Label-Free Proteomics Approach Characterizes Plasma Protein Signature of Donor Brain Death

Purpose Despite recent advances in donation after circulatory death, transplants from brain-dead donors remain the sole source in heart transplantation (HTx) worldwide. Due to organ shortage, marginal donors are increasingly used and the utilization of transplants becomes markedly more challenging. They undergo invariably brain death that induces a systemic cytokine and catecholamine storm that lead to systemic inflammation, labile hemodynamics, and organ hypoperfusion. Together, these can damage the heart and aggravate later occurring graft injury, and ultimately, compromise graft function. However, the effect of donor brain death on allografts is not well understood. Methods In a separate prospective, blinded single-center trial, we collected donor plasma samples and relevant clinical patient data from 50 HTx brain-dead donors and as controls plasma samples from age- and gender-matched 23 healthy volunteers. Quantitative label-free proteomics in high definition MSE mode (HDMSE) was carried out on the samples. Various statistical analyses were performed on quantitative proteomics data to obtain the most reliably distinguishing proteins, which classify the donors vs controls. Results With two or more unique proteins per identification, 463 proteins were quantified in our pilot study. A complete separation between donors and controls based on a set of 278 proteins (p-valuePeer reviewe

3 '-UTR poly(T/U) repeat of EWSR1 is altered in microsatellite unstable colorectal cancer with nearly perfect sensitivity

Approximately 15 % of colorectal cancers exhibit instability of short nucleotide repeat regions, microsatellites. These tumors display a unique clinicopathologic profile and the microsatellite instability status is increasingly used to guide clinical management as it is known to predict better prognosis as well as resistance to certain chemotherapeutics. A panel of five repeats determined by the National Cancer Institute, the Bethesda panel, is currently the standard for determining the microsatellite instability status in colorectal cancer. Recently, a quasimonomorphic mononucleotide repeat 16T/U at the 3' untranslated region of the Ewing sarcoma breakpoint region 1 gene was reported to show perfect sensitivity and specificity in detecting mismatch repair deficient colorectal, endometrial, and gastric cancers in two independent populations. To confirm this finding, we replicated the analysis in 213 microsatellite unstable colorectal cancers from two independent populations, 148 microsatellite stable colorectal cancers, and the respective normal samples by PCR and fragment analysis. The repeat showed nearly perfect sensitivity for microsatellite unstable colorectal cancer as it was altered in 212 of the 213 microsatellite unstable (99.5 %) and none of the microsatellite stable colorectal tumors. This repeat thus represents the first potential single marker for detecting microsatellite instability.Peer reviewe

Using honey to heal diabetic foot ulcers

Diabetic ulcers seem to be arrested in the inflammatory/proliferative stage of the healing process, allowing infection and inflammation to preclude healing. Antibiotic-resistant bacteria have become a major cause of infections, including diabetic foot infections. It is proposed here that the modern developments of an ancient and traditional treatment for wounds, dressing them with honey, provide the solution to the problem of getting diabetic ulcers to move on from the arrested state of healing. Honeys selected to have a high level of antibacterial activity have been shown to be very effective against antibiotic-resistant strains of bacteria in laboratory and clinical studies. The potent anti-inflammatory action of honey is also likely to play an important part in overcoming the impediment to healing that inflammation causes in diabetic ulcers, as is the antioxidant activity of honey. The action of honey in promotion of tissue regeneration through stimulation of angiogenesis and the growth of fibroblasts and epithelial cells, and its insulin-mimetic effect, would also be of benefit in stimulating the healing of diabetic ulcers. The availability of honey-impregnated dressings which conveniently hold honey in place on ulcers has provided a means of rapidly debriding ulcers and removing the bacterial burden so that good healing rates can be achieved with neuropathic ulcers. With ischemic ulcers, where healing cannot occur because of lack of tissue viability, these honey dressings keep the ulcers clean and prevent infection occurring

Effects of ash removal by agitated aqueous washing and sedimentation on the physico-chemical characteristics and fast pyrolysis of trommel fines

A pre-treated trommel fines feedstock (DPT) with 35.1 wt% ash content and particle size range of 0.5–2 mm was processed through two (100% distilled water and 1% surfactant in distilled water) aqueous agitated washing and sedimentation procedures for ash reduction prior to fast pyrolysis in a bubbling fluidized bed reactor. The washing process led to more than 36% reduction in the ash/inorganic contents of the DPT feedstock and yielded about 78 wt% of organic-rich feedstocks denoted as WPT1 and WPT2. Characterisation and fast pyrolysis of all three feedstocks was carried out to evaluate the effect of the washing process on their physico-chemical characteristics and yields of fast pyrolysis products. Results showed that the ash reduction led to increase in the volatile matter contents of the washed feedstocks by 20%, while reducing nitrogen contents. In addition, fast pyrolysis of the feedstocks showed improved yield of liquid and gas products, with a dramatic reduction of reaction water, indicating that the ash removal reduced the catalytic effect of the ash on water formation during the fast pyrolysis process. The major organic compounds in the liquid products included phenols and furans from biogenic fraction of the feedstock as well as aromatic hydrocarbons such as those obtained from pyrolysis of plastics. More importantly, the overall energy yields from the fast pyrolysis process increased by over 35% after washing the feedstock, with washing with only distilled water alone giving the highest energy yield of 93%. Hence, coupling the water-washing ash reduction process with fast pyrolysis appeared to be a suitable technology for valorising feedstocks with high ash contents such as trommel fines for energy and chemicals

C2-O-sLeX Glycoproteins Are E-Selectin Ligands that Regulate Invasion of Human Colon and Hepatic Carcinoma Cells

Similar to mechanisms of recruitment of activated leukocytes to inflamed tissues, selectins mediate adhesion and extravasation of circulating cancer cells. Our objective was to determine whether sialyl Lewis X modified core 2 O-glycans (C2-O-sLeX) present on colon and hepatic carcinoma cells promote their adhesion and invasion. We examined membrane expression of C2-O-sLeX, selectin binding, invasion of human colon and hepatic carcinoma cell lines, and mRNA levels of alpha-2,3 fucosyltransferase (FucT-III) and core 2 beta-1,6 N-acetylglucosaminyltransferase (C2GnT1) genes, necessary for C2-O-sLeX synthesis, by quantitative reverse-transcriptase (RT) PCR. Synthesis of core 2 branched O-glycans decorated by sLeX is dependent on C2GnT1 function and thus we determined enzyme activity of C2GnT1. The cell lines that expressed C2GnT1 and FucT-III mRNA by quantitative RT-PCR were highly positive for C2-O-sLeX by flow cytometry, and colon carcinoma cells possessed highly active C2GnT1 enzyme. Cells bound avidly to E-selection but not to P- and L-selectin. Gene knock-down of C2GnT1 in colon and hepatic carcinoma cells using short hairpin RNAs (shRNA) resulted in a 40–90% decrease in C2-O-sLeX and a 30–50% decrease in E-selectin binding compared to control cells. Invasion of hepatic and colon carcinoma cells containing C2GnT1 shRNA was significantly reduced compared to control cells in Matrigel assays and C2GnT1 activity was down-regulated in the latter cells. The sLeX epitope was predominantly distributed on core 2 O-glycans on colon and hepatic carcinoma cells. Our findings indicate that C2GnT1 gene expression and the resulting C2-O-sLeX carbohydrates produced mediate the adhesive and invasive behaviors of human carcinomas which may influence their metastatic potential

The high affinity selectin glycan ligand C2-O-sLex and mRNA transcripts of the core 2 β-1,6-N-acetylglusaminyltransferase (C2GnT1) gene are highly expressed in human colorectal adenocarcinomas

<p>Abstract</p> <p>Background</p> <p>The metastasis of cancer cells and leukocyte extravasation into inflamed tissues share common features. Specialized carbohydrates modified with sialyl Lewis x (sLe^x) antigens on leukocyte membranes are ligands for selectin adhesion molecules on activated vascular endothelial cells at inflammatory sites. The activity of the enzyme core 2 β1,6 <it>N</it>-acetylglucosaminyltransferase (C2GnT1) in leukocytes greatly increases their ability to bind to endothelial selectins. C2GnT1 is essential for the synthesis of core 2-branched O-linked carbohydrates terminated with sLe^x(C2-O-sLe^x). Our goal was to determine the expression profiles of C2-O-sLe^xin the malignant progression and metastasis of colorectal adenocarcinomas. The well characterized CHO-131 monoclonal antibody (mAb) specifically recognizes C2-O-sLe^xpresent in human leukocytes and carcinoma cells. Using CHO-131 mAb, we investigated whether C2-O-sLe^xwas present in 113 human primary colorectal adenocarcinomas, 10 colorectal adenomas, 46 metastatic liver tumors, 28 normal colorectal tissues, and 5 normal liver tissues by immunohistochemistry. We also examined mRNA levels of the enzyme core 2 β1,6-<it>N</it>-acetylglucosaminyltransferase (C2GnT1) in 20 well, 15 moderately, and 2 poorly differentiated colorectal adenocarcinomas, and in 5 normal colorectal tissues by using quantitative real-time polymerase chain reactions (RT-PCR).</p> <p>Results</p> <p>We observed high reactivity with CHO-131 mAb in approximately 70% of colorectal carcinomas and 87% of metastatic liver tumors but a lack of reactivity in colorectal adenomas and normal colonic and liver tissues. Positive reactivity with CHO-131 mAb was very prominent in neoplastic colorectal glands of well to moderately differentiated adenocarcinomas. The most intense staining with CHO-131 mAb was observed at the advancing edge of tumors with the deepest invasive components.</p> <p>Finally, we analyzed C2GnT1 mRNA levels in 37 colorectal adenocarcinomas and 5 normal colorectal tissues by RT-PCR. Significantly, we observed a greater than 15-fold increase in C2GnT1 mRNA levels in colorectal adenocarcinomas compared to normal colorectal tissues.</p> <p>Conclusion</p> <p>C2-O-sLe^x, detected by the CHO-131 mAb, is a tumor associated antigen whose expression is highly upregulated in colorectal adenocarcinomas and metastatic liver tumors compared to normal tissues. C2-O-sLe^xis a potentially useful early predictor of metastasis.</p

Cancer risk in MLH1, MSH2 and MSH6 mutation carriers; different risk profiles may influence clinical management

Background: Lynch syndrome (LS) is associated with a high risk for colorectal cancer (CRC) and extracolonic malignancies, such as endometrial carcinoma (EC). The risk is dependent of the affected mismatch repair gene. The aim of the present study was to calculate the cumulative risk of LS related cancers in proven MLH1, MSH2 and MSH6 mutation carriers.Methods: The studypopulation consisted out of 67 proven LS families. Clinical information including mutation status and tumour diagnosis was collected. Cumulative risks were calculated and compared using Kaplan Meier survival analysis.Results: MSH6 mutation carriers, both males and females had the lowest risk for developing CRC at age 70 years, 54% and 30% respectively and the age of onset was delayed by 3-5 years in males. With respect to endometrial carcinoma, female MSH6 mutation carriers had the highest risk at age 70 years (61%) compared to MLH1 (25%) and MSH2 (49%). Also, the age of EC onset was delayed by 5-10 years in comparison with MLH1 and MSH2.Conclusions: Although the cumulative lifetime risk of LS related cancer is similar, MLH1, MSH2 and MSH6 mutations seem to cause distinguishable cancer risk profiles. Female MSH6 mutation carriers have a lower CRC risk and a higher risk for developing endometrial carcinoma. As a consequence, surveillance colonoscopy starting at age 30 years instead of 20-25 years is more suitable. Also, prophylactic hysterectomy may be more indicated in female MSH6 mutation carriers compared to MLH1 and MSH2 mutation carriers

Comprehensive evaluation of coding region point mutations in microsatellite-unstable colorectal cancer

Microsatellite instability (MSI) leads to accumulation of an excessive number of mutations in the genome, mostly small insertions and deletions. MSI colorectal cancers (CRCs), however, also contain more point mutations than microsatellite-stable (MSS) tumors, yet they have not been as comprehensively studied. To identify candidate driver genes affected by point mutations in MSI CRC, we ranked genes based on mutation significance while correcting for replication timing and gene expression utilizing an algorithm, MutSigCV. Somatic point mutation data from the exome kit-targeted area from 24 exome-sequenced sporadic MSI CRCs and respective normals, and 12 whole-genome-sequenced sporadic MSI CRCs and respective normals were utilized. The top 73 genes were validated in 93 additional MSI CRCs. The MutSigCV ranking identified several well-established MSI CRC driver genes and provided additional evidence for previously proposed CRC candidate genes as well as shortlisted genes that have to our knowledge not been linked to CRC before. Two genes, SMARCB1 and STK38L, were also functionally scrutinized, providing evidence of a tumorigenic role, for SMARCB1 mutations in particular. © 2018 The Authors. Published under the terms of the CC BY 4.0 licensePeer reviewe

Annual outpatient hysteroscopy and endometrial sampling (OHES) in HNPCC/Lynch syndrome (LS)

Background: LS women have a 40-60 % lifetime risk of endometrial cancer (EC). Most international guidelines recommend screening. However, data on efficacy are limited. Purpose: To assess the performance of OHES for EC screening in LS and compare it with transvaginal ultrasound (TVS) alone. Methods: A prospective observational cohort study of LS women attending a tertiary high-risk familial gynaecological cancer clinic was conducted. LS women opting for EC screening underwent annual OHES and TVS. Histopathological specimens were processed using a strict protocol. Data of women screened between October 2007 and March 2010 were analysed from a bespoke database. Histology was used as the gold standard. Diagnostic accuracy of OHES was compared with TVS using specificity, and positive (PLR) and negative (NLR) likelihood ratios. Results: Forty-one LS women underwent 69 screens (41 prevalent, 28 incident). Four (three prevalent, one incident) women were detected to have EC/atypical endometrial hyperplasia (AEH), five had endometrial polyps and two had endometrial hyperplasia (EH) on OHES. TVS detected two of four EC/AEH. OHES had similar specificity of 89.8 % (CI 79.2, 96.2 %), but higher PLR 9.8 (CI 4.6, 21) and lower NLR (zero) compared to TVS: specificity 84.75 %(CI 73, 92.8 %), PLR 3.28 (CI 1.04, 10.35) and NLR 0.59 (CI 0.22, 1.58). No interval cancers occurred over a median follow-up of 22 months. The annual incidence was 3.57 % (CI 0.09, 18.35) for EC, 10.71 % (CI 2.27, 28.23) for polyps and 21.4 % (CI 8.3, 40.1) for any endometrial pathology. Conclusions: Our findings suggest that in LS, annual OHES is acceptable and has high diagnostic accuracy for EC/AEH screening. Larger international studies are needed for confirmation, given the relatively small numbers of LS women at individual centres. It reinforces the current recommendation that endometrial sampling is crucial when screening these women. © 2012 Springer-Verlag

Inactivation of promoter 1B of APC causes partial gene silencing: evidence for a significant role of the promoter in regulation and causative of familial adenomatous polyposis

Familial adenomatous polyposis (FAP) is caused by germline mutations in the adenomatous polyposis coli (APC) gene. Two promoters, 1A and 1B, have been recognized in APC, and 1B is thought to have a minor role in the regulation of the gene. We have identified a novel deletion encompassing half of this promoter in the largest family (Family 1) of the Swedish Polyposis Registry. The mutation leads to an imbalance in allele-specific expression of APC, and transcription from promoter 1B was highly impaired in both normal colorectal mucosa and blood from mutation carriers. To establish the significance of promoter 1B in normal colorectal mucosa (from controls), expression levels of specific transcripts from each of the promoters, 1A and 1B, were examined, and the expression from 1B was significantly higher compared with 1A. Significant amounts of transcripts generated from promoter 1B were also determined in a panel of 20 various normal tissues examined. In FAP-related tumors, the APC germline mutation is proposed to dictate the second hit. Mutations leaving two or three out of seven 20-amino-acid repeats in the central domain of APC intact seem to be required for tumorigenesis. We examined adenomas from mutation carriers in Family 1 for second hits in the entire gene without any findings, however, loss of the residual expression of the deleterious allele was observed. Three major conclusions of significant importance in relation to the function of APC can be drawn from this study; (i) germline inactivation of promoter 1B is disease causing in FAP; (ii) expression of transcripts from promoter 1B is generated at considerable higher levels compared with 1A, demonstrating a hitherto unknown importance of 1B; (iii) adenoma formation in FAP, caused by impaired function of promoter 1B, does not require homozygous inactivation of APC allowing for alternative genetic models as basis for adenoma formation