In a rat model of bypass DuraGraft ameliorates endothelial dysfunction of arterial grafts.

Coronary artery bypass surgery can result in endothelial dysfunction due to ischemia/reperfusion (IR) injury. Previous studies have demonstrated that DuraGraft helps maintain endothelial integrity of saphenous vein grafts during ischemic conditions. In this study, we investigated the potential of DuraGraft to mitigate endothelial dysfunction in arterial grafts after IR injury using an aortic transplantation model. Lewis rats (n = 7-9/group) were divided in three groups. Aortic arches from the control group were prepared and rings were immediately placed in organ baths, while the aortic arches of IR and IR + DuraGraft rats were preserved in saline or DuraGraft, respectively, for 1 h before being transplanted heterotopically. After 1 h after reperfusion, the grafts were explanted, rings were prepared, and mounted in organ baths. Our results demonstrated that the maximum endothelium-dependent vasorelaxation to acetylcholine was significantly impaired in the IR group compared to the control group, but DuraGraft improved it (control: 89 ± 2%; IR: 24 ± 1%; IR + DuraGraft: 48 ± 1%, p < 0.05). Immunohistochemical analysis revealed decreased intercellular adhesion molecule-1, 4-hydroxy-2-nonenal, caspase-3 and caspase-8 expression, while endothelial cell adhesion molecule-1 immunoreactivity was increased in the IR + DuraGraft grafts compared to the IR-group. DuraGraft mitigates endothelial dysfunction following IR injury in a rat bypass model. Its protective effect may be attributed, at least in part, to its ability to reduce the inflammatory response, oxidative stress, and apoptosis.

Pharmacological inhibition of the cysteine protease cathepsin C improves graft function after heart transplantation in rats.

BACKGROUND: Heart transplantation (HTX) is the standard treatment for end-stage heart failure. However, reperfusion following an ischemic period can contribute to myocardial injury. Neutrophil infiltration, along with the subsequent release of tissue-degrading neutrophil elastase (NE)-related serine proteases and oxygen-derived radicals, is associated with adverse graft outcomes. The inhibition of cathepsin C (CatC) has been shown to block NE-related protease activation. We hypothesized that the CatC inhibitor BI-9740 improves graft function after HTX. METHODS: In a rat model of HTX, the recipient Lewis rats were orally administered with either a placebo (n = 12) or BI-9740 (n = 11, 20 mg/kg) once daily for 12 days. Donor hearts from untreated Lewis rats were explanted, preserved in a cardioplegic solution, and subsequently heterotopically implanted. In vivo left-ventricular (LV) graft function was assessed after 1 h of reperfusion. The proteolytic activity of neutrophil serine proteases was determined in bone marrow lysates from BI-9740-treated and control rats. Additionally, myocardial morphological changes were examined, and heart samples underwent immunohistochemistry and western blot analysis. RESULTS: The NE-related proteolytic activity in bone marrow cell lysates was markedly decreased in the BI-9740-treated rats compared to those of the placebo group. Histopathological lesions, elevated CatC and myeloperoxidase-positive cell infiltration, and nitrotyrosine immunoreactivity with an increased number of poly(ADP-ribose) polymerase (PARP)-1-positive cells were lowered in the hearts of animals treated with BI-9740 compared to placebo groups. Regarding the functional parameters of the implanted graft, improvements were observed in both systolic function (LV systolic pressure 110 ± 6 vs 74 ± 6 mmHg; dP/dtmax 2782 ± 149 vs 2076 ± 167 mmHg/s, LV developed pressure, at an intraventricular volume of 200 µl, p < 0.05) and diastolic function in the hearts of BI-9740 treated animals compared with those receiving the only placebo. Furthermore, the administration of BI-9740 resulted in a shorter graft re-beating time compared to the placebo group. However, this study did not provide evidence of DNA fragmentation, the generation of both superoxide anions and hydrogen peroxide, correlating with the absence of protein alterations related to apoptosis, as evidenced by western blot in grafts after HTX. CONCLUSIONS: We provided experimental evidence that pharmacological inhibition of CatC improves graft function following HTX in rats.

Non-Hydrolysable Analogues of Cyclic and Branched Condensed Phosphates: Chemistry and Chemical Proteomics.

Studies into the biology of condensed phosphates almost exclusively cover linear polyphosphates. However, there is evidence for the presence of cyclic polyphosphates (metaphosphates) in organisms and for enzymatic digestion of branched phosphates (ultraphosphates) with alkaline phosphatase. Further research of non-linear condensed phosphates in biology would profit from interactome data of such molecules, however, their stability in biological media is limited. Here we present syntheses of modified, non-hydrolysable analogues of cyclic and branched condensed phosphates, called meta- and ultraphosphonates, and their application in a chemical proteomics approach using yeast cell extracts. We identify putative interactors with overlapping hits for structurally related capture compounds underlining the quality of our results. The datasets serve as starting point to study the biological relevance and functions of meta- and ultraphosphates. In addition, we examine the reactivity of meta- and ultraphosphonates with implications for their "hydrolysable" analogues: Efforts to increase the ring-sizes of meta- or cyclic ultraphosphonates revealed a strong preference to form trimetaphosphate-analogue structures by cyclization and/or ring-contraction. Using carbodiimides for condensation, the so far inaccessible dianhydro product of ultraphosphonate, corresponding to P4 O11 2- , was selectively obtained and then ring-opened by different nucleophiles yielding modified cyclic ultraphosphonates.

Stable Isotope Phosphate Labelling of Diverse Metabolites is Enabled by a Family of 18 O-Phosphoramidites.

Stable isotope labelling is state-of-the-art in quantitative mass spectrometry, yet often accessing the required standards is cumbersome and very expensive. Here, a unifying synthetic concept for 18 O-labelled phosphates is presented, based on a family of modified 18 O2 -phosphoramidite reagents. This toolbox offers access to major classes of biologically highly relevant phosphorylated metabolites as their isotopologues including nucleotides, inositol phosphates, -pyrophosphates, and inorganic polyphosphates. 18 O-enrichment ratios >95 % and good yields are obtained consistently in gram-scale reactions, while enabling late-stage labelling. We demonstrate the utility of the 18 O-labelled inositol phosphates and pyrophosphates by assignment of these metabolites from different biological matrices. We demonstrate that phosphate neutral loss is negligible in an analytical setup employing capillary electrophoresis electrospray ionisation triple quadrupole mass spectrometry.

The Aryne Phosphate Reaction.

Condensed phosphates are a critically important class of molecules in biochemistry. Non-natural analogues are important for various applications, such as single-molecule real-time DNA sequencing. Often, such analogues contain more than three phosphate units in their oligophosphate chain. Consequently, investigations into phosphate reactivity enabling new ways of phosphate functionalization and oligophosphorylation are essential. Here, we scrutinize the potential of phosphates to act as arynophiles, paving the way for follow-up oligophosphorylation reactions. The aryne phosphate reaction is a powerful tool to-depending on the perspective-(oligo)phosphorylate arenes or arylate (oligo-cyclo)phosphates. Based on Kobayashi-type o-silylaryltriflates, the aryne phosphate reaction enables rapid entry into a broad spectrum of arylated products, like monophosphates, diphosphates, phosphodiesters and polyphosphates. The synthetic potential of these new transformations is demonstrated by efficient syntheses of nucleotide analogues and an unprecedented one-flask octaphosphorylation.

Stable Isotope Phosphate Labelling of Diverse Metabolites is Enabled by a Family of 18O-Phosphoramidites.

Stable isotope labelling is state-of-the-art in quantitative mass spectrometry, yet often accessing the required standards is cumbersome and very expensive. Here, a unifying synthetic concept for 18O-labelled phosphates is presented, based on a family of modified 18O2-phosphoramidite reagents. This toolbox offers access to major classes of biologically highly relevant phosphorylated metabolites as their isotopologues including nucleotides, inositol phosphates, -pyrophosphates, and inorganic polyphosphates. 18O-enrichment ratios >95 % and good yields are obtained consistently in gram-scale reactions, while enabling late-stage labelling. We demonstrate the utility of the 18O-labelled inositol phosphates and pyrophosphates by assignment of these metabolites from different biological matrices. We demonstrate that phosphate neutral loss is negligible in an analytical setup employing capillary electrophoresis electrospray ionisation triple quadrupole mass spectrometry.

Lost in Condensation: Poly-, Cyclo-, and Ultraphosphates.

Much like linear, branched, and cyclic alkanes, condensed phosphates exist as linear, branched, and cyclic structures. Inasmuch as alkanes are the cornerstone of organic chemistry, generating an inexplorably large chemical space, a comparable richness in structures can be expected for condensed phosphates, as also for them the concepts of isomerism apply. Little of their chemical space has been charted, and only a few different synthesis methods are available to construct isomers of condensed phosphates. Here, we will discuss the application of phosphoramidites with one, two, or three P-N bonds that can be substituted selectively to access different condensed phosphates in a highly controllable manner. Work directed toward the further exploration of this chemical space will contribute to our understanding of the fundamental chemistry of phosphates.In biology, condensed phosphates play important roles in the form of inorganic representatives, such as pyrophosphate, polyphosphate, and cyclophosphate, and also in conjugation with organic molecules, such as esters and amidates. Phosphorus is one of the six biogenic elements; the omnipresence of phosphates in biology points toward their critical involvement in prebiotic chemistry and the emergence of life itself. Indeed, it is hard to imagine any life without phosphate. It is therefore desirable to achieve through synthesis a better understanding of the chemistry of the condensed phosphates to further explore their biology.There is a rich but underexplored chemistry of the family of condensed phosphates per se, which is further diversified by their conjugation to important biomolecules and metabolites. For example, proteins may be polyphosphorylated on lysins, a very recent addition to posttranslational modifications. Adenosine triphosphate, as a representative of the small molecules, on the other hand, is well known as the universal cellular energy currency. In this Account, we will describe our motivations and our approaches to construct, modify, and synthetically apply different representatives of the condensed phosphates. We also describe the generation of hybrids composed of cyclic and linear structures of different oxidation states and develop them into reagents of great utility. A pertinent example is provided in the step-economic synthesis of the magic spot nucleotides (p)ppGpp. Finally, we provide an overview of 31P NMR data collected over the years in our laboratories, helping as a waymarker for not getting lost in condensation.

The chemistry of branched condensed phosphates.

Condensed phosphates may exist as linear, cyclic or branched structures. Due to their important role in nature, linear polyphosphates have been well studied. In contrast, branched phosphates (ultraphosphates) remain largely uncharacterised, because they were already described in 1950 as exceedingly unstable in the presence of water, epitomized in the antibranching-rule. This rule lacks experimental backup, since, to the best of our knowledge, no rational synthesis of defined ultraphosphates is known. Consequently, detailed studies of their chemical properties, reactivity and potential biological relevance remain elusive. Here, we introduce a general synthesis of monodisperse ultraphosphates. Hydrolysis half-lives up to days call the antibranching-rule into question. We provide evidence for the interaction of an enzyme with ultraphosphates and discover a rearrangement linearizing the branched structure. Moreover, ultraphosphate can phosphorylate nucleophiles such as amino acids and nucleosides with implications for prebiotic chemistry. Our results provide an entry point into the uncharted territory of branched condensed phosphates.

Enhancing evidence-based medicine with natural language argumentative analysis of clinical trials.

In the latest years, the healthcare domain has seen an increasing interest in the definition of intelligent systems to support clinicians in their everyday tasks and activities. Among others, also the field of Evidence-Based Medicine is impacted by this twist, with the aim to combine the reasoning frameworks proposed thus far in the field with mining algorithms to extract structured information from clinical trials, clinical guidelines, and Electronic Health Records. In this paper, we go beyond the state of the art by proposing a new end-to-end pipeline to address argumentative outcome analysis on clinical trials. More precisely, our pipeline is composed of (i) an Argument Mining module to extract and classify argumentative components (i.e., evidence and claims of the trial) and their relations (i.e., support, attack), and (ii) an outcome analysis module to identify and classify the effects (i.e., improved, increased, decreased, no difference, no occurrence) of an intervention on the outcome of the trial, based on PICO elements. We annotated a dataset composed of more than 500 abstracts of Randomized Controlled Trials (RCT) from the MEDLINE database, leading to a labeled dataset with 4198 argument components, 2601 argument relations, and 3351 outcomes on five different diseases (i.e., neoplasm, glaucoma, hepatitis, diabetes, hypertension). We experiment with deep bidirectional transformers in combination with different neural architectures (i.e., LSTM, GRU and CRF) and obtain a macro F1-score of.87 for component detection and.68 for relation prediction, outperforming current state-of-the-art end-to-end Argument Mining systems, and a macro F1-score of.80 for outcome classification.

Human pluripotent stem cell-derived acinar/ductal organoids generate human pancreas upon orthotopic transplantation and allow disease modelling.

OBJECTIVE: The generation of acinar and ductal cells from human pluripotent stem cells (PSCs) is a poorly studied process, although various diseases arise from this compartment. DESIGN: We designed a straightforward approach to direct human PSCs towards pancreatic organoids resembling acinar and ductal progeny. RESULTS: Extensive phenotyping of the organoids not only shows the appropriate marker profile but also ultrastructural, global gene expression and functional hallmarks of the human pancreas in the dish. Upon orthotopic transplantation into immunodeficient mice, these organoids form normal pancreatic ducts and acinar tissue resembling fetal human pancreas without evidence of tumour formation or transformation. Finally, we implemented this unique phenotyping tool as a model to study the pancreatic facets of cystic fibrosis (CF). For the first time, we provide evidence that in vitro, but also in our xenograft transplantation assay, pancreatic commitment occurs generally unhindered in CF. Importantly, cystic fibrosis transmembrane conductance regulator (CFTR) activation in mutated pancreatic organoids not only mirrors the CF phenotype in functional assays but also at a global expression level. We also conducted a scalable proof-of-concept screen in CF pancreatic organoids using a set of CFTR correctors and activators, and established an mRNA-mediated gene therapy approach in CF organoids. CONCLUSIONS: Taken together, our platform provides novel opportunities to model pancreatic disease and development, screen for disease-rescuing agents and to test therapeutic procedures.

Alterations of the cytoskeleton in human cells in space proved by life-cell imaging.

Microgravity induces changes in the cytoskeleton. This might have an impact on cells and organs of humans in space. Unfortunately, studies of cytoskeletal changes in microgravity reported so far are obligatorily based on the analysis of fixed cells exposed to microgravity during a parabolic flight campaign (PFC). This study focuses on the development of a compact fluorescence microscope (FLUMIAS) for fast live-cell imaging under real microgravity. It demonstrates the application of the instrument for on-board analysis of cytoskeletal changes in FTC-133 cancer cells expressing the Lifeact-GFP marker protein for the visualization of F-actin during the 24(th) DLR PFC and TEXUS 52 rocket mission. Although vibration is an inevitable part of parabolic flight maneuvers, we successfully for the first time report life-cell cytoskeleton imaging during microgravity, and gene expression analysis after the 31(st) parabola showing a clear up-regulation of cytoskeletal genes. Notably, during the rocket flight the FLUMIAS microscope reveals significant alterations of the cytoskeleton related to microgravity. Our findings clearly demonstrate the applicability of the FLUMIAS microscope for life-cell imaging during microgravity, rendering it an important technological advance in live-cell imaging when dissecting protein localization.