate a combination of downstream pathways to direct cardiac morphogenesis.

The impact of IL-10 and IL-17 on myeloid-derived suppressor cells in vitro and in vivo in a murine model of asthma.

Myeloid-derived suppressor cells (MDSCs) hold promise for clinical applications due to their immunosuppressive properties, particularly in the context of inflammation. In the present study, the number and immunosuppressive activity of MDSCs isolated from naïve Il10-/-, Il17-/-, and WT mice as control, as well as from house dust mite extract (HDM)-induced asthmatic Il10-/- and Il17-/- mice, were investigated. IL-10 deficiency increased the number of polymorphonuclear (PMN)-MDSCs in the lung, spleen, and bone marrow, without concurrent impairment of their suppressive activity in vitro. In the asthma model, the IL-17 knockout was concomitant with a lower number and activity of monocytic (M)-MDSCs and an altered inflammatory reaction with impaired lung function. Additionally, we found a higher baseline inflammation of the Il17-/- mice in the lung, manifested in increased airway resistance. We conclude that the impact of IL-10 and IL-17 deficiency on MDSCs differs in the context of inflammation. Accordingly, the in vitro experiments demonstrated an increased number of PMN-MDSCs across tissues in Il10-/- mice, which indicates that IL-10 might serve a pivotal role in preserving immune homeostasis under physiological circumstances. In the context of HDM-induced airway inflammation, IL-17 was found to be an important player in the suppression of pulmonary inflammation and regulation of M-MDSCs.

BMP4 regulates asymmetric Pkd2 distribution in mouse nodal immotile cilia and ciliary mechanosensing required for left-right determination.

BACKGROUND: Mouse nodal immotile cilia mechanically sense the bending direction for left-right (L-R) determination and activate the left-side-specific signaling cascade, leading to increased Nodal activity. Asymmetric distribution of Pkd2, a crucial channel for L-R determination, on immotile cilia has been reported recently. However, the causal relationship between the asymmetric Pkd2 distribution and direction-dependent flow sensing is not well understood. Furthermore, the underlying molecular mechanism directing this asymmetric Pkd2 distribution remains unclear. RESULTS: The effects of several recombinant proteins and inhibitors on the Pkd2 distribution were analyzed using super-resolution microscopy. Notably, bone morphogenetic protein 4 (BMP4) affected the Pkd2 distribution. Additionally, three-dimensional manipulation of nodal immotile cilia using optical tweezers revealed that excess BMP4 caused defects in the mechanosensing ability of the cilia. CONCLUSIONS: Experimental data together with model calculations suggest that BMP4 regulates the asymmetric distribution of Pkd2 in nodal immotile cilia, thereby affecting the ability of these cilia to sense the bending direction for L-R determination. This study, for the first time, provides insight into the relationship between the asymmetric protein distribution in cilia and their function.

A Novel High-Content Screening Assay Identified Belinostat as Protective in a FSGS-Like Zebrafish Model.

BACKGROUND: FSGS affects the complex three-dimensional morphology of podocytes, resulting in loss of filtration barrier function and the development of sclerotic lesions. Therapies to treat FSGS are limited, and podocyte-specific drugs are unavailable. To address the need for treatments to delay or stop FSGS progression, researchers are exploring the repurposing of drugs that have been approved by the US Food and Drug Administration (FDA) for other purposes. METHODS: To identify drugs with potential to treat FSGS, we used a specific zebrafish screening strain to combine a high-content screening (HCS) approach with an in vivo model. This zebrafish screening strain expresses nitroreductase and the red fluorescent protein mCherry exclusively in podocytes (providing an indicator for podocyte depletion), as well as a circulating 78 kDa vitamin D-binding enhanced green fluorescent protein fusion protein (as a readout for proteinuria). To produce FSGS-like lesions in the zebrafish, we added 80 µ M metronidazole into the fish water. We used a specific screening microscope in conjunction with advanced image analysis methods to screen a library of 138 drugs and compounds (including some FDA-approved drugs) for podocyte-protective effects. Promising candidates were validated to be suitable for translational studies. RESULTS: After establishing this novel in vivo HCS assay, we identified seven drugs or compounds that were protective in our FSGS-like model. Validation experiments confirmed that the FDA-approved drug belinostat was protective against larval FSGS. Similar pan-histone deacetylase inhibitors also showed potential to reproduce this effect. CONCLUSIONS: Using an FSGS-like zebrafish model, we developed a novel in vivo HCS assay that identified belinostat and related pan-histone deacetylase inhibitors as potential candidates for treating FSGS.

Usability and usefulness of (electronic) patient identification systems-A cross-sectional evaluation in German-speaking radiation oncology departments.

PURPOSE: Patient misidentification in radiation oncology (RO) is a significant concern due to the potential harm to patient health and the burden on healthcare systems. Electronic patient identification systems (ePIS) are increasingly being used as an alternative or supplement to organizational systems (oPIS). The objective of this study was to assess the usability and usefulness of ePIS and oPIS in German-speaking countries. METHODS: A cross-sectional survey was designed by a group of experts from various professional backgrounds in RO. The survey consisted of 38 questions encompassing quantitative and qualitative data on usability, user experience, and usefulness of PIS. It was available between August and October 2022. RESULTS: Of 118 eligible participants, 37% had implemented some kind of ePIS. Overall, 22% of participants who use an oPIS vs. 10% of participants who use an ePIS reported adverse events in terms of patients' misidentification in the past 5 years. Frequent or very frequent drop-outs of electronic systems were reported by 31% of ePIS users. Users of ePIS significantly more often affirmed a positive cost-benefit ratio of ePIS as well as an improvement of workflow, whereas users of oPIS more frequently apprehended a decrease in staffs' attention through ePIS. The response rate was 8%. CONCLUSION: The implementation of ePIS can contribute to efficient PI and improved processes. Apprehensions by oPIS users and assessments of ePIS users differ significantly in aspects of the perceived usefulness of ePIS. However, technical problems need to be addressed to ensure the reliability of ePIS. Further research is needed to assess the impact of different PIS on patient safety in RO.

Inhibition of the glucocorticoid receptor attenuates proteinuric kidney diseases in multiple species.

BACKGROUND AND HYPOTHESIS: Glucocorticoids are the treatment of choice for proteinuric patients with minimal-change disease (MCD) and primary focal and segmental glomerulosclerosis (FSGS). Immunosuppressive as well as direct effects on podocytes are believed to mediate their actions. In this study, we analyzed the anti-proteinuric effects of inhibition of the glucocorticoid receptor (GR) in glomerular epithelial cells, including podocytes. METHODS: We employed genetic and pharmacological approaches to inhibit the GR. Genetically, we used Pax8-Cre/GRfl/fl mice to specifically inactivate the GR in kidney epithelial cells. Pharmacologically, we utilized a glucocorticoid antagonist called mifepristone. RESULTS: Genetic inactivation of GR, specifically in kidney epithelial cells, using Pax8-Cre/GRfl/fl mice, ameliorated proteinuria following protein overload. We further tested the effects of pharmacological GR inhibition in three models and species: the puromycin-aminonucleoside-induced nephrosis model in rats, the protein overload model in mice and the inducible transgenic NTR/MTZ zebrafish larvae with specific and reversible podocyte injury. In all three models, both pharmacological GR activation and inhibition consistently and significantly ameliorated proteinuria. Additionally, we translated our findings to humans, where three nephrotic adult patients with MCD or primary FSGS with contraindications or insufficient responses to corticosteroids, were treated with mifepristone. This treatment resulted in a clinically relevant reduction of proteinuria. CONCLUSIONS: Thus, across multiple species and proteinuria models, both genetic and pharmacological GR inhibition was at least as effective as pronounced GR activation. While, the mechanism remains perplexing, GR inhibition may be a novel and targeted therapeutic approach to treat glomerular proteinuria potentially bypassing adverse actions of steroids.

BDNF: mRNA expression in urine cells of patients with chronic kidney disease and its role in kidney function.

Podocyte loss and changes to the complex morphology are major causes of chronic kidney disease (CKD). As the incidence is continuously increasing over the last decades without sufficient treatment, it is important to find predicting biomarkers. Therefore, we measured urinary mRNA levels of podocyte genes NPHS1, NPHS2, PODXL and BDNF, KIM-1, CTSL by qRT-PCR of 120 CKD patients. We showed a strong correlation between BDNF and the kidney injury marker KIM-1, which were also correlated with NPHS1, suggesting podocytes as a contributing source. In human biopsies, BDNF was localized in the cell body and major processes of podocytes. In glomeruli of diabetic nephropathy patients, we found a strong BDNF signal in the remaining podocytes. An inhibition of the BDNF receptor TrkB resulted in enhanced podocyte dedifferentiation. The knockdown of the orthologue resulted in pericardial oedema formation and lowered viability of zebrafish larvae. We found an enlarged Bowman's space, dilated glomerular capillaries, podocyte loss and an impaired glomerular filtration. We demonstrated that BDNF is essential for glomerular development, morphology and function and the expression of BDNF and KIM-1 is highly correlated in urine cells of CKD patients. Therefore, BDNF mRNA in urine cells could serve as a potential CKD biomarker.

Myeloid-derived suppressor cells in influenza virus-induced asthma exacerbation.

Myeloid-derived suppressor cells (MDSCs) are a phenotypically heterogenous group of cells that potently suppress the immune response. A growing body of evidence supports the important role of MDSCs in a variety of lung diseases, such as asthma. However, the role of MDSCs in asthma exacerbation has so far not been investigated. Here, we studied the role of MDSCs in a murine model of influenza virus-induced asthma exacerbation. BALB/c mice were exposed to house dust mite (HDM) three times a week for a total of five weeks to induce a chronic asthmatic phenotype, which was exacerbated by additional exposure to the A/Hamburg/5/2009 hemagglutinin 1 neuraminidase 1 (H1N1) influenza virus. Induction of lung inflammatory features, production of T helper (Th) 1- and Th2- associated inflammatory cytokines in the lavage fluid and an increased airway hyper-responsiveness were observed, establishing the asthma exacerbation model. The number and activity of pulmonary M-MDSCs increased in exacerbated asthmatic mice compared to non-exacerbated asthmatic mice. Furthermore, depletion of MDSCs aggravated airway hyper-responsiveness in exacerbated asthmatic mice. These findings further denote the role of MDSCs in asthma and provide some of the first evidence supporting a potential important role of MDSCs in asthma exacerbation.

[Adjuvant radiation therapy for breast cancer]. / Adjuvante Strahlentherapie des Mammakarzinoms.

Adjuvant radiotherapy is an integral part of multimodal therapy for early breast cancer. It contributes to the reduction of local recurrences across all disease stages. (Moderate) hypofractionated whole-breast irradiation is the standard of care. In low-risk situations, partial breast irradiation can be an option. The indication for adjuvant radiotherapy after mastectomy or additional irradiation of regional lymph nodes depends on the patient's individual risk profile. Long-term results of treatment and further development of irradiation techniques now allow shorter, individualized and well-tolerated treatments with the aim of therapy de-escalation.

Linac-Based Ultrahypofractionated Partial Breast Irradiation (APBI) in Low-Risk Breast Cancer: First Results of a Monoinstitutional Observational Analysis.

Purpose: For adjuvant radiotherapy of low-risk breast cancer after breast-conserving surgery, there have been many trials of hypofractionation and partial breast irradiation (PBI) over the years, with proven mild long-term toxicity. The aim of this study was to introduce a short-course dose-adapted concept, proven in whole breast irradiation (WBI) for use in accelerated partial breast irradiation (APBI), while monitoring dosimetric data and toxicity. Methods: From April 2020 to March 2022, 61 patients with low-risk breast cancer or ductal carcinoma in situ (DCIS) were treated at a single institution with percutaneous APBI of 26 Gy in five fractions every other day after breast-conserving surgery. Dosimetric data for target volume and organs at risk were determined retrospectively. Acute toxicity was evaluated. Results: The target volume of radiotherapy comprised an average of 19% of the ipsilateral mamma. The burden on the heart and lungs was very low. The mean cardiac dose during irradiation of the left breast was only 0.6 Gy. Two out of three patients remained without any acute side effects. Conclusions: Linac-based APBI is an attractive treatment option for patients with low-risk breast cancer in whom neither WBI nor complete omission of radiotherapy appears to be an adequate alternative.

Generation of a human induced pluripotent stem cell line (UMGACBi001-A) from urine cells of a chronic kidney disease patient with hypertension, diabetic nephropathy and acute sepsis.

Chronic kidney disease is a major public health burden associated with a drastically reduced quality of living and life span that lacks suitable, individualized therapeutic strategies. Here we present a human induced pluripotent stem cell line (iPSC, UMGACBi001-A) reprogrammed from urine cells of an acute septic dialysis patient suffering from chronic kidney disease using non-integrating administration of RNAs. The generated iPSCs were positively characterized for typical morphology, pluripotency marker expression, directed differentiation potential, non-contamination, chromosomal consistency and donor identity. This iPSC-line can be a useful source for in vitro disease modelling and individualized therapeutic approaches.

The ShGlomAssay Combines High-Throughput Drug Screening With Downstream Analyses and Reveals the Protective Role of Vitamin D3 and Calcipotriol on Podocytes.

Chronic kidney disease (CKD) is a major public health burden affecting more than 500 million people worldwide. Podocytopathies are the main cause for the majority of CKD cases due to pathogenic morphological as well as molecular biological alterations of postmitotic podocytes. Podocyte de-differentiation is associated with foot process effacement subsequently leading to proteinuria. Since currently no curative drugs are available, high throughput screening methods using a small number of animals are a promising and essential tool to identify potential drugs against CKD in the near future. Our study presents the implementation of the already established mouse GlomAssay as a semi-automated high-throughput screening method-shGlomAssay-allowing the analysis of several hundreds of FDA-verified compounds in combination with downstream pathway analysis like transcriptomic and proteomic analyses from the same samples, using a small number of animals. In an initial prescreening we have identified vitamin D3 and its analog calcipotriol to be protective on podocytes. Furthermore, by using RT-qPCR, Western blot, and RNA sequencing, we found that mRNA and protein expression of nephrin, the vitamin D receptor and specific podocyte markers were significantly up-regulated due to vitamin D3- and calcipotriol-treatment. In contrast, kidney injury markers were significantly down-regulated. Additionally, we found that vitamin D3 and calcipotriol have had neither influence on the expression of the miR-21 and miR-30a nor on miR-125a/b, a miRNA described to regulate the vitamin D receptor. In summary, we advanced the established mouse GlomAssay to a semi-automated high-throughput assay and combined it with downstream analysis techniques by using only a minimum number of animals. Hereby, we identified the vitamin D signaling pathway as podocyte protective and to be counteracting their de-differentiation.

Evaluation of endogenous miRNA reference genes across different zebrafish strains, developmental stages and kidney disease models.

The majority of kidney diseases arise from the loss of podocytes and from morphological changes of their highly complex foot process architecture, which inevitably leads to a reduced kidney filtration and total loss of kidney function. It could have been shown that microRNAs (miRs) play a pivotal role in the pathogenesis of podocyte-associated kidney diseases. Due to their fully functioning pronephric kidney, larval zebrafish have become a popular vertebrate model, to study kidney diseases in vivo. Unfortunately, there is no consensus about a proper normalization strategy of RT-qPCR-based miRNA expression data in zebrafish. In this study we analyzed 9 preselected candidates dre-miR-92a-3p, dre-miR-206-3p, dre-miR-99-1, dre-miR-92b-3p, dre-miR-363-3p, dre-let-7e, dre-miR-454a, dre-miR-30c-5p, dre-miR-126a-5p for their capability as endogenous reference genes in zebrafish experiments. Expression levels of potential candidates were measured in 3 different zebrafish strains, different developmental stages, and in different kidney disease models by RT-qPCR. Expression values were analyzed with NormFinder, BestKeeper, GeNorm, and DeltaCt and were tested for inter-group differences. All candidates show an abundant expression throughout all samples and relatively high stability. The most stable candidate without significant inter-group differences was dre-miR-92b-3p making it a suitable endogenous reference gene for RT-qPCR-based miR expression zebrafish studies.

Identification of miR-16 as an endogenous reference gene for the normalization of urinary exosomal miRNA expression data from CKD patients.

Chronic kidney disease (CKD) is a severe disorder with an increasing incidence worldwide. An early detection may help to prevent its progression and to minimize the risk of cardiovascular diseases as one of the major comorbidities. Recently, extracellular miRNAs like urinary exosomal miRNAs became of great interest as non-invasive biomarkers which can be determined by RT-qPCR. But until now, there is no consensus regarding the normalization of miRNAs isolated from body fluids. The present study analyzed the miRNAs miR-16, miR-92a, miR-21, miR-124a and the small nuclear RNA RNU6B for their applicability as an endogenous reference gene in expression studies of exosomal miRNAs isolated from CKD patients. For this purpose, miRNA expression levels were determined by RT-qPCR after the isolation of urinary exosomes from 33 CKD patients and from 5 healthy controls. Expression data was analyzed with the normalization determination software NormFinder, BestKeeper, GeNorm and DeltaCt. Our results revealed an abundant expression of the four candidate miRNAs in urinary exosomes and no detectable expression of RNU6B. We identified miR-16 as the most stable endogenous reference gene in our data set, making it a suitable endogenous reference gene for miRNA studies of urinary exosomes derived from CKD patients.

Recent advances in microwave-assisted combinatorial synthesis and library generation.

Progress and developments made in microwave-assisted combinatorial synthesis and library production since 2002 are reviewed. The use of microwave technology in both solution and solid phase synthesis is discussed with special reference to agrochemical applications where appropriate.

A communication-theory based view on telemedical communication.

Communication theory based analysis sheds new light on the use of health telematics. This analysis of structures in electronic medical communication shows communicative structures with special features. Current and evolving telemedical applications are analyzed. The methodology of communicational theory (focusing on linguistic pragmatics) is used to compare it with its conventional counterpart. The semiotic model, the roles of partners, the respective message and their relation are discussed. Channels, sender, addressee, and other structural roles are analyzed for different types of electronic medical communication. The communicative processes are shown as mutual, rational action towards a common goal. The types of communication/texts are analyzed in general. Furthermore the basic communicative structures of medical education via internet are presented with their special features. The analysis shows that electronic medical communication has special features compared to everyday communication: A third participant role often is involved: the patient. Messages often are addressed to an unspecified partner or to an unspecified partner within a group. Addressing in this case is (at least partially) role-based. Communication and message often directly (rather than indirectly) influence actions of the participants. Communication often is heavily regulated including legal implications like liability, and more. The conclusion from the analysis is that the development of telemedical applications so far did not sufficiently take communicative structures into consideration. Based on these results recommendations for future developments of telemedical applications/services are given.

ProSAR: a new methodology for combinatorial library design.

A method is introduced for performing reagent selection for chemical library design based on topological (2D) pharmacophore fingerprints. Optimal reagent selection is achieved by optimizing the Shannon entropy of the 2D pharmacophore distribution for the reagent set. The method, termed ProSAR, is therefore expected to enumerate compounds that could serve as a good starting point for deriving a structure activity relationship (SAR) in combinatorial library design. This methodology is exemplified by library design examples where the active compounds were already known. The results show that most of the pharmacophores on the substituents for the active compounds are covered by the designed library. This strategy is further expanded to include product property profiles for aqueous solubility, hERG risk assessment, etc. in the optimization process so that the reagent pharmacophore diversity and the product property profile are optimized simultaneously via a genetic algorithm. This strategy is applied to a two-dimensional library design example and compared with libraries designed by a diversity based strategy which minimizes the average ensemble Tanimoto similarity. Our results show that by using the PSAR methodology, libraries can be designed with simultaneously good pharmacophore coverage and product property profile.