An in-depth investigation of the microbiota and its virulence factors associated with severe udder cleft dermatitis lesions.

Udder cleft dermatitis (UCD) is a skin condition affecting the anterior parts of the udder in dairy cattle. In the present study, we aimed to shed light on the microbiota in severe UCD lesions versus healthy udder skin by putting forward a taxonomic and functional profile based on a virulence factor analysis. Through shotgun metagenomic sequencing, we found a high proportion of bacteria in addition to a low abundance of archaea. A distinct clustering of healthy udder skin versus UCD lesion samples was shown by applying principal component analysis and (sparse) partial least squares analysis on the metagenomic data. Proteobacteria, Bacillota, and Actinomycetota were among the most abundant phyla in healthy udder skin samples. In UCD samples, Bacteroidota was the most abundant phylum. At genus level, Bifidobacterium spp. had the highest relative abundance in healthy skin samples, whereas Porphyromonas spp. and Corynebacterium spp. had the highest relative abundance in UCD samples. In the differential abundance analysis, Porphyromonas spp. and Bacteroides spp. were significantly differentially abundant in UCD samples, whereas Bifidobacterium spp., Staphylococcus sp. AntiMn-1, and Staphylococcus equorum were more commonly found in healthy samples. Moreover, the abundance of several treponeme phylotypes was significantly higher in lesion samples. The streptococcal cysteine protease speB was among the most abundant virulence factors present in severe UCD lesions, while a plethora of virulence factors such as the antitoxin relB were downregulated, possibly contributing to creating the ideal wound climate for the dysbiotic community. Network analysis showed healthy lesion samples had a large network ofpositive, correlations between the abundances of beneficial species such as Aerococcus urinaeequi and Bifidobacterium angulatum, indicating that the healthy skin microbiome forms an active protective bacterial network, which is disrupted in case of UCD. In UCD samples, a smaller microbial network mainly consisting of positive correlations between the abundances of Bacteroides fragilis and anaerobic Bacteroidota was exposed. Moreover, a high correlation between the taxonomic data and virulence factors was revealed, concurrently with 2 separate networks of microbes and virulence factors. One network, matching with the taxonomic findings in the healthy udder skin samples, showcased a community of harmless or beneficial bacteria, such as Bifidobacterium spp. and Butyrivibrio proteoclasticus, associated with hcnB, hcnC, relB, glyoxalase, and cupin 2. The other network, corresponding to UCD samples, consisted of pathogenic or facultative pathogenic and mainly anaerobic bacteria such as Treponema spp., Mycoplasmopsis spp., and bovine gammaherpesvirus 4, that correlated with virulence factors SpvB, fhaB, and haemagglutination activity domain-associated factor. Our results point toward a dysbiotic community with a notable decrease in diversity and evenness, with a loss of normal skin inhabitants and innocuous or useful species making way for predominantly anaerobic, facultative pathogens. The shift in the abundance of virulence factors such as fhaB and SpvB could play a role in the manifestation of a local micro-environment favorable to the microbiome associated with udder skin lesions. Lastly, the presence of specific networks between microbial species, and between microbes and virulence factors was shown.

Comparative study of preimplantation development following distinct assisted oocyte activation protocols in a PLC-zeta knockout mouse model.

Mammalian fertilization encompasses a series of Ca2+ oscillations initiated by the sperm factor phospholipase C zeta (PLCζ). Some studies have shown that altering the Ca2+ oscillatory regime at fertilization affects preimplantation blastocyst development. However, assisted oocyte activation (AOA) protocols can induce oocyte activation in a manner that diverges profoundly from the physiological Ca2+ profiling. In our study, we used the newly developed PLCζ-null sperm to investigate the independent effect of AOA on mouse preimplantation embryogenesis. Based on previous findings, we hypothesized that AOA protocols with Ca2+ oscillatory responses might improve blastocyst formation rates and differing Ca2+ profiles might alter blastocyst transcriptomes. A total of 326 MII B6D2F1-oocytes were used to describe Ca2+ profiles and to compare embryonic development and individual blastocyst transcriptomes between four control conditions: C1 (in-vivo fertilization), C2 (ICSI control sperm), C3 (parthenogenesis) and C4 (ICSI-PLCζ-KO sperm) and four AOA groups: AOA1 (human recombinant PLCζ), AOA2 (Sr2+), AOA3 (ionomycin) and AOA4 (TPEN). All groups revealed remarkable variations in their Ca2+ profiles; however, oocyte activation rates were comparable between the controls (91.1% ± 13.8%) and AOA (86.9% ± 11.1%) groups. AOA methods which enable Ca2+ oscillatory responses (AOA1: 41% and AOA2: 75%) or single Ca2+ transients (AOA3: 50%) showed no significantly different blastocyst rates compared to ICSI control group (C2: 70%). In contrast, we observed a significant decrease in compaction (53% vs. 83%) and blastocyst rates (41% vs. 70%) in the absence of an initial Ca2+ trigger (AOA4) compared with the C2 group. Transcription profiles did not identify significant differences in gene expression levels between the ICSI control group (C2) and the four AOA groups.

Transcriptional landscape changes during human embryonic stem cell derivation.

STUDY QUESTION: What are the transcriptional changes occurring during the human embryonic stem cell (hESC) derivation process, from the inner cell mass (ICM) to post-ICM intermediate stage (PICMI) to hESC stage, that have downstream effects on pluripotency states and differentiation? SUMMARY ANSWER: We reveal that although the PICMI is transcriptionally similar to the hESC profile and distinct from ICM, it exhibits upregulation of primordial germ cell (PGC) markers, dependence on leukemia inhibitory factor (LIF) signaling, upregulation of naïve pluripotency-specific signaling networks and appears to be an intermediate switching point from naïve to primed pluripotency. WHAT IS KNOWN ALREADY: It is currently known that the PICMI exhibits markers of early and late-epiblast stage. It is suggested that hESCs acquire primed pluripotency features due to the upregulation of post-implantation genes in the PICMI which renders them predisposed towards differentiation cues. Despite this current knowledge, the transcriptional landscape changes during hESC derivation from ICM to hESC and the effect of PICMI on pluripotent state is still not well defined. STUDY DESIGN, SIZE, DURATION: To gain insight into the signaling mechanisms that may govern the ICM to PICMI to hESC transition, comparative RNA sequencing (RNA-seq) analysis was performed on preimplantation ICMs, PICMIs and hESCs in biological and technical triplicates (n = 3). PARTICIPANTS/MATERIALS, SETTING, AND METHODS: Primed hESCs (XX) were maintained in feeder-free culture conditions on Matrigel for two passages and approximately 50 cells were collected in biological and technical triplicates (n = 3). For ICM sample collection, Day 3, frozen-thawed human embryos were cultured up to day five blastocyst stage and only good quality blastocysts were subjected to laser-assisted micromanipulation for ICM collection (n = 3). Next, day six expanded blastocysts were cultured on mouse embryonic fibroblasts and manual dissection was performed on the PICMI outgrowths between post-plating Day 6 and Day 10 (n = 3). Sequencing of these samples was performed on NextSeq500 and statistical analysis was performed using edgeR (false discovery rate (FDR) < 0.05). MAIN RESULTS AND THE ROLE OF CHANCE: Comparative RNA-seq data analysis revealed that 634 and 560 protein-coding genes were significantly up and downregulated in hESCs compared to ICM (FDR < 0.05), respectively. Upon ICM to PICMI transition, 471 genes were expressed significantly higher in the PICMI compared to ICM, while 296 genes were elevated in the ICM alone (FDR < 0.05). Principle component analysis showed that the ICM was completely distinct from the PICMI and hESCs while the latter two clustered in close proximity to each other. Increased expression of E-CADHERIN1 (CDH1) in ICM and intermediate levels in the PICMI was observed, while CDH2 was higher in hESCs, suggesting a role of extracellular matrix components in facilitating pluripotency transition during hESC derivation. The PICMI also showed regulation of naïve-specific LIF and bone morphogenetic protein signaling, differential regulation of primed pluripotency-specific fibroblast growth factor and NODAL signaling pathway components, upregulation of phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway (PI3K/AKT/mTORC), as well as predisposition towards the germ cell lineage, further confirmed by gene ontology analysis. Hence, the data suggest that the PICMI may serve as an intermediate pluripotency stage which, when subjected to an appropriate culture niche, could aid in enhancing naïve hESC derivation and germ cell differentiation efficiency. LARGE-SCALE DATA: Gene Expression Omnibus (GEO) Accession number GSE119378. LIMITATIONS, REASONS FOR CAUTION: Owing to the limitation in sample availability, the sex of ICM and PICMI have not been taken into consideration. Obtaining cells from the ICM and maintaining them in culture is not feasible as it will hamper the formation of PICMI and hESC derivation. Single-cell quantitative real-time PCR on low ICM and PICMI cell numbers, although challenging due to limited availability of human embryos, will be advantageous to further corroborate the RNA-seq data on transcriptional changes during hESC derivation process. WIDER IMPLICATIONS OF THE FINDINGS: We elucidate the dynamics of transcriptional network changes from the naïve ICM to the intermediate PICMI stage and finally the primed hESC lines. We provide an in-depth understanding of the PICMI and its role in conferring the type of pluripotent state which may have important downstream effects on differentiation, specifically towards the PGC lineage. This knowledge contributes to our limited understanding of the true nature of the human pluripotent state in vitro. STUDY FUNDING/COMPETING INTEREST(S): This research is supported by the Concerted Research Actions funding from Bijzonder Onderzoeksfonds University Ghent (BOF GOA 01G01112).The authors declare no conflict of interest.

Chromosomal mosaicism in human blastocysts: the ultimate challenge of preimplantation genetic testing?

STUDY QUESTION: To what extent does a trophectoderm (TE) biopsy reliably reflect the chromosomal constitution of the inner cell mass (ICM) in human blastocysts? SUMMARY ANSWER: Concordance between TE and ICM was established in 62.1% of the embryos analysed. WHAT IS KNOWN ALREADY: Next generation sequencing (NGS) platforms have recently been optimised for preimplantation genetic testing for aneuploidies (PGT-A). However, higher sensitivity has led to an increase in reports of chromosomal mosaicism within a single TE biopsy. This has raised substantial controversy surrounding the prevalence of mosaicism in human blastocysts and the clinical implications of heterogeneity between the TE and ICM. STUDY DESIGN, SIZE, DURATION: To define the distribution and rate of mosaicism in human blastocysts, we assessed chromosomal profiles of the ICM and multiple TE portions obtained from the same embryo. We evaluated donated embryos with an unknown chromosomal profile (n = 34), as well as PGT-A blastocysts, previously diagnosed as abnormal or mosaic (n = 24). Our intra-embryo comparison included a total of 232 samples, obtained from 58 embryos. PARTICIPANTS/MATERIALS, SETTING, METHODS: Four embryo samples, including the ICM and three distinct TE portions, were acquired from good quality blastocysts by micromanipulation. Whole genome amplification (WGA), followed by NGS was performed on all embryo segments. Profiles were compared between samples from the same embryo, while the results from pretested blastocysts were further correlated to the original report. The embryos investigated in our untested group were obtained from good prognosis patients (n = 25), with maternal age ranging from 23 to 39 years. For the pretested embryo group, maternal age ranged from 23 to 40 years (n = 18). MAIN RESULTS AND THE ROLE OF CHANCE: We uncover chromosomal mosaicism, involving both numerical and structural aberrations, in up to 37.9% of the blastocysts analysed. Within the untested group, the overall concordance between the ICM and all TE portions was 55.9%. A normal ICM was detected in 20.6% of blastocysts for which at least one TE portion showed a chromosomal aberration. Conversely, 17.6% of embryos presented with mosaic or uniform abnormalities within the ICM, while showing normal or mosaic TE profiles. For the pretested blastocysts, the overall concordance between the ICM and all TE samples was 70.8%. However, 50% of embryos previously diagnosed with mosaicism did not confirm the original diagnosis. Notably, 31.3% of embryos with a mosaic aberration reported in the original TE biopsy, revealed a euploid profile in the ICM and all three TE samples. Taken together, concordance between the ICM and all TE portions was established in 62.1% of blastocysts, across both embryo groups. Finally, we could not observe a significant effect of age on embryo mosaicism (P = 0.101 untested group; P = 0.7309 pretested group). Similarly, ICM and TE quality were not found to affect the occurrence of chromosomal mosaicism (P = 0.718 and P = 0.462 untested group; P = 1.000 and P = 0.2885 pretested group). LARGE SCALE DATA: All data that support the findings of this study are available online in Vivar (http://cmgg.be/vivar) upon request. LIMITATIONS, REASONS FOR CAUTION: Evaluating biological variation in some instances remains challenging. The technological limitations of sampling mitotic errors that lead to mosaicism, as well as WGA artefacts, warrant careful interpretation. WIDER IMPLICATIONS OF THE FINDINGS: Our results highlight the complex nature of genetic (in)stability during early ontogenesis and indicate that blastocysts harbour a higher rate of chromosomal mosaicism than may have been anticipated. Moreover, our findings reveal an overall high diagnostic sensitivity and relatively low specificity in the context of PGT-A. This suggests that a considerable proportion of embryos are potentially being classified as clinically unsuitable. Ultimately, more precise quantification will benefit the clinical management of embryo mosaicism. STUDY FUNDING/COMPETING INTEREST(S): M.P. is supported by the Special Research Fund, Bijzonder Onderzoeksfonds (BOF01D08114). J.T. and L.D. are supported by the agency for innovation through science (131673, 141441). B.H. and this research are supported by the Special Research Fund, Bijzonder Onderzoeksfonds (BOF15/GOA/011). The authors declare no competing interests. TRIAL REGISTRATION NUMBER: Not applicable.

Direct comparison of distinct naive pluripotent states in human embryonic stem cells.

Until recently, human embryonic stem cells (hESCs) were shown to exist in a state of primed pluripotency, while mouse embryonic stem cells (mESCs) display a naive or primed pluripotent state. Here we show the rapid conversion of in-house-derived primed hESCs on mouse embryonic feeder layer (MEF) to a naive state within 5-6 days in naive conversion media (NCM-MEF), 6-10 days in naive human stem cell media (NHSM-MEF) and 14-20 days using the reverse-toggle protocol (RT-MEF). We further observe enhanced unbiased lineage-specific differentiation potential of naive hESCs converted in NCM-MEF, however, all naive hESCs fail to differentiate towards functional cell types. RNA-seq analysis reveals a divergent role of PI3K/AKT/mTORC signalling, specifically of the mTORC2 subunit, in the different naive hESCs. Overall, we demonstrate a direct evaluation of several naive culture conditions performed in the same laboratory, thereby contributing to an unbiased, more in-depth understanding of different naive hESCs.

Minimizing technical variation during sample preparation prior to label-free quantitative mass spectrometry.

Sample preparation is the crucial starting point to obtain high-quality mass spectrometry data and can be divided into two main steps in a bottom-up proteomics approach: cell/tissue lysis with or without detergents and a(n) (in-solution) digest comprising denaturation, reduction, alkylation, and digesting of the proteins. Here, some important considerations, among others, are that the reagents used for sample preparation can inhibit the digestion enzyme (e.g., 0.1% sodium dodecyl sulfate [SDS] and 0.5 M guanidine HCl), give rise to ion suppression (e.g., polyethylene glycol [PEG]), be incompatible with liquid chromatography-tandem mass spectrometry (LC-MS/MS) (e.g., SDS), and can induce additional modifications (e.g., urea). Taken together, all of these irreproducible effects are gradually becoming a problem when label-free quantitation of the samples is envisioned such as during the increasingly popular high-definition mass spectrometry (HDMS(E)) and sequential window acquisition of all theoretical fragment ion spectra (SWATH) data-independent acquisition strategies. Here, we describe the detailed validation of a reproducible method with sufficient protein yield for sample preparation without any known LC-MS/MS interfering substances by using 1% sodium deoxycholate (SDC) during both cell lysis and in-solution digest.

Combined prevalence of inherited skeletal disorders in dog breeds in Belgium.

Canine hip dysplasia (CHD), canine elbow dysplasia (CED), and humeral head osteochondrosis (HHOC) are inherited traits with uneven incidence in dog breeds. Knowledge of the combined prevalence of these three disorders is necessary to estimate the effect of the currently applied breeding strategies, in order to improve the genetic health of the population. Official screening results of the Belgian National Committee for Inherited Skeletal Disorders (NCSID) revealed that an average of 31.8% (CHD, CED, or both; n = 1273 dogs) and 47.2% (CHD, CED, HHOC, or a combination of these three diseases; n = 250 dogs) of dogs are mildly to severely affected by at least one skeletal disorder. According to the current breeding recommendations in some dog breeds in Belgium, these animals should be restricted (mild signs) or excluded (moderate to severe signs) from breeding. The introduction of genetic parameters, such as estimated breeding values, might create a better approach to gradually reduce the incidence of these complex inherited joint disorders, without compromising genetic population health.

The effects of positioning, reason for screening and the referring veterinarian on prevalence estimates of canine hip dysplasia.

Although the prevalence of canine hip dysplasia (HD) has been the subject of a number of published studies, estimates vary widely. This study evaluated several possible causes for these differences. Sixty Belgian, Dutch and German veterinarians were asked to submit all hip radiographs obtained for screening purposes (irrespective of HD status) over a 2-year period, resulting in a database of 583 dogs. Each set of radiographs was accompanied by information on the reason for screening (breeding soundness examination, clinical complaint, assistance dogs, or other reasons), and dog breed, date of birth and age. Dog positioning exerted an effect at multiple levels. The agreement among different observers regarding correct or incorrect positioning was limited and incorrect positioning itself reduced the inter-observer agreement for radiographic hip conformation. Dysplastic dogs were more commonly positioned incorrectly than non-dysplastic dogs. The clinical complaint population had a high prevalence of dysplastic dogs (>70%) compared with the breeding population (11%) and the assistance dogs (6%). There was a significantly lower prevalence of HD among cases referred by veterinarians who frequently submitted hip-extended radiographs for evaluation (P = 0.002) compared to those who refer less frequently. However, this was likely to be selection bias, as radiographs that were from dogs suspected to be dysplastic were not submitted by frequent senders. The prevalence of dysplastic dogs varied widely between breeds (16.7-71.4%). Dogs diagnosed with dysplasia were significantly older than dogs considered healthy (P = 0.001) and dogs classified as borderline dysplastic (P = 0.035). Inter-observer agreement for hip conformation was moderately low, resulting in >7% variation in prevalence estimates for dysplasia.

Reduced amounts and abnormal forms of phospholipase C zeta (PLCzeta) in spermatozoa from infertile men.

BACKGROUND: In mammals, oocyte activation at fertilization is thought to be induced by the sperm-specific phospholipase C zeta (PLCzeta). However, it still remains to be conclusively shown that PLCzeta is the endogenous agent of oocyte activation. Some types of human infertility appear to be caused by failure of the sperm to activate and this may be due to specific defects in PLCzeta. METHODS AND RESULTS: Immunofluorescence studies showed PLCzeta to be localized in the equatorial region of sperm from fertile men, but sperm deficient in oocyte activation exhibited no specific signal in this same region. Immunoblot analysis revealed reduced amounts of PLCzeta in sperm from infertile men, and in some cases, the presence of an abnormally low molecular weight form of PLCzeta. In one non-globozoospermic case, DNA analysis identified a point mutation in the PLCzeta gene that leads to a significant amino acid change in the catalytic region of the protein. Structural modelling suggested that this defect may have important effects upon the structure and function of the PLCzeta protein. cRNA corresponding to mutant PLCzeta failed to induce calcium oscillations when microinjected into mouse oocytes. Injection of infertile human sperm into mouse oocytes failed to activate the oocyte or trigger calcium oscillations. Injection of such infertile sperm followed by two calcium pulses, induced by assisted oocyte activation, activated the oocytes without inducing the typical pattern of calcium oscillations. CONCLUSIONS: Our findings illustrate the importance of PLCzeta during fertilization and suggest that mutant forms of PLCzeta may underlie certain types of human male infertility.

The HCV serum proteome: a search for fibrosis protein markers.

Liver fibrosis/cirrhosis is a serious health issue in hepatitis C virus (HCV-) infected patients and is currently diagnosed by the invasive liver biopsy. The aim of this study was to find useful fibrosis markers in HCV-patients' sera of different fibrosis degrees (METAVIR F0-F4) based on proteomics. Serum proteome profiles were created by two-dimensional gel electrophoresis. Profiles were analysed between different degrees of fibrosis (F0-F4) and between early (F0F1) and late (F2F3F4) fibrosis by univariate analyses (P

Deguelin inhibits expression of IkappaBalpha protein and induces apoptosis of B-CLL cells in vitro.

We investigated if deguelin, a naturally occurring rotenoid, was able to inhibit nuclear factor kappa B (NF-kappaB)-binding protein (IkappaBalpha) expression and to induce apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) cells in vitro. Deguelin-induced cell death in the majority of B-CLL cells and was found to be more toxic toward B-CLL cells than to the normal mononuclear or B-cells, suggesting selectivity towards the malignant cells. Deguelin was found to reduce IkappaBalpha protein expression, and thus interacts with the NFkappaB pathway. The induced apoptosis was characterized by processing of caspase-9 and -3 and poly-(ADP)-ribose-polymerase cleavage. Exposure of B-CLL cells to deguelin resulted in Bcl2-associated protein (Bax) conformational changes and downregulation of the key survival protein myeloid cell leukemia sequence 1 (Mcl-1), which is associated with response to treatment in B-CLL patients. Deguelin retained its ability to induce apoptosis in B-CLL cells in the presence of interleukin-4, a pro-survival cytokine in B-CLL, and when cultured with 50% human serum. These data indicate that deguelin is able to induce apoptosis in B-CLL cells in the presence of pro-survival signals and thus merits further investigation for clinical application either as a single agent or in combination with other anticancer agents.

Edelfosine protects precultured heart fragments against the invasion of malignant cells through altered sialylation.

1-O-octadecyl-2-O-methylglycero-3-phosphocholine (ET-18-OMe)-treated precultured heart fragments (PHF) are resistant to the invasion of malignant cells. Previous studies have demonstrated that this effect is due to the alterations of the N-linked glycoproteins in PHF after 48-h ET-18-OMe treatment. Moreover, the observed effect was still present seven days after ET-18-OMe was omitted. The present study reveals that approximately 13.4% of the administered ET-18-OMe was taken up by PHF and about 75% of the initial uptake was still present after ET-18-OMe was removed. In addition, we found significant changes in the sialic acid content and sialyltransferase activities in both conditions. Overall, these results clearly demonstrate that the uptake and retention of ET-18-OMe are responsible for the resistance to the invasion of malignant cells due to the altered sialylation of the cell surface glycoproteins in PHF.

Analysis of melphalan adducts of 2'-deoxynucleotides in calf thymus DNA hydrolysates by capillary high-performance liquid chromatography-electrospray tandem mass spectrometry.

Melphalan is a bifunctional alkylating agent that covalently binds with intracellular nucleophilic sites. A methodology using electrospray mass spectrometry was developed to detect and identify DNA adducts. Alkylation sites within a particular nucleotide were examined using electrospray tandem mass spectrometry hyphenated to capillary liquid chromatography in combination with a column switching system. In the reaction mixtures resulting from the interaction of 2'-deoxynucleotides and melphalan several base-aklylated adducts were found. In the case of 2'-deoxyadenosine monophosphate, thymidine monophosphate and 2'-deoxyguanosine phosphate alkylation was observed in the mononucleotide reaction mixtures but not in the DNA-hydrolysates. Calf thymus DNA was reacted in vitro with melphalan. The DNA pellet was isolated and enzymatically hydrolyzed with the aid of Nuclease P1. In this hydrolysate both mono-alkylated 2'-deoxynucleotides and dinucleotides were found. The most important adduct found was identified as the N-7 alklylated dGMP adduct. The alkylated dinucleotides were identified as a pdApdT/melphalan and pdGpdC/melphalan the latter being the most important.

Analysis of the DNA adducts of phenyl glycidyl ether in a calf thymus DNA hydrolysate by capillary zone electrophoresis-electrospray mass spectrometry: evidence for phosphate alkylation.

Calf thymus DNA was reacted in vitro with phenyl glycidyl ether (PGE) and was hydrolysed enzymatically, to the 5'-monophosphate nucleotides using deoxyribonuclease I (DNA-ase I) and nuclease P1. The adducts were concentrated using solid phase extraction (SPE), on a polystyrene divinylbenzene copolymer in order to remove the unmodified nucleotides. The adducts could be identified using capillary zone electrophoresis-electrospray tandem mass spectrometry (CZE ES-MS/MS), using sample stacking. In addition to the base alkylated 2'-deoxynucleotides present in the DNA-hydrolysate, also phosphate alkylated 2'-deoxynucleotide adducts were identified for TMP and dAMP. An additional adduct, dUMP alkylated on the uridine moiety was found originating from the hydrolytic deamination of dCMP alkylated on N3 of the cytosine moiety. Enzymatic hydrolysis using nuclease P1 was incomplete as shown by the presence of dinucleotides alkylated on the base moiety. They were successfully hydrolysed to the corresponding 2'-deoxynucleotides by snake venom phosphodiesterase (SVP). Data are shown indicating that alkylations on the pyrimidine bases were more resistant to enzymatic hydrolysis with nuclease P1 than the purine alkylated products.

Analysis of the DNA damage induced by phenyl glycidyl ether using capillary zone electrophoresis-electrospray mass spectrometry.

The in vitro adduct formation between phenyl glycidyl ether (PGE) and calf thymus DNA was investigated. Agarose slab gel electrophoresis of DNA incubated with PGE revealed that nearly all high-molecular-weight species were degraded after 10 h of incubation. After DNA precipitation the reaction products present in the supernatant were subjected to a solid-phase extraction on a polystyrene divinylbenzene copolymer, enabling analysis on capillary zone electrophoresis (CZE), using sample stacking. These reaction products were mainly produced during the first 10 h of incubation, indicating that these products result from the DNA degradation. On the other hand, analysis of the adducts present in the enzymatic digest of the DNA pellet revealed that these adducts were formed only after 10 h of incubation. The reaction products present in the DNA supernatant were identified by on-line coupling of CZE to electrospray tandem mass spectrometry. Three major reaction products resulted from phosphate alkylation, as proven by the analysis of the corresponding low-energy CAD product ion mass spectra. This phosphate alkylation results in phosphotriesters which readily hydrolyze, resulting in DNA strand breaks.