Loss of mismatch repair signaling impairs the WNT-bone morphogenetic protein crosstalk and the colonic homeostasis.

The fine balance between proliferation, differentiation, and apoptosis in the colonic epithelium is tightly controlled by the interplay between WNT, Notch, and bone morphogenetic protein (BMP) signaling. How these complex networks coordinate the colonic homeostasis, especially if cancer predisposing mutations such as mutations in the DNA mismatch repair (MMR) are present, is unclear. Inactivation of the MMR system has long been linked to colorectal cancer; however, little is known about its role in the regulation of the colonic homeostasis. It has been shown that loss of MMR promotes the proliferation of colon epithelial cells that renders them highly susceptible to transformation. The mechanism through which MMR mediates this effect, yet, remains to be determined. Using an MMR-deficient mouse model, we show that increased methylation of Dickkopf1 impacts its expression, and consequently, the ability to negatively regulate WNT signaling. As a result, excessive levels of active ß-catenin promote strong crypt progenitor-like phenotype and abnormal proliferation. Under these settings, the development and function of the goblet cells are affected. MMR-deficient mice have fewer goblet cells with enlarged mucin-loaded vesicles. We further show that MMR inactivation impacts the WNT-BMP signaling crosstalk.

Determining lower threshold concentrations for synergistic effects.

Though only occurring rarely, synergistic interactions between chemicals in mixtures have long been a point of focus. Most studies analyzing synergistic interactions used unrealistically high chemical concentrations. The aim of the present study is to determine the threshold concentration below which proven synergists cease to act as synergists towards the aquatic crustacean Daphnia magna. To do this, we compared several approaches and test-setups to evaluate which approach gives the most conservative estimate for the lower threshold for synergy for three known azole synergists. We focus on synergistic interactions between the pyrethroid insecticide, alpha-cypermethrin, and one of the three azole fungicides prochloraz, propiconazole or epoxiconazole measured on Daphnia magna immobilization. Three different experimental setups were applied: A standard 48h acute toxicity test, an adapted 48h test using passive dosing for constant chemical exposure concentrations, and a 14-day test. Synergy was defined as occuring in mixtures where either EC50 values decreased more than two-fold below what was predicted by concentration addition (horizontal assessment) or as mixtures where the fraction of immobile organisms increased more than two-fold above what was predicted by independent action (vertical assessment). All three tests confirmed the hypothesis of the existence of a lower azole threshold concentration below which no synergistic interaction was observed. The lower threshold concentration, however, decreased with increasing test duration from 0.026±0.013µM (9.794±4.897µgL-1), 0.425±0.089µM (145.435±30.46µgL-1) and 0.757±0.253µM (249.659±83.44µgL-1) for prochloraz, propiconazole and epoxiconazole in standard 48h toxicity tests to 0.015±0.004µM (5.651±1.507µgL-1), 0.145±0.025µM (49.619±8.555µgL-1) and 0.122±0.0417µM (40.236±13.75µgL-1), respectively, in the 14-days tests. Testing synergy in relation to concentration addition provided the most conservative values. The threshold values for the vertical assessments in tests where the two could be compared were in general 1.2 to 4.7 fold higher than the horizontal assessments. Using passive dosing rather than dilution series or spiking did not lower the threshold significantly. Below the threshold for synergy, slight antagony could often be observed. This is most likely due to induction of enzymes active in metabolization of alpha-cypermethrin. The results emphasize the importance of test duration when assessing synergy, but also show that azole concentrations within the typically monitored range of up to 0.5µgL-1 are not likely to cause severe synergy concerning Daphnia magna immobilization.

Mutations in the bacterial ribosomal protein l3 and their association with antibiotic resistance.

Different groups of antibiotics bind to the peptidyl transferase center (PTC) in the large subunit of the bacterial ribosome. Resistance to these groups of antibiotics has often been linked with mutations or methylations of the 23S rRNA. In recent years, there has been a rise in the number of studies where mutations have been found in the ribosomal protein L3 in bacterial strains resistant to PTC-targeting antibiotics but there is often no evidence that these mutations actually confer antibiotic resistance. In this study, a plasmid exchange system was used to replace plasmid-carried wild-type genes with mutated L3 genes in a chromosomal L3 deletion strain. In this way, the essential L3 gene is available for the bacteria while allowing replacement of the wild type with mutated L3 genes. This enables investigation of the effect of single mutations in Escherichia coli without a wild-type L3 background. Ten plasmid-carried mutated L3 genes were constructed, and their effect on growth and antibiotic susceptibility was investigated. Additionally, computational modeling of the impact of L3 mutations in E. coli was used to assess changes in 50S structure and antibiotic binding. All mutations are placed in the loops of L3 near the PTC. Growth data show that 9 of the 10 mutations were well accepted in E. coli, although some of them came with a fitness cost. Only one of the mutants exhibited reduced susceptibility to linezolid, while five exhibited reduced susceptibility to tiamulin.

[A rare form of twin-twin transfusion syndrome].

Twin anaemia-polycythaemia sequence (TAPS) is a rare form of feto-fetal transfusion between monochorionic twins. It occurs spontaneously or after laser surgery for twin-twin transfusion syndrome. TAPS is characterized by a large inter-twin haemoglobin difference and can be detected both ante- and postnatally. This is a case report of TAPS, detected antenatally by a routine scanning of monochorionic twins in gestational week 29.

Pesticide cocktails can interact synergistically on aquatic crustaceans.

BACKGROUND, AIM AND SCOPE: The ergosterol biosynthesis-inhibiting (EBI) fungicide prochloraz can enhance the effect of other pesticides in a range of animal species. Approximately 50% of the fungicides used in Denmark are EBI fungicides. Hence, if they all have synergising potential, a risk assessment of pesticide mixtures based on additivity might not suffice. This study investigates the synergising potential of six different EBI fungicides representing the imidazoles (prochloraz), the triazoles (epoxiconazole, propiconazole and tebuconazole), the piperidines (fenpropidin) and the morpholines (fenpropimorph) together with the pyrethroid insecticide alpha-cypermethrin. MATERIALS AND METHODS: Tests were made on the aquatic crustacean Daphnia magna. Mixtures of each of the fungicides were tested together with the insecticide both at a 50:50% effect mixture ratio and, subsequently, in a ray design including five mixture ratios. The results were tested against the concentration addition reference model using dose-response surface analyses. RESULTS: The results of the binary dose-response surface studies showed that mixtures with prochloraz increased toxicity up to 12-fold compared with what was expected using the reference model concentration addition (CA). Epoxiconazole and propiconazole enhanced toxicity up to six and sevenfold, respectively. Fenpropimorph showed antagonism, whilst mixtures with tebuconazole and fenpropidin did not deviate statistically from CA. CONCLUSIONS: Hence, it can be concluded that both imidazoles and some, but not all, triazoles can enhance the effect of a pyrethroid insecticide towards D. magna substantially. Epoxiconazole and propiconazole are often sprayed out together with pyrethroids in tank mixtures. The extent to which this might create unforeseen ecological problems is discussed.