Loss of HR6B ubiquitin-conjugating activity results in damaged synaptonemal complex structure and increased crossing-over frequency during the male meiotic prophase.

The ubiquitin-conjugating enzymes HR6A and HR6B are the two mammalian homologs of Saccharomyces cerevisiae RAD6. In yeast, RAD6 plays an important role in postreplication DNA repair and in sporulation. HR6B knockout mice are viable, but spermatogenesis is markedly affected during postmeiotic steps, leading to male infertility. In the present study, increased apoptosis of HR6B knockout primary spermatocytes was detected during the first wave of spermatogenesis, indicating that HR6B performs a primary role during the meiotic prophase. Detailed analysis of HR6B knockout pachytene nuclei showed major changes in the synaptonemal complexes. These complexes were found to be longer. In addition, we often found depletion of synaptonemal complex proteins from near telomeric regions in the HR6B knockout pachytene nuclei. Finally, we detected an increased number of foci containing the mismatc

Collagen Peptides in Urine: A New Promising Biomarker for the Detection of Colorectal Liver Metastases

Introduction:For both patients and the outpatient clinic the frequent follow-up visits after a resection of colorectal cancer (CRC) are time consuming and due to large patient numbers expensive. Therefore it is important to develop an effective non-invasive test for the detection of colorectal liver metastasis (CRLM) which could be used outside the hospital. The urine proteome is known to provide detailed information for monitoring changes in the physiology of humans. Urine collection is non-invasive and urine naturally occurring peptides (NOPs) have the advantage of being easily accessible without labour-intensive sample preparation. These advantages make it potentially useful for a quick and reliable application in clinical settings. In this study, we will focus on the identification and validation of urine NOPs to discriminate patients with CRLM from healthy controls.Materials and Methods:Urine samples were collected from 24 patients with CRLM and 25 healthy controls. In the first part of the study, samples were measured with a nano liquid chromatography (LC) system (Thermo Fisher Scientific, Germaring, Germany) coupled on-line to a hybrid linear ion trap/Orbitrap mass spectrometer (LTQ-Orbitrap-XL, Thermo Fisher Scientific, Bremen, Germany). A discovery set was used to construct the model and consecutively the validation set, being independent from the discovery set, to check the acquired model. From the peptides which were selected, multiple reaction monitoring (MRM's) were developed on a UPLC-MS/MS system.Results:Seven peptides were selected and applied in a discriminant analysis a sensitivity of 84.6% and a specificity of 92.3% were established (Canonical correlation:0.797, Eigenvalue:1.744, F:4.49, p:0.005). The peptides AGPP(-OH)GEAGKP(-OH)GEQGVP(-OH)GDLGA P(-OH)GP and KGNSGEP(-OH)GAPGSKGDTGAKGEP(-OH)GPVG were selected for further quantitative analysis which showed a sensitivity of 88% and a specificity of 88%.Conclusion:Urine proteomic analysis revealed two very promising peptides, both part from collagen type 1, AGPP(-OH)GEAGKP(-OH)GEQGVP(-OH)GDLGAP(-OH)GP and KGNSGEP(-OH)GAPGSKGDTGAKGEP(-OH)GPVG which could detect CRLM in a non-invasive manner

The ubiquitin-conjugating DNA repair enzyme is a maternal factor essential for early embryonic development in mice

The Saccharomyces cerevisiae RAD6 protein is required for a surprising diversity of cellular processes, including sporulation and replicational damage bypass of DNA lesions. In mammals, two RAD6-related genes, HR6A and HR6B, encode highly homologous proteins. Here, we describe the phenotype of cells and mice deficient for the mHR6A gene. Just like mHR6B knockout mouse embryonic fibroblasts, mHR6A-deficient cells appear to have normal DNA damage resistance properties, but mHR6A knockout male and female mice display a small decrease in body weight. The necessity for at least one functional mHR6A (X-chromosomal) or mHR6B (autosomal) allele in all somatic cell types is supported by the fact that neither animals lacking both proteins nor females with only one intact mHR6A allele are viable. In striking contrast to mHR6B knockout males, which show a severe spermatogenic defect, mHR6A knockout males are normally fertile. However, mHR6A knockout females fail to produce offspring despite a normal ovarian histology and ovulation. The absence of mHR6A in oocytes prevents development beyond the embryonic two-cell stage but does not result in an aberrant methylation pattern of histone H

Cell-free microRNAs as early predictors of graft viability during ex vivo normothermic machine perfusion of human donor livers

Background: Cell-free microRNAs (miRs) have emerged as early and sensitive biomarkers for tissue injury and function. This study aimed to investigate whether the release of hepatocyte-derived microRNAs (HDmiRs) and cholangiocyte-derived miRs (CDmiRs) correlates with hepato-cholangiocellular injury and function during oxygenated, normothermic machine perfusion (NMP) of human liver grafts. Methods: Donor livers (n = 12), declined for transplantation, were subjected to oxygenated NMP (6 hours) after a period of static cold storage (median 544 minutes (IQR 421-674)). Perfusate and bile samples were analyzed by qRT-PCR for HDmiR-122 and CDmiR-222. Spearman correlations were performed between miR levels and currently available indicators and classic markers. Results: Both HDmiR-122 and CDmiR-222 levels in perfusate at 30 minutes of NMP strongly correlated with hepatocyte injury (peak perfusate AST) and cholangiocyte injury (peak biliary LDH). In bile, only CDmiR-222 correlated with these injury markers. For hepato-cholangiocellular function, both miRs in perfusate correlated with total bilirubin, while HDmiR-122 (in perfusate) and CDmiR-222 (in bile) correlated with bicarbonate secretion. Both the relative ratio of HDmiR-122/CDmiR-222 and AST in perfusate at 30 minutes significantly correlated with cumulative bile production, but only the relative ratio was predictive of histopathological injury after 6 hours NMP. Conclusion: Early levels of HDmiR-122 and CDmiR-222, in perfusate and/or bile, are predictive of excretory functions and hepato-cholangiocellular injury after 6 hours NMP. These miRs may represent new biomarkers for graft viability and function during machine perfusion

Fast, robust and effective decellularization of whole human livers using mild detergents and pressure controlled perfusion

Human whole-liver perfusion-decellularization is an emerging technique for producing bio-scaffolds for tissue engineering purposes. The native liver extracellular matrix (ECM) provides a superior microenvironment for hepatic cells in terms of adhesion, survival and function. However, current decellularization protocols show a high degree of variation in duration. More robust and effective protocols are required, before human decellularized liver ECM can be considered for tissue engineering applications. The aim of this study is to apply pressure-controlled perfusion and test the efficacy of two different detergents in porcine and human livers. To test this, porcine livers were decellularized using two different protocols; a triton-x-100 (Tx100)-only protocol (N = 3) and a protocol in which Tx100 was combined with SDS (N = 3) while maintaining constant pressure of 120 mm Hg. Human livers (N = 3) with different characteristics (age, weight and fat content) discarded for transplantation were decellularized using an adapted version of the Tx-100-only protocol. Decellularization efficacy was determined by histology and analysis of DNA and RNA content. Furthermore, the preservation of ECM components was assessed. After completing the perfusion cycles with detergents the porcine li

Human Bile Contains Cholangiocyte Organoid-Initiating Cells Which Expand as Functional Cholangiocytes in Non-canonical Wnt Stimulating Conditions

Diseases of the bile duct (cholangiopathies) remain a common indication for liver transplantation, while little progress has been made over the last decade in understanding the underlying pathophysiology. This is largely due to lack of proper in vitro model systems to study cholangiopathies. Recently, a culture method has been developed that allows for expansion of human bile duct epithelial cells grown as extrahepatic cholangiocyte organoids (ncECOs) in non-canonical Wnt-stimulating conditions. These ncECOs closely resemble cholangiocytes in culture and have shown to efficiently repopulate collagen scaffolds that could act as functional biliary tissue in mice. Thus far, initiation of ncECOs required tissue samples, thereby limiting broad patient-specific applications. Here, we report that bile fluid, which can be less invasively obtained and with low risk for the patients, is an alternative source for culturing ncECOs. Further characterization showed that bile-derived cholangiocyte organoids (ncBCOs) are highly similar to ncECOs obtained from bile duct tissue biopsies. Compared to the previously reported bile-cholangiocyte organoids cultured in canonical Wnt-stimulation conditions, ncBCOs have superior function of cholangiocyte ion channels and are able to respond to secretin and somatostatin. In conclusion, bile is a new, less invasive, source for patient-derived cholangiocyte organoids and makes their regenerative medicine applications more safe and feasible

Human extrahepatic and intrahepatic cholangiocyte organoids show region-specific differentiation potential and model cystic fibrosis-related bile duct disease

The development, homeostasis, and repair of intrahepatic and extrahepatic bile ducts are thought to involve distinct mechanisms including proliferation and maturation of cholangiocyte and progenitor cells. This study aimed to characterize human extrahepatic cholangiocyte organoids (ECO) using canonical Wnt-stimulated culture medium previously developed for intrahepatic cholangiocyte organoids (ICO). Paired ECO and ICO were derived from common bile duct and liver tissue, respectively. Characterization showed both organoid types were highly similar, though some differences in size and gene expression were observed. Both ECO and ICO have cholangiocyte fate differentiation capacity. However, unlike ICO, ECO lack the potential for differentiation towards a hepatocyte-like fate. Importantly, ECO derived from a cystic fibrosis patient showed n

Evaluation of RNA isolation methods for microRNA quantification in a range of clinical biofluids

Background: Extracellular microRNAs (miRNAs), released from cells into biofluids, have emerged as promising biomarkers for diagnostic and prognostic purposes. Several RNA isolation methods are available for the analysis of these cell-free miRNAs by RT-qPCR. Not all methods, however, are equally suitable for different biofluids. Here, we extracted total RNA from four very diverse biofluids: serum, urine, bile, and graft preservation fluid (perfusate). Four different protocols were used: a phenol-chloroform extraction and alcohol precipitation in combination with a precipitation carrier (QP) and three different column-based isolation methods, one with phenol-chloroform extraction (RN) and two without (NG and CU). For this range of clinical biofluid samples, we evaluated the potential of these different RNA isolation methods assessing recovery efficiency and the co-purification of RT-qPCR inhibiting compounds. Results: Differences were observed between each of the RNA isolation methods in the recovery of cel-miR-39, a synthetic miRNA spiked in during the workup procedure, and for endogenous miRNAs. Co-purification of heparin, a known RT-qPCR inhibitor, was assessed usin

Expression of the ubiquitin-conjugating DNA repair enzymes HHR6A and B suggests a role in spermatogenesis and chromatin modification.

RAD6, a member of the expanding family of ubiquitin-conjugating (E2) enzymes, functions in the so-called "N-rule" protein breakdown pathway of Saccharomyces cerevisiae. In vitro, the protein can attach one or multiple ubiquitin (Ub) moieties to histones H2A and B and trigger their E3-dependent degradation. Rad6 mutants display a remarkably pleiotropic phenotype, implicating the protein in DNA damage-induced mutagenesis, postreplication repair, repression of retrotransposition, and sporulation. RAD6 transcription is strongly induced upon UV exposure and in meiosis, suggesting that it is part of a damage-induced response pathway and that it is involved in meiotic recombination. It is postulated that the protein exerts its functions by modulating chromatin structure. Previously, we have cloned two human homologs of this gene (designated HHR6A and HHR6B) and demonstrated that they partially complement the yeast defect. Here we present a detailed characterisation of their expression at the transcript and protein levels. Both HHR6 proteins, resolved by 2-dimensional immunoblot analysis, are expressed in all mammalian tissues and cell types examined, indicating that both genes are functional and constitutively expressed. Although the proteins are highly conserved, the UV induction present in yeast is not preserved, pointing to important differences in damage response between yeast and mammals. Absence of alterations in HHR6 transcripts or protein upon heat shock and during the cell cycle suggests that the proteins are not involved in stress response or cell cycle regulation. Elevated levels of HHR6 transcripts and proteins were found in testis. Enhanced HHR6 expression did not coincide with meiotic recombination but with the replacement of histones by transition proteins. Immunohistochemistry demonstrated that the HHR6 proteins are located in the nucleus, consistent with a functional link with chromatin. Electron microscopy combined with immunogold labeling revealed a preferential localisation of HHR6 in euchromatin areas, suggesting that the protein is associated with transcriptionally active regions. Our findings support the idea that both HHR6 genes have overlapping, constitutive functions related to chromatin confo