Enzymatic debridement in scalds is not as effective as in flame burns regarding additional eschar excision: A retrospective matched-control study.

OBJECTIVE: Enzymatic debridement of burn eschar became an accepted and widely used technique for burn wound treatment over the last years. However, this practice is not exempt from failure and recent experimental studies indicate that it may not be as efficient in scalds as in flame burns. METHODS: Patients that were admitted to the burn intensive care unit between June 2017 and February 2021 and received enzymatic debridement within the first 72 h after scald and flame burn were included. Patients with scald burns were matched regarding age, sex and per cent total body surface area (%TBSA) burned in a 1:2 ratio with patients presenting with flame burns. RESULTS: Eighteen patients with scald burns were matched with 36 with flame burns. After matching, both groups were similar in terms of age (flame burns 44.5 ± 21.1 years vs. scald 41.8 ± 22.6 years, p = 0.666), and %TBSA burned (11.0 ± 8.2% vs. 10.6 ± 9.6%, p = 0.851). Patients with scald burns significantly more often underwent further surgical eschar excision compared to controls (scald 16 (88.9%) vs. flame 19 (52.8%), p = 0.016). Length of stay per %TBSA was significantly longer in scald burns (scald 7.8 ± 9.2 days vs. flame 3.7 ± 3.8, p = 0.013). CONCLUSION: This study indicates that enzymatic debridement may not be as effective in scalds as in flame burns. It was shown that patients with scalds and subsequent enzymatic debridement more frequently underwent additional surgical intervention and that the size of the transplanted area was larger compared to control. Moreover, those patients had a longer length of stay at the hospital per %TBSA burned.

5-Methylcytosine DNA glycosylase participates in the genome-wide loss of DNA methylation occurring during mouse myoblast differentiation.

Changes in gene expression during mouse myoblast differentiation were monitored by DNA microarray hybridisation. Four days after the onset of differentiation 2.37% of the genes increased in activity from a value of zero, whereas during the same time 1.68% of total genes had decreased expression. During the first 24 h of differentiation an average of 700 000 CpG sites per haploid genome were demethylated. Maximal loss of DNA methylation is attained after 2 days of differentiation, followed by a gradual remethylation. The highest demethylation is observed in highly repeated DNA sequences, followed by single copy sequences. When DNA replication is inhibited by aphidicolin or L-mimosine this genome-wide demethylation is still observed. During the first 3 h of differentiation there is an increase in the number of hemimethylated CpG sites, which disappear rapidly during the course of genome-wide hypomethylation. Transfection of cells with an antisense morpholino oligonucleotide to 5-methylcytosine DNA glycosylase (G/T mismatch DNA glycosylase) decreases both the activity of the enzyme and genome-wide demethylation. It is concluded that the genome-wide loss of DNA methylation in differentiating mouse myoblasts occurs in part by formation of hemimethylated CpG sites, which can serve as the substrate for 5-methylcytosine-DNA glycosylase.

A re-investigation of the ribonuclease sensitivity of a DNA demethylation reaction in chicken embryo and G8 mouse myoblasts.

Recently published results (Nucleic Acids Res. 26, 5573-5580, 1998) suggest that the ribonuclease sensitivity of the DNA demethylation reaction may be an experimental artifact due to the possible tight binding of the nucleases to the methylated DNA substrate. Using an improved protocol we show for two different systems that demethylation of hemimethylated DNA is indeed sensitive to micrococcal nuclease, requires RNA and is not an experimental artifact. The purified 5-MeC-DNA glycosylase from chicken embryos and G8 mouse myoblasts was first incubated for 5 min at 37 degrees C with micrococcal nuclease in the presence of Ca2+ in the absence of the DNA substrate. Upon blocking the nuclease activity by the addition of 25 mM EGTA, the DNA demethylation reaction was initiated by adding the labeled hemimethylated DNA substrate to the reaction mixture. Under these conditions the DNA demethylation reaction was abolished. In parallel controls, where the purified 5-MeC-DNA glycosylase was pre-incubated at 37 degrees C with the nuclease, Ca2+ and EGTA or with the nuclease and EGTA, RNA was not degraded and no inhibition of the demethylation reaction was obtained. As has already been shown for chicken embryos, the loss of 5-MeC-DNA glycosylase activity from G8 myoblasts following nuclease treatment can also be restored by the addition of synthetic RNA complementary to the methylated strand of the substrate DNA. No reactivation of 5-MeC-DNA glycosylase is obtained by complementation with a random RNA sequence, the RNA sequence complementary to the non-methylated strand or DNA, thus ruling out a non-specific competition of the RNA for the binding of the nuclease to the labeled DNA substrate.

Mechanism of active DNA demethylation during embryonic development and cellular differentiation in vertebrates.

Incubation of hemimethylated and labelled oligodeoxynucleotides with nuclear extracts from differentiating chicken embryos and mouse myoblasts resulted in the replacement of m5C by C. One of the enzymes involved is m5CpG endonuclease. It cleaves only m5CpG and not, m5CpT, m5CpA, m5CpC or m6ApT. The enzyme is not sequence specific and catalyses the reaction in the presence of high concentrations of EDTA or EGTA.

Transient DNA demethylation in differentiating mouse myoblasts correlates with higher activity of 5-methyldeoxycytidine excision repair.

It has been recently shown that in developing chicken embryonic nuclear extracts there is a 5-methyldeoxycytidine excision repair activity (Jost, J. P. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4684-4688). We show that in differentiating mouse myoblasts, a similar enzymatic reaction may be responsible for the genome-wide DNA demethylation (up to 50% of all CmCGG) occurring between the 3rd and 5th days of differentiation. Furthermore, in differentiating myoblasts, there is first a 50% transient decrease in DNA methyltransferase activity and a 90% drop in the rate of DNA synthesis, followed by an increase in 5-methyl-CpG endonuclease and 5-methyldeoxycytidine excision repair activities. As tested in vitro, the maximal activity of the 5-methyldeoxycytidine excision repair coincides with the maximal in vivo genome-wide DNA demethylation. We also find that 3-aminobenzamide, a potent inhibitor of ADP-ribosyltransferase, blocks the differentiation of myoblasts, the 5-methyldeoxycytidine excision repair activity, and the genome-wide demethylation.