Effects of TET2 mutations on DNA methylation in chronic myelomonocytic leukemia

TET2 enzymatically converts 5-methyl-cytosine to 5-hydroxymethyl-cytosine, possibly leading to loss of DNA methylation. TET2 mutations are common in myeloid leukemia and were proposed to contribute to leukemogenesis through DNA methylation. To expand on this concept, we studied chronic myelomonocytic leukemia (CMML) samples. TET2 missense or nonsense mutations were detected in 53% (16/30) of patients. In contrast, only 1/30 patient had a mutation in IDH1 or IDH2, and none of them had a mutation in DNMT3A in the sites most frequently mutated in leukemia. Using bisulfite pyrosequencing, global methylation measured by the LINE-1 assay and DNA methylation levels of 10 promoter CpG islands frequently abnormal in myeloid leukemia were not different between TET2 mutants and wild-type CMML cases. This was also true for 9 out of 11 gene promoters reported by others as differentially methylated by TET2 mutations. We found that two non-CpG island promoters, AIM2 and SP140, were hypermethylated in patients with mutant TET2. These were the only two gene promoters (out of 14,475 genes) previously found to be hypermethylated in TET2 mutant cases. However, total 5-methyl-cytosine levels in TET2 mutant cases were significantly higher than TET2 wild-type cases (median = 14.0% and 9.8%, respectively) (p = 0.016). Thus, TET2 mutations affect global methylation in CMML but most of the changes are likely to be outside gene promoters.Key words: TET2, DNA methylation, mutation, CMML, promoter     

Minocycline inhibits alkali burn-induced corneal neovascularization in mice

The purpose of this study was to investigate the effects of minocycline on alkali burn-induced corneal neovascularization (CNV). A total of 105 mice treated with alkali burns were randomly divided into three groups to receive intraperitoneal injections of either phosphate buffered saline (PBS) or minocycline twice a day (60 mg/kg or 30 mg/kg) for 14 consecutive days. The area of CNV and corneal epithelial defects was measured on day 4, 7, 10, and14 after alkali burns. On day 14, a histopathological examination was performed to assess morphological change and the infiltration of polymorphonuclear neutrophils (PMNs). The mRNA expression levels of vascular endothelial growth factor (VEGF) and its receptors (VEGFRs), basic fibroblast growth factor (bFGF), matrix metalloproteinases (MMPs), interleukin-1α, 1β, 6 (IL-1α, IL-1β, IL-6) were analyzed using real-time quantitative polymerase chain reaction. The expression of MMP-2 and MMP-9 proteins was determined by gelatin zymography. In addition, enzyme-linked immunosorbent assay was used to analyze the protein levels of VEGFR1, VEGFR2, IL-1β and IL-6. Minocycline at a dose of 60 mg/kg or 30 mg/kg significantly enhanced the recovery of the corneal epithelial defects more than PBS did. There were significant decreases of corneal neovascularization in the group of high-dosage minocycline compared with the control group at all checkpoints. On day 14, the infiltrated PMNs was reduced, and the mRNA expression of VEGFR1, VEGFR2, bFGF, IL-1β, IL-6, MMP-2, MMP-9, -13 as well as the protein expression of VEGFR2, MMP-2, -9, IL-1β, IL-6 in the corneas were down-regulated with the use of 60 mg/kg minocycline twice a day. Our results showed that the intraperitoneal injection of minocycline (60 mg/kg b.i.d.) can significantly inhibit alkali burn-induced corneal neovascularization in mice, possibly by accelerating corneal wound healing and by reducing the production of angiogenic factors, inflammatory cytokines and MMPs.     

The strongest resistance of Staphylococcus aureus to erythromycin is caused by decreasing uptake of the antibiotic into the cells

The consequence of excessive use of macrolides is a high occurrence of mechanisms responsible for resistance to these drugs. Of 97 erythromycin-resistant bacterial strains gathered in the Wroc?aw area in Poland, 60% exhibited very high resistance, and those with the inducible MLS_B (macrolide-lincosamide-streptogramin B) resistance phenotype predominated. Direct genetic investigation revealed that the erm genes coding for ribosomal methylases are the most frequently occurring erythromycin resistance-determining genes. No genetic resistance determinant was detected in 13% of the erythromycin-resistant strains. The efflux mechanism occurs in strains isolated from the nasopharyngeal cavity twice as often as in those isolated from other material, where the mechanism connected with target site modification predominates. Measurements of radiolabelled antibiotic accumulation inside bacterial cells revealed that in highly resistant strains (MIC > 1024 ??g/ml), an important factor responsible for the resistance is the permeability barrier at the cell wall level. This would be a hitherto unknown mechanism of resistance to erythromycin in Staphylococcus aureus.     

How nuclei of Giardia pass through cell differentiation: semi-open mitosis followed by nuclear interconnection

Differentiation into infectious cysts (encystation) and multiplication of pathogenic trophozoites after hatching from the cyst (excystation) are fundamental processes in the life cycle of the human intestinal parasite Giardia intestinalis. During encystation, a bi-nucleated trophozoite transforms to a dormant tetra-nucleated cyst enveloped by a protective cyst wall. Nuclear division during encystation is not followed by cytokinesis. In contrast to the well-studied mechanism of cyst wall formation, information on nuclei behavior is incomplete and basic cytological data are lacking. Here we present evidence that (1) the nuclei divide by semi-open mitosis during early encystment; (2) the daughter nuclei coming from different parent nuclei are always arranged in pairs; (3) in both pairs, the nuclei are interconnected via bridges formed by fusion of their nuclear envelopes; (4) each interconnected nuclear pair is associated with one basal body tetrad of the undivided diplomonad mastigont; and (5) the interconnection between nuclei persists through the cyst stage being a characteristic feature of encysted Giardia. Based on the presented results, a model of nuclei behavior during Giardia differentiation is proposed.     

Gemin5 proteolysis reveals a novel motif to identify L protease targets

Translation of picornavirus RNA is governed by the internal ribosome entry site (IRES) element, directing the synthesis of a single polyprotein. Processing of the polyprotein is performed by viral proteases that also recognize as substrates host factors. Among these substrates are translation initiation factors and RNA-binding proteins whose cleavage is responsible for inactivation of cellular gene expression. Foot-and-mouth disease virus (FMDV) encodes two proteases, L(pro) and 3C(pro). Widespread definition of L(pro) targets suffers from the lack of a sufficient number of characterized substrates. Here, we report the proteolysis of the IRES-binding protein Gemin5 in FMDV-infected cells, but not in cells infected by other picornaviruses. Proteolysis was specifically associated with expression of L(pro), yielding two stable products, p85 and p57. In silico search of putative L targets within Gemin5 identified two sequences whose potential recognition was in agreement with proteolysis products observed in infected cells. Mutational analysis revealed a novel L(pro) target sequence that included the RKAR motif. Confirming this result, the Fas-ligand Daxx, was proteolysed in FMDV-infected and L(pro)-expressing cells. This protein carries a RRLR motif whose substitution to EELR abrogated L(pro) recognition. Thus, the sequence (R)(R/K)(L/A)(R) defines a novel motif to identify putative targets of L(pro) in host factors.     

Experimental Administration of the Probiotic Escherichia coli Strain Nissle 1917 Results in Decreased Diversity of E. coli Strains in Pigs

The strain Escherichia coli Nissle 1917 (EcN) is widely used as an efficient probiotic in therapy and prevention of human infectious diseases, especially of the intestinal system. Concurrently, small adult pigs are being used as experimental omnivore models to study human gastrointestinal functions. EcN bacteria were applied to 6 adult healthy female pigs in a 2-week trial. 6 Control animals remained untreated. Altogether, 164 and 149 bacterial strains were isolated from smear samples taken from gastrointestinal mucosa in the experimental and control group, respectively. Each individual E. coli strain was then tested for the presence of 29 bacteriocin-encoding determinants as well as for DNA markers of A, B1, B2 and D phylogenetic groups. A profound reduction of E. coli genetic variance (from 32 variants to 13 ones, P?=?0.0006) was found in the experimental group, accompanied by a lower incidence of bacteriocin producers in the experimental group when compared to control (21.3 and 34.9%, respectively; P?=?0.007) and by changes in the incidence of individual bacteriocin types. The experimental administration of EcN strain was not sufficient for stable colonization of porcine gut, but induced significant changes in the enterobacterial microbiota.     

Transglycosylation specificity of Acremonium sp. α-rhamnosyl-β-glucosidase and its application to the synthesis of the new fluorogenic substrate 4-methylumbelliferyl-rutinoside

Transglycosylation potential of the fungal diglycosidase α-rhamnosyl-β-glucosidase was explored. The biocatalyst was shown to have broad acceptor specificity toward aliphatic and aromatic alcohols. This feature allowed the synthesis of the diglycoconjugated fluorogenic substrate 4-methylumbelliferyl-rutinoside. The synthesis was performed in one step from the corresponding aglycone, 4-methylumbelliferone, and hesperidin as rutinose donor. 4-Methylumbelliferyl-rutinoside was produced in an agitated reactor using the immobilized biocatalyst with a 16% yield regarding the sugar acceptor. The compound was purified by solvent extraction and silica gel chromatography. MALDI-TOF/TOF data recorded for the [M+Na](+) ions correlated with the theoretical monoisotopic mass (calcd [M+Na](+): 507.44 m/z; obs. [M+Na](+): 507.465 m/z). 4-Methylumbelliferyl-rutinoside differs from 4-methylumbelliferyl-glucoside in the rhamnosyl substitution at the C-6 of glucose, and this property brings about the possibility to explore in nature the occurrence of endo-β-glucosidases by zymographic analysis.