CFTR is a tumor suppressor gene in murine and human intestinal cancer.

This corrects the article DOI: 10.1038/onc.2015.483.

CFTR is a tumor suppressor gene in murine and human intestinal cancer.

CFTR, the cystic fibrosis (CF) gene, encodes for the CFTR protein that plays an essential role in anion regulation and tissue homeostasis of various epithelia. In the gastrointestinal (GI) tract CFTR promotes chloride and bicarbonate secretion, playing an essential role in ion and acid-base homeostasis. Cftr has been identified as a candidate driver gene for colorectal cancer (CRC) in several Sleeping Beauty DNA transposon-based forward genetic screens in mice. Further, recent epidemiological and clinical studies indicate that CF patients are at high risk for developing tumors in the colon. To investigate the effects of CFTR dysregulation on GI cancer, we generated Apc(Min) mice that carried an intestinal-specific knockout of Cftr. Our results indicate that Cftr is a tumor suppressor gene in the intestinal tract as Cftr mutant mice developed significantly more tumors in the colon and the entire small intestine. In Apc(+/+) mice aged to ~1 year, Cftr deficiency alone caused the development of intestinal tumors in >60% of mice. Colon organoid formation was significantly increased in organoids created from Cftr mutant mice compared with wild-type controls, suggesting a potential role of Cftr in regulating the intestinal stem cell compartment. Microarray data from the Cftr-deficient colon and the small intestine identified dysregulated genes that belong to groups of immune response, ion channel, intestinal stem cell and other growth signaling regulators. These associated clusters of genes were confirmed by pathway analysis using Ingenuity Pathway Analysis and gene set enrichment analysis (GSEA). We also conducted RNA Seq analysis of tumors from Apc(+/+) Cftr knockout mice and identified sets of genes dysregulated in tumors including altered Wnt ß-catenin target genes. Finally we analyzed expression of CFTR in early stage human CRC patients stratified by risk of recurrence and found that loss of expression of CFTR was significantly associated with poor disease-free survival.

The role of KCNQ1 in mouse and human gastrointestinal cancers.

Kcnq1, which encodes for the pore-forming α-subunit of a voltage-gated potassium channel, was identified as a gastrointestinal (GI) tract cancer susceptibility gene in multiple Sleeping Beauty DNA transposon-based forward genetic screens in mice. To confirm that Kcnq1 has a functional role in GI tract cancer, we created Apc(Min) mice that carried a targeted deletion mutation in Kcnq1. Results demonstrated that Kcnq1 is a tumor suppressor gene as Kcnq1 mutant mice developed significantly more intestinal tumors, especially in the proximal small intestine and colon, and some of these tumors progressed to become aggressive adenocarcinomas. Gross tissue abnormalities were also observed in the rectum, pancreas and stomach. Colon organoid formation was significantly increased in organoids created from Kcnq1 mutant mice compared with wild-type littermate controls, suggesting a role for Kcnq1 in the regulation of the intestinal crypt stem cell compartment. To identify gene expression changes due to loss of Kcnq1, we carried out microarray studies in the colon and proximal small intestine. We identified altered genes involved in innate immune responses, goblet and Paneth cell function, ion channels, intestinal stem cells, epidermal growth factor receptor and other growth regulatory signaling pathways. We also found genes implicated in inflammation and in cellular detoxification. Pathway analysis using Ingenuity Pathway Analysis and Gene Set Enrichment Analysis confirmed the importance of these gene clusters and further identified significant overlap with genes regulated by MUC2 and CFTR, two important regulators of intestinal homeostasis. To investigate the role of KCNQ1 in human colorectal cancer (CRC), we measured protein levels of KCNQ1 by immunohistochemistry in tissue microarrays containing samples from CRC patients with liver metastases who had undergone hepatic resection. Results showed that low expression of KCNQ1 expression was significantly associated with poor overall survival.

Primary infrarenal aortic stenting with or without iliac stenting for isolated and aortoiliac stenoses: single-centre experience with long-term follow-up.

PURPOSE: The purpose of this study was to evaluate the technical success, complications, long-term clinical outcome, and patency after primary infrarenal aortic stenting for aortic and aortoiliac stenosis. Between January 1999 and January 2006, 22 consecutive patients underwent endovascular treatment because of infrarenal aortic stenosis with and without common iliac stenosis (10 men; mean age 64 ± 14 years). Eleven (11 of 22) patients had an isolated aortic stenosis, whereas 11 of 22 had aortic stenosis that extended into the common iliac arteries (CIAs). Thirteen patients were Rutherford classification type 3, and 9 patients were type 4. Statistical analysis included paired Student t test and Kaplan-Meier life table analysis; p < 0.05 was considered significant. Technical and initial clinical success was achieved in all patients. There were three (14 %) procedure-related complications, which included two access-point pseudoaneurysms and one non-flow-limiting left external iliac dissection. Patients were followed-up for a mean period of 88 months (range 60-132). Mean preprocedure ankle brachial pressure indexes (ABPI) were 0.60 ± -0.15 (right) and 0.61 ± -0.16 (left). After the procedure they were 0.86 ± -0.07 (right) and 0.90 ± -0.09 (left). The increase in ABPI was significant (p < 0.05), and this continued throughout follow-up. Four (18 %) patients had recurrence of symptoms during follow-up. These occurred at 36, 48, 48, and 50 months after the original procedure. All four patients were successfully treated with repeat angioplasty procedures. There was a significant difference in primary patency between isolated aortic stenosis (100 %) and aortoiliac stenosis (60 %) (p = 0.031). Cumulative follow-up was 1920 months yielding a reintervention rate of 0.025/events/year. CONCLUSION: Primary stenting of infrarenal stenosis is safe and successful with a low reintervention rate. It should be considered as first-line treatment for patients with infrarenal aortic stenotic disease.

Replacement tunnelled dialysis catheters for haemodialysis access: Same site, new site, or exchange - a multivariate analysis and risk score.

AIM: To identify variables related to complications following tunnelled dialysis catheter (TDC) replacement and stratifying the risk to reduce morbidity in patients with end-stage renal disease. MATERIALS AND METHODS: One hundred and forty TDCs (Split Cath, medCOMP) were replaced in 140 patients over a 5 year period. Multiple variables were retrospectively collected and analysed to stratify the risk and to predict patients who were more likely to suffer from complications. Multivariate regression analysis was used to identify variables predictive of complications. RESULTS: There were six immediate complications, 42 early complications, and 37 late complications. Multivariate analysis revealed that variables significantly associated to complications were: female sex (p = 0.003; OR 2.9); previous TDC in the same anatomical position in the past (p = 0.014; OR 4.1); catheter exchange (p = 0.038; OR 3.8); haemoglobin <11 g/dl (p = 0.033; OR 3.6); albumin <30 g/l (p = 0.007; OR 4.4); prothrombin time >15 s (p = 0.002; OR 4.1); and C-reactive protein >50 mg/l (p = 0.007; OR 4.6). A high-risk score, which used the values from the multivariate analysis, predicted 100% of the immediate complications, 95% of the early complications, and 68% of the late complications. CONCLUSION: Patients can now be scored prior to TDC replacement. A patient with a high-risk score can be optimized to reduce the chance of complications. Further prospective studies to confirm that rotating the site of TDC reduces complications are warranted as this has implications for current guidelines.

Recent research on fumonisins: a review.

Fumonisins are well known mycotoxins produced by Fusarium verticillioides, F. proliferatum and other Fusarium species. Many new fumonisins and fumonisin-like compounds have been detected by mass spectrometry in cultures of F. verticillioides. Recently, fumonisins B(2) and B(4) were produced by Aspergillus niger isolated from coffee and fumonisin B(2) in A. niger from grapes. Fumonisin B(2) was itself detected in coffee beans, wine and beer, adding to the list of foodstuffs and feedstuffs other than corn (maize) and sorghum in which fumonisins have been found in recent years. Fumonisin B(1) (FB(1)) can bind to proteins (PB FB(1)) and to other matrix components during food processing involving heat. The occurrence of bound fumonisins in processed corn foods is common. Another type of binding (or association) relates to observed instability of fumonisins in rice flour, corn starch and corn meal at room temperature; this can affect the immunoaffinity column clean-up procedure in analysis of naturally contaminated starch-containing corn foods for fumonisins. The occurrence of N-fatty acylated fumonisin derivatives in retail fried corn foods has also been demonstrated. Bioaccessibility of free FB(1) and total bound FB(1) (TB FB(1)) present in corn flakes has been estimated by in vitro digestion experiments. Intentional binding of fumonisins to cholestyramine has been demonstrated in vivo and is a potential means of detoxification of animal feed.

Application of isotope dilution mass spectrometry: determination of ochratoxin A in the Canadian Total Diet Study.

Analytical methods are generally developed and optimized for specific commodities. Total Diet Studies, representing typical food products 'as consumed', pose an analytical challenge since every food product is different. In order to address this technical challenge, a selective and sensitive analytical method was developed suitable for the quantitation of ochratoxin A (OTA) in Canadian Total Diet Study composites. The method uses an acidified solvent extraction, an immunoaffinity column (IAC) for clean-up, liquid chromatography-tandem mass spectrometry (LC-MS/MS) for identification and quantification, and a uniformly stable isotope-labelled OTA (U-[(13)C(20)]-OTA) as an internal recovery standard. Results are corrected for this standard. The method is accurate (101% average recovery) and precise (5.5% relative standard deviation (RSD)) based on 17 duplicate analysis of various food products over 2 years. A total of 140 diet composites were analysed for OTA as part of the Canadian Total Diet Study. Samples were collected at retail level from two Canadian cities, Quebec City and Calgary, in 2008 and 2009, respectively. The results indicate that 73% (102/140) of the samples had detectable levels of OTA, with some of the highest levels of OTA contamination found in the Canadian bread supply.

Ochratoxin A in cocoa and chocolate sampled in Canada.

In order to determine the levels of ochratoxin A (OTA) in cocoa and cocoa products available in Canada, a previously published analytical method, with minor modifications to the extraction and immunoaffinity clean-up and inclusion of an evaporation step, was initially used (Method I). To improve the low method recoveries (46-61%), 40% methanol was then included in the aqueous sodium bicarbonate extraction solvent (pH 7.8) (Method II). Clean-up was on an Ochratest™ immunoaffinity column and OTA was determined by liquid chromatography (LC) with fluorescence detection. Recoveries of OTA from spiked cocoa powder (0.5 and 5 ng g(-1)) were 75-84%; while recoveries from chocolate were 93-94%. The optimized method was sensitive (limit of quantification (LOQ) = 0.07-0.08 ng g(-1)), accurate (recovery = 75-94%) and precise (coefficient of variation (CV) < 5%). It is applicable to cocoa and chocolate. Analysis of 32 samples of cocoa powder (16 alkalized and 16 natural) for OTA showed an incidence of 100%, with concentrations ranging from 0.25 to 7.8 ng g(-1); in six samples the OTA level exceeded 2 ng g(-1), the previously considered European Union limit for cocoa. The frequency of detection of OTA in 28 chocolate samples (21 dark or baking chocolate and seven milk chocolate) was also 100% with concentrations ranging from 0.05 to 1.4 ng g(-1); one sample had a level higher than the previously considered European Union limit for chocolate (1 ng g(-1)).

Surveys of rice sold in Canada for aflatoxins, ochratoxin A and fumonisins.

Approximately 200 samples of rice (including white, brown, red, black, basmati and jasmine, as well as wild rice) from several different countries, including the United States, Canada, Pakistan, India and Thailand, were analysed for aflatoxins, ochratoxin A (OTA) and fumonisins by separate liquid chromatographic methods in two different years. The mean concentrations for aflatoxin B(1) (AFB(1)) were 0.19 and 0.17 ng g(-1) with respective positive incidences of 56% and 43% (≥ the limit of detection (LOD) of 0.002 ng g(-1)). Twenty-three samples analysed in the second year also contained aflatoxin B(2) (AFB(2)) at levels ≥LOD of 0.002 ng g(-1). The five most contaminated samples in each year contained 1.44-7.14 ng AFB(1) g(-1) (year 1) and 1.45-3.48 ng AFB(1) g(-1) (year 2); they were mostly basmati rice from India and Pakistan and black and red rice from Thailand. The average concentrations of ochratoxin A (OTA) were 0.05 and 0.005 ng g(-1) in year 1 and year 2, respectively; incidences of samples containing ≥LOD of 0.05 ng g(-1) were 43% and 1%, respectively, in the 2 years. All positive OTA results were confirmed by LC-MS/MS. For fumonisins, concentrations of fumonisin B(1) (FB(1)) averaged 4.5 ng g(-1) in 15 positive samples (≥0.7 ng g(-1)) from year 1 (n = 99); fumonisin B(2) (FB(2)) and fumonisin B(3) (FB(3)) were also present (≥1 ng g(-1)). In the second year there was only one positive sample (14 ng g(-1) FB(1)) out of 100 analysed. All positive FB(1) results were confirmed by LC-MS/MS.

Sampling of cereals and cereal-based foods for the determination of ochratoxin A: an overview.

The mycotoxin ochratoxin A (OTA) is known to be heterogeneously distributed both intrinsically (from one individual food item to the next) as well as distributionally (throughout a sample of individual food items) in cereals and cereal-based foods. Therefore, proper sampling and sample comminution are special challenges, but are prerequisites for obtaining sound analytical data. This paper outlines the issue of the sampling process for cereals and cereal-based foods, starting with the planning phase, followed by the sampling step itself and the formation of analytical samples. The sampling of whole grain and retail-level cereal-based foods will be discussed. Furthermore, possibilities to reduce sampling variance are presented.

Determination of the cyanobacterial toxin cylindrospermopsin in algal food supplements.

For the analysis of blue-green algal food supplements for cylindrospermopsin (CYN), a C18 solid-phase extraction column and a polygraphitized carbon solid-phase extraction column in series was an effective procedure for the clean-up of extracts. Determination of CYN was by liquid chromatography with ultraviolet light detection. At extract spiking levels of CYN equivalent to 25-500 µg g(-1), blue-green algal supplement recoveries were in the range 70-90%. CYN was not detected in ten samples of food supplements and one chocolate product, all containing blue-green algae. The limit of detection for the method was 16 µg g(-1), and the limit of quantification was 52 µg g(-1).

Clinical outcomes following endovascular treatment of the malfunctioning autologous dialysis fistula.

BACKGROUND: There is limited long-term prospective data on the use of endovascular techniques and the use of thrombolysis in malfunctioning autologous haemodialysis fistulas. PURPOSE: Prospective assessment of clinical outcomes following angioplasty with or without low-dose thrombolysis was undertaken in patients who presented with malfunctioning autologous haemodialysis fistulas. METHODS: Consecutive patients referred to our department over a 6-month period were included. Twenty-five patients underwent percutaneous intervention by angioplasty alone (n = 14), angioplasty and stent (n = 2), thrombolysis alone (n = 2), angioplasty, thrombolysis and stent (n = 2) and angioplasty and thrombolysis (n = 5). Patients underwent clinical follow-up and were reviewed at 6, 12, 18 and 24 months to determine fistula status. Thirty-day mortality in the group was two patients. Statistical analysis was performed with Mann-Whitney, chi-squared and Kruskal-Wallis tests. Kaplan-Meier curves were constructed to compare primary and secondary patency rates. RESULTS: Technical success and initial clinical success rates were 88% and 76%, respectively. Primary and secondary clinical success rates at 6 months were 68% and 72%, at 12 months were 68% and 72%, at 18 months were 60% and 68% and at 24 months were 52% and 68%, respectively. There were no major complications following interventional procedures. There were four minor complications. After an initially successful procedure, five patients required subsequent intervention during the follow-up period. The overall fistula event rate was very low (five per 600 patient months or 0.0996 per access year) with a fistula loss rate of 0.14 per access year. CONCLUSIONS: Our results confirm that excellent clinical results can be achieved by percutaneous endovascular treatment in malfunctioning autologous fistulas, justifying their continued use as first-line management.

Bioaccessibility of total bound fumonisin from corn flakes.

Fumonisin B1 (FB1) is often found as a natural contaminant of corn and corn-based food. Several publications have demonstrated the presence of fumonisin bound to proteins and to other compounds of the matrix. In spite of the low oral bioavailability of FB1 in rats, pigs, chickens, cows, and monkeys, FB1 can cause agriculturally significant disease and possibly human cancer. The aim of this work was to determine the bioaccessibility of total bound FB1 (TB FB1) (percentage of TB FB1, released from corn flakes to the chyme) after in vitro digestion. Two samples of corn flakes washed with solvents were incubated with gastrointestinal tract solutions simulating saliva plus stomach and duodenal juices. After hydrolysis of the chyme with KOH, TB FB1 was determined as hydrolyzed FB1 (HFB1). The bioaccessibility of TB FB1 in chyme from corn flakes was 37-64%, indicating that these derivatives should be considered in evaluation of exposure to fumonisin.

Phytophthora multivora sp. nov., a new species recovered from declining Eucalyptus, Banksia, Agonis and other plant species in Western Australia.

A new Phytophthora species, isolated from rhizosphere soil of declining or dead trees of Eucalyptus gomphocephala, E. marginata, Agonis flexuosa, and another 13 plant species, and from fine roots of E. marginata and collar lesions of Banksia attenuata in Western Australia, is described as Phytophthora multivora sp. nov. It is homothallic and produces semipapillate sporangia, smooth-walled oogonia containing thick-walled oospores, and paragynous antheridia. Although morphologically similar to P. citricola, phylogenetic analyses of the ITS and cox1 gene regions demonstrate that P. multivora is unique. Phytophthora multivora is pathogenic to bark and cambium of E. gomphocephala and E. marginata and is believed to be involved in the decline syndrome of both eucalypt species within the tuart woodland in south-west Western Australia.

Mycotoxins in breakfast cereals from the Canadian retail market: a 3-year survey.

One hundred and fifty-six samples of breakfast cereals were collected from the Canadian retail marketplace over a 3-year period. The samples were analysed for the mycotoxins deoxynivalenol, nivalenol, HT-2 toxin, zearalenone, ochratoxin A, and fumonisins B1 and B2 to contribute to dietary exposure estimates in support of the development of Canadian guidelines for selected mycotoxins in foods. The samples included corn-, oat-, wheat- and rice-based cereals, as well as mixed-grain cereals, and were primarily from North American processors. Overall, deoxynivalenol was the most frequently detected mycotoxin--it was detected in over 40% of all samples analysed. Fumonisins and ochratoxin A were each detected in over 30% of all samples. Zearalenone was detected in over 20% of all samples. Nivalenol and HT-2 toxin were each detected in only one sample. The survey clearly demonstrated regular occurrence of low levels of multiple mycotoxins in breakfast cereals on the Canadian market.

Mycotoxins in botanicals and dried fruits: a review.

Botanicals are used in many countries for medicinal and general health-promoting purposes. Numerous natural occurrences of mycotoxins in botanicals and dried fruits have been reported. Aflatoxins or ochratoxin A (OTA) have been found in botanicals such as ginseng, ginger, liquorice, turmeric, and kava-kava in the USA, Spain, Argentina, India, and some other countries, while fumonisins have been found in medicinal wild plants in South Africa and in herbal tea and medicinal plants in Turkey. Zearalenone was identified in ginseng root. Dried fruits can be contaminated with aflatoxins, OTA, kojic acid, and, occasionally, with patulin or zearalenone. One main area of concern is aflatoxins in dried figs; bright greenish yellow fluorescence under ultraviolet light is associated with aflatoxin contamination. OTA in dried vine fruits (raisins, sultanas, and currants) is another concern. There are also reports of aflatoxins in raisins and OTA in dried figs, apricots, dried plums (prunes), dates, and quince. Maximum permitted levels in the European Union include 4 microg kg(-1) for total aflatoxins in dried fruit intended for direct consumption and 10 microg kg(-1) for OTA in dried vine fruit. This review discusses the occurrence of mycotoxins in botanicals and dried fruits and analytical issues such as sampling, sample preparation, and methods for analysis. Fungal contamination of these products, the influence of sorting, storage, and processing, and prevention are also considered.

Analysis of ergot alkaloids - a review.

Methods for detection and determination of ergot alkaloids in grains, grasses, feeds and grain foods are reviewed. They incorporate simple detection procedures - colorimetry, thin layer chromatography and enzyme-linked immunosorbent assay - or instrumental procedures such as liquid chromatography with fluorescence, mass spectrometric (MS) or MS/MS detection, capillary zone electrophoresis, and direct MS/MS.

Effect ofin vitro digestion on fumonisin B1 in corn flakes.

Low levels of fumonisins have been found frequently in corn based breakfast cereals and can occur bound to protein and other matrix components.In vitro digestion of two samples of corn flakes was carried out under "fed conditions." Fumonisins were measured as o-phthaldialdehyde/mercaptoethanol derivatives by LC-fluorescence. One sample of corn flakes (FN12) had high concentrations of fumonisin B1 (FB) (average 125 ng/g) and total bound FB1, (TB FB1) (average 92 ng/g) and the other (FN11) had a low level of free FB1 (average 29 ng/g) and no detectable TB FB1. After incubation of the samples with gastrointestinal tract solutions simulating saliva plus stomach and duodenal juices, chyme was analysed for FB1, hydrolyzed FB1 (HFB1) and partially hydrolyzed fumonisin B1 (PHFB1). The bioaccessibility (percentage of FB1 released from corn flakes into chyme) was 38-78% for incurred FB1 in FN12, 8-54% for incurred plus spiked FB1 in FN12, and 19-66% for incurred plus spiked FB1 in FN11. HFB1 and PHFB1 were not detected. If free FB1 was first extracted from sample FN12, no FB1 was detected in the chyme, indicating no contribution from TB FB1. Concentrations were corrected for method recovery of FB1 or, for bound FB1, partial method recovery of HFB1.

Survey of breakfast and infant cereals for aflatoxins B1, B2, G1 and G2.

Three hundred and forty-nine breakfast and infant cereal samples were collected at retail level across Canada from 2002 to 2005. They included rice-, soy-, barley-based and mixed-grain infant cereals, corn-, wheat-, rice-based and mixed-grain breakfast cereals, and were analysed for aflatoxins B1, B2, G1 and G2 using a modified AOAC International official method. An immunoaffinity column was used for the cleanup and purification of extracts. Determination of aflatoxins was by LC using post-column derivatization with pyridinium hydrobromide perbromide and fluorescence detection. Results indicated that 50% of both breakfast and infant cereals had detectable levels (limit of detection = 0.002 ng g-1) of aflatoxin B1, which is the most toxic of the four toxins. The levels found varied from 0.002 to 1.00 ng g-1 for aflatoxin B1, from 0.002 to 0.14 ng g-1 for aflatoxin B2, from 0.008 to 0.27 ng g-1 for aflatoxin G1, and from 0.008 to 0.048 ng g-1 for aflatoxin G2. Only 4% of the breakfast cereals and 1% of the infant cereals had aflatoxin B1 levels exceeding 0.1 ng g-1, which is the European Union maximum limit for aflatoxin B1 in baby foods and processed cereal-based foods for infants and young children.