Rapid eradication of colon carcinoma by Clostridium perfringens Enterotoxin suicidal gene therapy

Background Bacterial toxins have evolved to an effective therapeutic option for cancer therapy. The Clostridium perfringens enterotoxin (CPE) is a pore- forming toxin with selective cytotoxicity. The transmembrane tight junction proteins claudin-3 and -4 are known high affinity CPE receptors. Their expression is highly upregulated in human cancers, including breast, ovarian and colon carcinoma. CPE binding to claudins triggers membrane pore complex formation, which leads to rapid cell death. Previous studies demonstrated the anti-tumoral effect of treatment with recombinant CPE-protein. Our approach aimed at evaluation of a selective and targeted cancer gene therapy of claudin-3- and/or claudin-4- expressing colon carcinoma in vitro and in vivo by using translation optimized CPE expressing vector. Methods In this study the recombinant CPE and a translation optimized CPE expressing vector (optCPE) was used for targeted gene therapy of claudin-3 and/or -4 overexpressing colon cancer cell lines. All experiments were performed in the human SW480, SW620, HCT116, CaCo-2 and HT-29 colon cancer and the isogenic Sk-Mel5 and Sk-Mel5 Cldn-3-YFP melanoma cell lines. Claudin expression analysis was done at protein and mRNA level, which was confirmed by immunohistochemistry. The CPE induced cytotoxicity was analyzed by the MTT cytotoxicity assay. In addition patient derived colon carcinoma xenografts (PDX) were characterized and used for the intratumoral in vivo gene transfer of the optCPE expressing vector in PDX bearing nude mice. Results Claudin-3 and -4 overexpressing colon carcinoma lines showed high sensitivity towards both recCPE application and optCPE gene transfer. The positive correlation between CPE cytotoxicity and level of claudin expression was demonstrated. Transfection of optCPE led to targeted, rapid cytotoxic effects such as membrane disruption and necrosis in claudin overexpressing cells. The intratumoral optCPE in vivo gene transfer led to tumor growth inhibition in colon carcinoma PDX bearing mice in association with massive necrosis due to the intratumoral optCPE expression. Conclusions This novel approach demonstrates that optCPE gene transfer represents a promising and efficient therapeutic option for a targeted suicide gene therapy of claudin-3 and/or claudin-4 overexpressing colon carcinomas, leading to rapid and effective tumor cell killing in vitro and in vivo

Minimizing ergot infection in hybrid rye by a SMART breeding approach

In Hybridsorten bei Winterroggen führt das Restorergen Rfp1 zu einer vollständigen Restauration der männlichen Fertilität und trägt dazu bei, die Kontamination des Erntegutes mit Mutterkorn zu minimieren. Wir beschreiben Ergebnisse zur Validierung neuer Rfp1-Selektionsmarker an Elitezuchtmaterial des Roggens. Für alle per molekularer Markeranalyse genotypisierten Individuen wurde eine perfekte Übereinstimmung zwischen der postulierten genetischen Konstitution am Restorerlocus Rfp1 und dem Pollenschüttungsvermögen in den Testkreuzungsnachkommenschaften beobachtet. Rekombination zwischen den untersuchten Rfp1-Markern zeigt, dass die neuen Selektionsmarker dazu geeignet sind, rekombinativ verkleinerte, Rfp1-tragende Genomsegmente zu identifizieren. Es konnte gezeigt werden, dass die mit Rfp1 assoziierten, eng gekoppelten Markerallele diagnostisch für Rfp1 sind. Die vorgestellten Ergebnisse belegen, dass die neuen Selektionsmarker eine effiziente Genotypisierung aktueller Elitezuchtlinien im Hinblick auf das Rfp1-Restorergen ermöglichen. Für die mit Rfp1 assoziierten Selektionsmarker konnte in der vorliegenden Studie auch Kopplung mit dem Restorergen Rfc1 nachgewiesen werden. Diese Kopplungsbeziehungen lassen die validierten Marker daher für eine indirekte Selektion von Maintainer-Genotypen des C-Plasmas geeignet erscheinen. Die beobachtete Kopplung von STS-Markern zu Rfp1 bzw. Rfc1 bestätigt frühere Vermutungen, dass in dieser Region auf Chromosom 4RL entweder mehrere Restorergene lokalisiert sind oder es sich bei den betreffenden Restorergenen um Allele desselben Genortes handelt.The restorer gene Rfp1 results in an almost complete restoration of male fertility in hybrid rye varieties and, thus, contributes to minimize harvest contamination with ergot. Here, we report on the validation of recently established Rfp1 markers located on chromosome 4RL in elite breeding lines of rye. The Rfp1 genotypes, as deduced by molecular-marker analysis, perfectly corresponded with the degree of male fertility assessed in test crosses of individual genotpes with male sterile testers. Recombination could be observed between Rfp1 markers indicating their potential to reduce the donor chromosome segment carrying Rfp1. These marker alleles proved to be diagnostic for Rfp1 in current breeding lines. Taken together, results presented qualify the novel markers as efficent molecular tools to assess the restorer gene Rfp1 in elite breeding lines of rye. In addition, we have observed linkage of the Rfp1 markers to the restorer gene Rfc1. Thus, the validated markers should be applicable for marker-assisted selection strategies of maintainer genotypes of the male sterility inducing C cytoplasm, which occur at low frequency in European rye populations as well. The observed linkage of the STS markers to both Rfp1 and Rfc1 supports the assumption that the restorer genes identified on chromosome 4RL are either alleles of a single restorer gene or represent different linked genes located in this sub-genomic region

Peri-operative red blood cell transfusion in neonates and infants: NEonate and Children audiT of Anaesthesia pRactice IN Europe: A prospective European multicentre observational study

BACKGROUND: Little is known about current clinical practice concerning peri-operative red blood cell transfusion in neonates and small infants. Guidelines suggest transfusions based on haemoglobin thresholds ranging from 8.5 to 12 g dl-1, distinguishing between children from birth to day 7 (week 1), from day 8 to day 14 (week 2) or from day 15 (≥week 3) onwards. OBJECTIVE: To observe peri-operative red blood cell transfusion practice according to guidelines in relation to patient outcome. DESIGN: A multicentre observational study. SETTING: The NEonate-Children sTudy of Anaesthesia pRactice IN Europe (NECTARINE) trial recruited patients up to 60 weeks' postmenstrual age undergoing anaesthesia for surgical or diagnostic procedures from 165 centres in 31 European countries between March 2016 and January 2017. PATIENTS: The data included 5609 patients undergoing 6542 procedures. Inclusion criteria was a peri-operative red blood cell transfusion. MAIN OUTCOME MEASURES: The primary endpoint was the haemoglobin level triggering a transfusion for neonates in week 1, week 2 and week 3. Secondary endpoints were transfusion volumes, 'delta haemoglobin' (preprocedure - transfusion-triggering) and 30-day and 90-day morbidity and mortality. RESULTS: Peri-operative red blood cell transfusions were recorded during 447 procedures (6.9%). The median haemoglobin levels triggering a transfusion were 9.6 [IQR 8.7 to 10.9] g dl-1 for neonates in week 1, 9.6 [7.7 to 10.4] g dl-1 in week 2 and 8.0 [7.3 to 9.0] g dl-1 in week 3. The median transfusion volume was 17.1 [11.1 to 26.4] ml kg-1 with a median delta haemoglobin of 1.8 [0.0 to 3.6] g dl-1. Thirty-day morbidity was 47.8% with an overall mortality of 11.3%. CONCLUSIONS: Results indicate lower transfusion-triggering haemoglobin thresholds in clinical practice than suggested by current guidelines. The high morbidity and mortality of this NECTARINE sub-cohort calls for investigative action and evidence-based guidelines addressing peri-operative red blood cell transfusions strategies. TRIAL REGISTRATION: ClinicalTrials.gov, identifier: NCT02350348

Brasetto hybrid winter rye

Brasetto is one of the first two hybrid winter (fall) rye (Secale cereale L.) cultivars to be registered for production in Canada. In registration trials, its grain yield was 42 percent higher than the mean of the check entries and 23 percent higher than the highest yielding reference cultivar, Hazlet. It has a very high falling number, which is of special interest in the food market. Except for lower grain protein content and a slightly higher ergot (Claviceps purpurea (Fr.) Tul.) level, the rest of its measured agronomic and quality characters have been better than or within the range of the Fall Rye Co-operative Registration Trial check cultivars in Canada. Its expected endâ use will be primarily for food and feed grain.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Additional file 4: Figure S3. of Rapid eradication of colon carcinoma by Clostridium perfringens Enterotoxin suicidal gene therapy

Influence of optCPE in vivo gene transfer on body weight. Body weight of Co7515* PDX bearing mice was measured during tumor growth inhibition. In all animals no systemic toxicities, such as body weight loss, were observed, which strongly indicates the safety of this gene therapeutic approach. (JPG 283 kb

Additional file 2: Figure S1. of Rapid eradication of colon carcinoma by Clostridium perfringens Enterotoxin suicidal gene therapy

Knockdown of claudin-3 and -4 leads to reduced CPE activity in human colon cancer cells. a Sequences of used short interfering RNA (siRNA) targeting claudin-3 and -4. b Western blot analysis for claudin-3 and claudin-4 gene expression in human colon cancer cell lines SW480 (left panel) and HCT116 (right panel) 72 h after siRNA treatment, showing an efficient down-regulation of both with two independent siRNA compared to control (siCo). c Specific toxin responsiveness of claudin-3 and -4 down-regulated colon cancer cells. 72 h after siRNA transfection tumor cells were treated with recCPE at indicated concentrations for another 72 h. The cytotoxicity was determined by MTT assay and compared to siCo treated cells. A significantly reduced responsiveness (*** P < 0.0001) was demonstrated in both colon cancer cell lines, SW480 (left panel) and HCT116 (right panel). All assays were performed in two independent experiments and are expressed as mean percent of untreated control. Bars: SD. Level of significance was calculated by 2way-ANOVA (Bonferroni posttest). b Cytotoxicity of optCPE gene transfer in siRNA treated colon cancer cells and proof of claudin specificity. The siCldn3 + siCldn4 treated SW480 and HCT116 cells were transfected with optCPE construct 72 h after siRNA treatment. MTT assay was performed 72 h after CPE treatment and a significantly reduced CPE mediated cytotoxicity was observed in down-regulated SW480 (left panel) and also in HCT116 (right panel) cells compared to siCo treated cells. All assays were performed in two independent experiments and expressed as survival in optical density [OD]. Bars: SD. Level of significance was calculated by nonparametric, unpaired students t-test, *** P < 0.0001. Both assays demonstrate high selectivity of CPE on claudin-3 and -4 as down-regulated cells remain unaffected. (JPG 600 kb

A translocation breakpoint cluster disrupts the newly defined 3´end of the SNURF-SNRPN transcription unit on chromosome 15

Balanced translocations affecting the paternal copy of 15q11–q13 are a rare cause of Prader–Willi syndrome (PWS) or PWS-like features. Here we report on the cytogenetic and molecular characterization of a de novo balanced reciprocal translocation t(X;15)(q28;q12) in a female patient with atypical PWS. The translocation breakpoints in this patient and two previously reported patients map 70–80 kb distal to the SNURF-SNRPN gene and define a breakpoint cluster region. The breakpoints disrupt one of several hitherto unknown 3′ exons of this gene. Using RT–PCR we demonstrate that sequences distal to the breakpoint, including the recently identified C/D box small nucleolar RNA (snoRNA) gene cluster HBII-85 as well as IPW and PAR1, are not expressed in the patient. Our data suggest that lack of expression of these sequences contributes to the PWS phenotype

Towards a Biohybrid Lung: Endothelial Cells Promote Oxygen Transfer through Gas Permeable Membranes

In patients with respiratory failure, extracorporeal lung support can ensure the vital gas exchange via gas permeable membranes but its application is restricted by limited long-term stability and hemocompatibility of the gas permeable membranes, which are in contact with the blood. Endothelial cells lining these membranes promise physiological hemocompatibility and should enable prolonged application. However, the endothelial cells increase the diffusion barrier of the blood-gas interface and thus affect gas transfer. In this study, we evaluated how the endothelial cells affect the gas exchange to optimize performance while maintaining an integral cell layer. Human umbilical vein endothelial cells were seeded on gas permeable cell culture membranes and cultivated in a custom-made bioreactor. Oxygen transfer rates of blank and endothelialized membranes in endothelial culture medium were determined. Cell morphology was assessed by microscopy and immunohistochemistry. Both setups provided oxygenation of the test fluid featuring small standard deviations of the measurements. Throughout the measuring range, the endothelial cells seem to promote gas transfer to a certain extent exceeding the blank membranes gas transfer performance by up to 120%. Although the underlying principles hereof still need to be clarified, the results represent a significant step towards the development of a biohybrid lung

A translocation breakpoint cluster disrupts the newly defined 3′ end of the SNURF-SNRPN transcription unit on chromosome 15

Balanced translocations affecting the paternal copy of 15q11-q13 are a rare cause of Prader-Willi syndrome(PWS) or PWS-like features. Here we report on the cytogenetic and molecular characterization of a denovo balanced reciprocal translocation t(X;15)(q28;q12) in a female patient with atypical PWS. The translocation breakpoints in this patient and two previously reported patients map 70-80 kb distal to the SNURF-SNRPN gene and define a breakpoint cluster region. The breakpoints disrupt one of several hitherto unknown 3′ exons of this gene. Using RT-PCR we demonstrate that sequences distal to the breakpoint, including the recently identified C/D box small nucleolar RNA (snoRNA) gene cluster HBII-85 as well as IPW and PAR1, are not expressed in the patient. Our data suggest that lack of expression of these sequences contributes to the PWS phenotyp