Microhomology-mediated DNA strand annealing and elongation by human DNA polymerases λ and β on normal and repetitive DNA sequences

‘Classical' non-homologous end joining (NHEJ), dependent on the Ku70/80 and the DNA ligase IV/XRCC4 complexes, is essential for the repair of DNA double-strand breaks. Eukaryotic cells possess also an alternative microhomology-mediated end-joining (MMEJ) mechanism, which is independent from Ku and DNA ligase 4/XRCC4. The components of the MMEJ machinery are still largely unknown. Family X DNA polymerases (pols) are involved in the classical NHEJ pathway. We have compared in this work, the ability of human family X DNA pols β, λ and μ, to promote the MMEJ of different model templates with terminal microhomology regions. Our results reveal that DNA pol λ and DNA ligase I are sufficient to promote efficient MMEJ repair of broken DNA ends in vitro, and this in the absence of auxiliary factors. However, DNA pol β, not λ, was more efficient in promoting MMEJ of DNA ends containing the (CAG)n triplet repeat sequence of the human Huntingtin gene, leading to triplet expansion. The checkpoint complex Rad9/Hus1/Rad1 promoted end joining by DNA pol λ on non-repetitive sequences, while it limited triplet expansion by DNA pol β. We propose a possible novel role of DNA pol β in MMEJ, promoting (CAG)n triplet repeats instabilit

Impact of deoxynivalenol on rainbow trout: Growth performance, digestibility, key gene expression regulation and metabolism

The impact of deoxynivalenol (DON) on rainbow trout, Oncorhynchus mykiss, is mainly characterised by impaired growth performance and reduced feed intake, usually with the total absence of any visible clinical signs. Despite the high concentrations of DON in the present study (up to 11,412 ± 1141 μg kg−1), no clinical signs (except anorexia at the higher DON dosage) were observed, which confirms the difficulties of diagnosing DON ingestion. Compared to the control group, the proteolytic enzyme activities (pepsin, trypsin and chymotrypsin) in trout were altered by DON ingestion. However, it was not clear if the observed impact on digestive enzymes was due to the direct action of DON, or a consequence of the lower feed intake determined for DON-treated animals. The impact of DON on the abundance of specific measured mRNA transcripts was unexpected with higher expression levels for insulin-like growth factors, igf1 and igf2, which are directly related to elevated insulin levels in plasma. This can also in part be influenced by the trypsin activity and by npy, given its higher mRNA expression levels. The apparent digestibility of dry matter, protein and energy was not affected by dietary levels of DON, however, nutrient retention, protein, fat and energy retention were significantly affected in animals fed DON. Adenylate cyclase-activating polypeptide (PACAP) expression seems to play an important role in controlling feed intake in DON fed trout. In the present study, we have shown for the first time that DON is metabolized to DON-3-sulfate in trout. DON-3-sulfate is much less toxic than DON, which helps to explain the lack of clinical signs in fish fed DON. Being a novel metabolite identified in trout makes it a potential biomarker of DON exposure. Suppression of appetite due to DON contamination in feeds might be a defense mechanism in order to decrease the exposure of the animal to DON, therefore reducing the potential negative impacts of DON

Toolbox for the Extraction and Quantification of Ochratoxin A and Ochratoxin Alpha Applicable for Different Pig and Poultry Matrices

Ochratoxin A (OTA) is one of the major mycotoxins causing severe effects on the health of humans and animals. Ochratoxin alpha (OTα) is a metabolite of OTA, which is produced through microbial or enzymatic hydrolysis, and one of the preferred routes of OTA detoxification. The methods described here are applicable for the extraction and quantification of OTA and OTα in several pig and poultry matrices such as feed, feces/excreta, urine, plasma, dried blood spots, and tissue samples such as liver, kidney, muscle, skin, and fat. The samples are homogenized and extracted. Extraction is either based on a stepwise extraction using ethyl acetate/sodium hydrogencarbonate/ethyl acetate or an acetonitrile/water mixture. Quantitative analysis is based on reversed-phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Method validation was successfully performed and the linearity, limit of quantification, accuracy, precision as well as the stability of the samples, were evaluated. The analyte recovery of the spiked samples was between 80 and 120% (80–150% for spiked concentrations ≤ 1 ng/g or ng/mL) and the relative standard deviation was ≤ 15%. Therefore, we provide a toolbox for the extraction and quantification of OTA and OTα in all relevant pig and poultry matrices

Comparison study of two fumonisin-degrading enzymes for detoxification in piglets

Fumonisins (FBs), particularly fumonisin B1 (FB1) and fumonisin B2 (FB2) produced mainly by Fusarium verticillioide and Fusarium proliferatum, are common contaminants in animal feed and pose a serious threat to both animal and human health. The use of microbial enzymes to efficiently and specifically convert fumonisins into non-toxic or low-toxic metabolites has emerged as the most promising approach. However, most of the available enzymes have only been evaluated in vitro and lack systematic evaluation in vivo. In this study, the detoxification efficacy of two carboxylesterases, FumD (FUMzyme®) and FumDSB, was evaluated comparatively in piglets. The results show that feeding piglets 4.4 mg/kg FBs-contaminated diets for 32 days did not significantly affect the average daily gain, organ indices, and immunoglobulins of the piglets. However, a significant reduction (21.2%) in anti-inflammatory cytokine interleukin-4 was observed in the FBs group, and supplementation with FUMzyme® and FumDSB significantly increased interleukin-4 by 62.1% and 28.0%, respectively. In addition, FBs-contaminated diets resulted in a 3-fold increase in the serum sphinganine/sphingosine (Sa/So) ratio, which is a specific biomarker that has been used to accurately reflect fumonisin levels. The serum Sa/So ratio was significantly reduced by 48.8% after the addition of FUMzyme®, and was insignificantly reduced by 8.2% in the FumDSB group. These results suggested that FUMzyme was more effective than FumDSB in mitigating FBs toxicity in piglets by down-regulating the Sa/So ratio

Complete switch from α\alpha-2,3- to α\alpha-2,6-regioselectivity in Pasteurella dagmatis β\beta- d -galactoside sialyltransferase by active-site redesign

Structure-guided active-site redesign of a family GT-80 β-D-galactoside sialyltransferase (from Pasteurella dagmatis) to change enzyme regioselectivity from α-2,3 in the wild type to α-2,6 in a P7H–M117A double mutant is reported. Biochemical data for sialylation of lactose together with protein crystal structures demonstrate highly precise enzyme engineering

Effect of Lipid Particle Biogenesis on the Subcellular Distribution of Squalene in the Yeast Saccharomyces cerevisiae*

Squalene belongs to the group of isoprenoids and is a precursor for the synthesis of sterols, steroids, and ubiquinons. In the yeast Saccharomyces cerevisiae, the amount of squalene can be increased by variation of growth conditions or by genetic manipulation. In this report, we show that a hem1Δ mutant accumulated a large amount of squalene, which was stored almost exclusively in cytoplasmic lipid particles/droplets. Interestingly, a strain bearing a hem1Δ deletion in a dga1Δlro1Δare1Δare2Δ quadruple mutant background (QMhem1Δ), which is devoid of the classical storage lipids, triacylglycerols and steryl esters, and lacks lipid particles, accumulated squalene at similar amounts as the hem1Δ mutant in a wild type background. In QMhem1Δ, however, increased amounts of squalene were found in cellular membranes, especially in microsomes. The fact that QMhem1Δ did not form lipid particles indicated that accumulation of squalene solely was not sufficient to initiate proliferation of lipid particles. Most importantly, these results also demonstrated that (i) squalene was not lipotoxic under the conditions tested, and (ii) organelle membranes in yeast can accommodate relatively large quantities of this non-polar lipid without compromising cellular functions. In summary, localization of squalene as described here can be regarded as an unconventional example of non-polar lipid storage in cellular membranes

Microhomology-mediated DNA strand annealing and elongation by human DNA polymerases λ and β on normal and repetitive DNA sequences

'Classical' non-homologous end joining (NHEJ), dependent on the Ku70/80 and the DNA ligase IV/XRCC4 complexes, is essential for the repair of DNA double-strand breaks. Eukaryotic cells possess also an alternative microhomology-mediated end-joining (MMEJ) mechanism, which is independent from Ku and DNA ligase 4/XRCC4. The components of the MMEJ machinery are still largely unknown. Family X DNA polymerases (pols) are involved in the classical NHEJ pathway. We have compared in this work, the ability of human family X DNA pols β, λ and μ, to promote the MMEJ of different model templates with terminal microhomology regions. Our results reveal that DNA pol λ and DNA ligase I are sufficient to promote efficient MMEJ repair of broken DNA ends in vitro, and this in the absence of auxiliary factors. However, DNA pol β, not λ, was more efficient in promoting MMEJ of DNA ends containing the (CAG)n triplet repeat sequence of the human Huntingtin gene, leading to triplet expansion. The checkpoint complex Rad9/Hus1/Rad1 promoted end joining by DNA pol λ on non-repetitive sequences, while it limited triplet expansion by DNA pol β. We propose a possible novel role of DNA pol β in MMEJ, promoting (CAG)n triplet repeats instability