Normothermic liver machine perfusion as a dynamic platform for regenerative purposes. What does the future have in store for us?

Liver transplantation has become an immense success; nevertheless, far more recipients are registered on waiting lists than there are available donor livers for transplantation. High-risk, extended criteria donor livers are increasingly used to reduce the discrepancy between organ demand and supply. Especially for high-risk livers, dynamic preservation using machine perfusion can decrease post-transplantation complications and may increase donor liver utilization by resuscitation and viability testing before transplantation. To further increase the availability of donor livers suitable for transplantation, new strategies are required that make it possible to use organs that are initially too damaged to be transplanted. With the current progress in experimental liver transplantation research, (long-term) normothermic machine perfusion may be used in the future as a dynamic platform for regenerative medicine approaches, enabling repair and regeneration of injured donor livers. Currently explored therapeutics such as defatting cocktails, ribonucleic acid interference, senolytics, and stem cell therapy may assist in the repair and/or regeneration of injured livers before transplantation. This review will provide a forecast of the future utility of normothermic machine perfusion for repair and regeneration of damaged donor livers to ultimately decrease the imbalance between donor liver demand and supply

Super-paramagnetic clustering of yeast gene expression profiles

High-density DNA arrays, used to monitor gene expression at a genomic scale, have produced vast amounts of information which require the development of efficient computational methods to analyze them. The important first step is to extract the fundamental patterns of gene expression inherent in the data. This paper describes the application of a novel clustering algorithm, Super-Paramagnetic Clustering (SPC) to analysis of gene expression profiles that were generated recently during a study of the yeast cell cycle. SPC was used to organize genes into biologically relevant clusters that are suggestive for their co-regulation. Some of the advantages of SPC are its robustness against noise and initialization, a clear signature of cluster formation and splitting, and an unsupervised self-organized determination of the number of clusters at each resolution. Our analysis revealed interesting correlated behavior of several groups of genes which has not been previously identified

The international normalised ratio to monitor coagulation factor production during normothermic machine perfusion of human donor livers

Background: Normothermic machine perfusion (NMP) of donor livers allows for new diagnostic and therapeutic strategies. As the liver produces most of the haemostatic proteins, coagulation assays such as the International Normalised Ratio (INR) performed in perfusate may be useful to assess hepatocellular function of donor livers undergoing NMP. However, high concentrations of heparin and low levels of fibrinogen may affect coagulation assays.Methods: Thirty donor livers that underwent NMP were retrospectively included in this study, of which 18 were subsequently transplanted. We measured INRs in perfusate in presence or absence of exogenously added fibrinogen and/or polybrene. Additionally, we prospectively included 14 donor livers that underwent NMP (of which 11 were transplanted) and measured INR using both a laboratory coagulation analyser and a point-of-care device.Results: In untreated perfusate samples, the INR was above the detection limit in all donor livers. Addition of both fibrinogen and polybrene was required for adequate INR assessment. INRs decreased over time and detectable perfusate INR values were found in 17/18 donor livers at the end of NMP. INR results were similar between the coagulation analyser and the point-of-care device, but did not correlate with established hepatocellular viability criteria.Conclusions: Most of the donor livers that were transplanted showed a detectable perfusate INR at the end of NMP, but samples require processing to allow for INR measurements using laboratory coagulation analysers. Point-of-care devices bypass this need for processing. The INR does not correlate with established viability criteria and might therefore have additional predictive value.</p

Finite-size scaling investigation of the liquid-liquid critical point in ST2 water and its stability with respect to crystallization.

The liquid-liquid critical point scenario of water hypothesizes the existence of two metastable liq- uid phases low-density liquid (LDL) and high-density liquid (HDL) deep within the supercooled region. The hypothesis originates from computer simulations of the ST2 water model, but the stabil- ity of the LDL phase with respect to the crystal is still being debated. We simulate supercooled ST2 water at constant pressure, constant temperature, and constant number of molecules N for N ≤ 729 and times up to 1 μs. We observe clear differences between the two liquids, both structural and dynamical. Using several methods, including finite-size scaling, we confirm the presence of a liquid-liquid phase transition ending in a critical point. We find that the LDL is stable with respect to the crystal in 98% of our runs (we perform 372 runs for LDL or LDL-like states), and in 100% of our runs for the two largest system sizes (N = 512 and 729, for which we perform 136 runs for LDL or LDL-like states). In all these runs, tiny crystallites grow and then melt within 1 μs. Only for N ≤ 343 we observe six events (over 236 runs for LDL or LDL-like states) of spontaneous crystal- lization after crystallites reach an estimated critical size of about 70 ± 10 molecules

Restoration of Bile Duct Injury of Donor Livers During Ex Situ Normothermic Machine Perfusion

BACKGROUND: End-ischemic ex situ normothermic machine perfusion (NMP) enables assessment of donor livers prior to transplantation. The objective of this study was to provide support for bile composition as a marker of biliary viability and to investigate whether bile ducts of high-risk human donor livers already undergo repair during NMP.METHODS: Forty-two livers that were initially declined for transplantation were included in our NMP clinical trial. After NMP, livers were either secondary declined (n = 17) or accepted for transplantation (n = 25) based on the chemical composition of bile and perfusate samples. Bile duct biopsies were taken before and after NMP and assessed using an established histological injury severity scoring system and a comprehensive immunohistochemical assessment focusing on peribiliary glands (PBGs), vascular damage, and regeneration.RESULTS: Bile ducts of livers that were transplanted after viability testing during NMP showed better preservation of PBGs, (micro)vasculature, and increased cholangiocyte proliferation, compared with declined livers. Biliary bicarbonate, glucose, and pH were confirmed as accurate biomarkers of bile duct vitality. In addition, we found evidence of PBG-based progenitor cell differentiation toward mature cholangiocytes during NMP.CONCLUSIONS: Favorable bile chemistry during NMP correlates well with better-preserved biliary microvasculature and PBGs, with a preserved capacity for biliary regeneration. During NMP, biliary tree progenitor cells start to differentiate toward mature cholangiocytes, facilitating restoration of the ischemically damaged surface epithelium.</p

Sequential Hypothermic and Normothermic Machine Perfusion Enables Safe Transplantation of High-risk Donor Livers

Ex situ normothermic machine perfusion (NMP) is increasingly used for viability assessment of high-risk donor livers, whereas dual hypothermic oxygenated machine perfusion (DHOPE) reduces ischemia-reperfusion injury. We aimed to resuscitate and test the viability of initially-discarded, high-risk donor livers using sequential DHOPE and NMP with two different oxygen carriers: an artificial hemoglobin-based oxygen carrier (HBOC) or red blood cells (RBC). In a prospective observational cohort study of 54 livers that underwent DHOPE-NMP, the first 18 procedures were performed with a HBOC-based perfusion solution and the subsequent 36 procedures were performed with an RBC-based perfusion solution for the NMP phase. All but one livers were derived from extended criteria donation after circulatory death donors, with a median donor risk index of 2.84 (IQR 2.52-3.11). After functional assessment during NMP, 34 livers (63% utilization), met the viability criteria and were transplanted. One-year graft and patient survival were 94% and 100%, respectively. Post-transplant cholangiopathy occurred in 1 patient (3%). There were no significant differences in utilization rate and post-transplant outcomes between the HBOC and RBC group. Ex situ machine perfusion using sequential DHOPE-NMP for resuscitation and viability assessment of high-risk donor livers results in excellent transplant outcomes, irrespective of the oxygen carrier used

Dissecting complex transcriptional responses using pathway-level scores based on prior information

<p>Abstract</p> <p>Background</p> <p>The genomewide pattern of changes in mRNA expression measured using DNA microarrays is typically a complex superposition of the response of multiple regulatory pathways to changes in the environment of the cells. The use of prior information, either about the function of the protein encoded by each gene, or about the physical interactions between regulatory factors and the sequences controlling its expression, has emerged as a powerful approach for dissecting complex transcriptional responses.</p> <p>Results</p> <p>We review two different approaches for combining the noisy expression levels of multiple individual genes into robust pathway-level differential expression scores. The first is based on a comparison between the distribution of expression levels of genes within a predefined gene set and those of all other genes in the genome. The second starts from an estimate of the strength of genomewide regulatory network connectivities based on sequence information or direct measurements of protein-DNA interactions, and uses regression analysis to estimate the activity of gene regulatory pathways. The statistical methods used are explained in detail.</p> <p>Conclusion</p> <p>By avoiding the thresholding of individual genes, pathway-level analysis of differential expression based on prior information can be considerably more sensitive to subtle changes in gene expression than gene-level analysis. The methods are technically straightforward and yield results that are easily interpretable, both biologically and statistically.</p

cAMP/PKA signaling balances respiratory activity with mitochondria dependent apoptosis via transcriptional regulation

Background Appropriate control of mitochondrial function, morphology and biogenesis are crucial determinants of the general health of eukaryotic cells. It is therefore imperative that we understand the mechanisms that co-ordinate mitochondrial function with environmental signaling systems. The regulation of yeast mitochondrial function in response to nutritional change can be modulated by PKA activity. Unregulated PKA activity can lead to the production of mitochondria that are prone to the production of ROS, and an apoptotic form of cell death. Results We present evidence that mitochondria are sensitive to the level of cAMP/PKA signaling and can respond by modulating levels of respiratory activity or committing to self execution. The inappropriate activation of one of the yeast PKA catalytic subunits, Tpk3p, is sufficient to commit cells to an apoptotic death through transcriptional changes that promote the production of dysfunctional, ROS producing mitochondria. Our data implies that cAMP/PKA regulation of mitochondrial function that promotes apoptosis engages the function of multiple transcription factors, including HAP4, SOK2 and SCO1. Conclusions We propose that in yeast, as is the case in mammalian cells, mitochondrial function and biogenesis are controlled in response to environmental change by the concerted regulation of multiple transcription factors. The visualization of cAMP/TPK3 induced cell death within yeast colonies supports a model that PKA regulation plays a physiological role in coordinating respiratory function and cell death with nutritional status in budding yeast

Integrative Analysis of the Mitochondrial Proteome in Yeast

In this study yeast mitochondria were used as a model system to apply, evaluate, and integrate different genomic approaches to define the proteins of an organelle. Liquid chromatography mass spectrometry applied to purified mitochondria identified 546 proteins. By expression analysis and comparison to other proteome studies, we demonstrate that the proteomic approach identifies primarily highly abundant proteins. By expanding our evaluation to other types of genomic approaches, including systematic deletion phenotype screening, expression profiling, subcellular localization studies, protein interaction analyses, and computational predictions, we show that an integration of approaches moves beyond the limitations of any single approach. We report the success of each approach by benchmarking it against a reference set of known mitochondrial proteins, and predict approximately 700 proteins associated with the mitochondrial organelle from the integration of 22 datasets. We show that a combination of complementary approaches like deletion phenotype screening and mass spectrometry can identify over 75% of the known mitochondrial proteome. These findings have implications for choosing optimal genome-wide approaches for the study of other cellular systems, including organelles and pathways in various species. Furthermore, our systematic identification of genes involved in mitochondrial function and biogenesis in yeast expands the candidate genes available for mapping Mendelian and complex mitochondrial disorders in humans

BayesPI - a new model to study protein-DNA interactions: a case study of condition-specific protein binding parameters for Yeast transcription factors

<p>Abstract</p> <p>Background</p> <p>We have incorporated Bayesian model regularization with biophysical modeling of protein-DNA interactions, and of genome-wide nucleosome positioning to study protein-DNA interactions, using a high-throughput dataset. The newly developed method (BayesPI) includes the estimation of a transcription factor (TF) binding energy matrices, the computation of binding affinity of a TF target site and the corresponding chemical potential.</p> <p>Results</p> <p>The method was successfully tested on synthetic ChIP-chip datasets, real yeast ChIP-chip experiments. Subsequently, it was used to estimate condition-specific and species-specific protein-DNA interaction for several yeast TFs.</p> <p>Conclusion</p> <p>The results revealed that the modification of the protein binding parameters and the variation of the individual nucleotide affinity in either recognition or flanking sequences occurred under different stresses and in different species. The findings suggest that such modifications may be adaptive and play roles in the formation of the environment-specific binding patterns of yeast TFs and in the divergence of TF binding sites across the related yeast species.</p