A case of nimesulide induced toxic epidermal necrolysis

Toxic epidermal necrolysis (TEN) is a rare life-threatening drug-induced mucocutaneous skin disease with a mortality rate of approximately 30%. Nimesulide is a preferential cyclo-oxygenase (COX-2) inhibitor which is frequently used for its antipyretic, anti-inflammatory and analgesic activity. Here, we report a case of nimesulide induced toxic epidermal necrolysis in a 57 years old male patient. This patient was admitted in the hospital with symptoms of epidermal sloughing and fluid filled blisters all over the body following over the counter intake of nimesulide for fever. The drug was promptly stopped, and patient was managed with steroids, antibiotics and other adequate supportive measures. The patient showed significant recovery following stoppage of drug and adequate management. This case highlights the importance of nimesulide and other NSAIDs as possible cause of TEN

Biochemical characterisation of TET DNA hydroxylases

Methylation of DNA in CpG dinucleotide plays an important role in mammalian development. The recent discovery of TET enzymes showed that DNA demethylation can occur through stepwise oxidation of 5-methylcytosine (5mC) to 5-hyrdoxymethylcytosine (5hmC), 5-formylcytosine (5fC) and finally to 5-carboxylcytosine (5-caC) followed by the removal of the higher oxidised bases by Thymine DNA glycosylase (TDG) and base excision repair mechanism. Genetic studies revealed that the TET enzymes are involved in numerous biological processes such as transcriptional regulation, hematopoietic stem cell differentiation, embryonic, primodial germ cells (PGCs) development and are commonly misregulated in cancer. While the biological functions of TET enzymes have been studied extensively, very little is known about their biochemical properties. In this body of work, the biochemistry of TET enzymes was investigated in detail with the focus on their catalytic and kinetic behaviour, which would allow us to understand the molecular mechanisms of TET enzymes. First of all, an in vitro system including a novel plate assay to quantify the oxidation products catalysed by TET enzymes, was established. As a proof of principle, several analogues of α-Ketoglutarate, the intermediates of citric acid cycle (oncometabolites) were tested. Moreover, the effect of divalent metal ions was tested both in vitro and in vivo and it was demonstrated that the addition of nickel ions to mammalian cells decreased the level of 5hmC through inhibition of TET enzymes by displacing the Fe2+ from the catalytic centre. Furthermore, using detailed biochemical studies, it was demonstrated that ascorbic acid (AscA) modulates the activity of TET enzymes through efficient recycling of Fe2+, which challenges the existing view of AscA as a bound cofactor of TET enzymes. Finally, using biochemical approach followed by next generation sequencing and bioinformatics analysis, the catalytic behaviour of TET enzymes on single molecule level was elucidated. Using linear double stranded DNA containing multiple 5hmC-substrates in different flanking sequence context, it was shown that mammalian TET enzymes oxidize 5hmC substrates in both CG and non-CG context. Importantly, both mammalian TET enzymes and Naegleria gruberi Tet1 like dioxygenase (nTet) showed a strong and distinct flanking sequence preference. In addition, it was shown that TET enzymes (both mammalian and nTet) might catalyse the substrates on DNA in distributive manner

Synthetic epigenetics-towards intelligent control of epigenetic states and cell identity

Epigenetics is currently one of the hottest topics in basic and biomedical research. However, to date, most of the studies have been descriptive in nature, designed to investigate static distribution of various epigenetic modifications in cells. Even though tremendous amount of information has been collected, we are still far from the complete understanding of epigenetic processes, their dynamics or even their direct effects on local chromatin and we still do not comprehend whether these epigenetic states are the cause or the consequence of the transcriptional profile of the cell. In this review, we try to define the concept of synthetic epigenetics and outline the available genome targeting technologies, which are used for locus-specific editing of epigenetic signals. We report early success stories and the lessons we have learned from them, and provide a guide for their application. Finally, we discuss existing limitations of the available technologies and indicate possible areas for further development

Antimicrobial Prophylaxis in Instrumented Spinal Fusion Surgery: A Comparative Analysis of 24-Hour and 72-Hour Dosages

Study DesignProspective study.PurposeTo compare the efficacy of 24-hour and 72-hour antibiotic prophylaxis in preventing surgical site infections (SSIs).Overview of LiteratureAntimicrobial prophylaxis in surgical practice has become a universally accepted protocol for minimizing postoperative complications related to infections. Although prophylaxis is an accepted practice, a debate exists with regard to the antibiotic type and its administration duration for various surgical procedures.MethodsOur institute is a tertiary care hospital with more than 100 spinal surgeries per year for various spine disorders in the department of orthopedics. We conducted this prospective study in our department from June 2012 to January 2015. A total of 326 patients were enrolled in this study, with 156 patients in the 72-hour antibiotic prophylaxis group (group A) and 170 patients in the 24-hour group (group B). Cefazolin was the antibiotic used in both groups. Two surgeons were involved in conducting all the spinal procedures. Our study compared SSIs among patients undergoing instrumented spinal fusion.ResultsThe overall rate of SSIs was 1.8% with no statistical difference between the two groups.ConclusionsThe 24-hour antimicrobial prophylaxis is as effective as the 72-hour dosage in instrumented spinal fusion surgery

Retinol and ascorbate drive erasure of epigenetic memory and enhance reprogramming to naïve pluripotency by complementary mechanisms

Epigenetic memory, in particular DNA methylation, is established during development in differentiating cells and must be erased to create naïve (induced) pluripotent stem cells. The ten-eleven translocation (TET) enzymes can catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidized derivatives, thereby actively removing this memory. Nevertheless, the mechanism by which the TET enzymes are regulated, and the extent to which they can be manipulated, are poorly understood. Here we report that retinoic acid (RA) or retinol (vitamin A) and ascorbate (vitamin C) act as modulators of TET levels and activity. RA or retinol enhances 5hmC production in naïve embryonic stem cells by activation of TET2 and TET3 transcription, whereas ascorbate potentiates TET activity and 5hmC production through enhanced Fe2+ recycling, and not as a cofactor as reported previously. We find that both ascorbate and RA or retinol promote the derivation of induced pluripotent stem cells synergistically and enhance the erasure of epigenetic memory. This mechanistic insight has significance for the development of cell treatments for regenenerative medicine, and enhances our understanding of how intrinsic and extrinsic signals shape the epigenome

TET-mediated DNA hydroxymethylation is negatively influenced by the PARP-dependent PARylation

Background Poly(ADP-ribosyl)ation (PARylation), a posttranslational modification introduced by PARP-1 and PARP-2, has first been implicated in DNA demethylation due to its role in base excision repair. Recent evidence indicates a direct influence of PARP-dependent PARylation on TET enzymes which catalyse hydroxymethylation of DNA—the first step in DNA demethylation. However, the exact nature of influence that PARylation exerts on TET activity is still ambiguous. In our recent study, we have observed a negative influence of PARP-1 on local TET-mediated DNA demethylation of a single gene and in this study, we further explore PARP–TET interplay. Results Expanding on our previous work, we show that both TET1 and TET2 can be in vitro PARylated by PARP-1 and PARP-2 enzymes and that TET1 PARylation negatively affects the TET1 catalytic activity in vitro. Furthermore, we show that PARylation inhibits TET-mediated DNA demethylation at the global genome level in cellulo. Conclusions According to our findings, PARP inhibition can positively influence TET activity and therefore affect global levels of DNA methylation and hydroxymethylation. This gives a strong rationale for future examination of PARP inhibitors' potential use in the therapy of cancers characterised by loss of 5-hydroxymethylcytosine

Pronounced sequence specificity of the TET enzyme catalytic domain guides its cellular function

TET (ten-eleven translocation) enzymes catalyze the oxidation of 5-methylcytosine bases in DNA, thus driving active and passive DNA demethylation. Here, we report that the catalytic domain of mammalian TET enzymes favor CGs embedded within basic helix-loop-helix and basic leucine zipper domain transcription factor–binding sites, with up to 250-fold preference in vitro. Crystal structures and molecular dynamics calculations show that sequence preference is caused by intrasubstrate interactions and CG flanking sequence indirectly affecting enzyme conformation. TET sequence preferences are physiologically relevant as they explain the rates of DNA demethylation in TET-rescue experiments in culture and in vivo within the zygote and germ line. Most and least favorable TET motifs represent DNA sites that are bound by methylation-sensitive immediate-early transcription factors and octamer-binding transcription factor 4 (OCT4), respectively, illuminating TET function in transcriptional responses and pluripotency support

Enzymatic hydroxylation and excision of extended 5-methylcytosine analogues

Methylation of cytosine to 5-methylcytosine (mC) is a prevalent reversible epigenetic mark in vertebrates established by DNA methyltransferases (MTases); the methylation mark can be actively erased via a multi-step demethylation mechanism involving oxidation by Ten-eleven translocation (TET) enzyme family dioxygenases, excision of the latter oxidation products by thymine DNA (TDG) or Nei-like 1 (NEIL1) glycosylases followed by base excision repair to restore the unmodified state. Here we probed the activity of the mouse TET1 (mTET1) and Naegleria gruberi TET (nTET) oxygenases with DNA substrates containing extended derivatives of the 5-methylcytosine carrying linear carbon chains and adjacent unsaturated C-C bonds. We found that the nTET and mTET1 enzymes were active on modified mC residues in single-stranded and double-stranded DNA in vitro, while the extent of the reactions diminished with the size of the extended group. Iterative rounds of nTET hydroxylations of ssDNA proceeded with high stereo specificity and included not only the natural alpha position but also the adjoining carbon atom in the extended side chain. The regioselectivity of hydroxylation was broken when the reactive carbon was adjoined with an sp1 or sp2 system. We also found that NEIL1 but not TDG was active with bulky TET-oxidation products. These findings provide important insights into the mechanism of these biologically important enzymatic reactions

Retinol and ascorbate drive erasure of epigenetic memory and enhance reprogramming to naïve pluripotency by complementary mechanisms

Epigenetic memory, in particular DNA methylation, is established during development in differentiating cells and must be erased to create naïve (induced) pluripotent stem cells. The ten-eleven translocation (TET) enzymes can catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidized derivatives, thereby actively removing this memory. Nevertheless, the mechanism by which the TET enzymes are regulated, and the extent to which they can be manipulated, are poorly understood. Here we report that retinoic acid (RA) or retinol (vitamin A) and ascorbate (vitamin C) act as modulators of TET levels and activity. RA or retinol enhances 5hmC production in naïve embryonic stem cells by activation of TET2 and TET3 transcription, whereas ascorbate potentiates TET activity and 5hmC production through enhanced Fe2+ recycling, and not as a cofactor as reported previously. We find that both ascorbate and RA or retinol promote the derivation of induced pluripotent stem cells synergistically and enhance the erasure of epigenetic memory. This mechanistic insight has significance for the development of cell treatments for regenenerative medicine, and enhances our understanding of how intrinsic and extrinsic signals shape the epigenome