The structure of insect DNA methyltransferase 2 (DNMT2) DNA binding domain is responsible for the non-CpG methylation in insect genomes

Alignment of vertebrate and invertebrate DNA methyltransferases; 2 (Dnmt2) evidenced an over-all evolutionary conservation of these proteins. However, alignment revealed a vertebrate-specific stretch of about forty amino acids located between the catalytic motif VIII and the target recognition domain that is constantly absent from insect homologues. The analysis of the three-dimensional structure of DNA methyltransferase indicated that this vertebrate specific Dnmt2 portion is located at the DNA binding domain whose structure is essential for the discrimination of the proper target sequence. Insect Dnmt2 enzymes are, therefore, devoid of a portion of the DNA binding domain suggesting that this structural change may alter the methylation target of insect Dnmt2 Making cytosine methylation not limited to the vertebrate canonical CpG but extended to cytosine residues belonging to other dinucleotides

Analysis of heterochromatic epigenetic markers in the holocentric chromosomes of the aphid Acyrthosiphon pisum

Monomethylated-K9 H3 histones (Me9H3) and heterochromatin protein 1 (HP1) are reported as heterochromatin markers in several eukaryotes possessing monocentric chromosomes. In order to confirm that these epigenetic markers are evolutionary conserved, we sequenced the HP1 cDNA and verified the distribution of Me9H3 histones and HP1 in the holocentric chromosomes of the aphid Acyrthosiphon pisum. Sequencing indicates that A. pisum HP1 cDNA (called ApHP1) is 1623 bp long, including a 170 bp long 5’UTR and a 688 bp long 3’UTR. The ApHP1 protein consists in 254 amino acidic residues, has a predicted molecular mass of 28 kDa and a net negative charge. At the structural level, it shows an N terminal chromo domain and a chromo shadow domain at the C terminus linked by a short hinge region. At the cytogenetic level, ApHP1 is located exclusively in the heterochromatic regions of the chromosomes. The same heterochromatic regions were labelled after immuno-staining with antibodies against Me9H3 histones confirming that Hp1 and Me9H3 co-localize at heterochromatic chromosomal areas. Surprisingly, aphid heterochromatin lacks DNA methylation and methylated cytosine residues were mainly spread at euchromatic regions. Finally, the absence of DNA methylation is observed also in aphid rDNA genes that have been repeatedly described as mosaic of methylated and unmethylated units in vertebrates

DNA methylation of fly genes and transposons

The use of anti-5-methylcytosine antibodies in affinity columns allowed the identification of methylated sequences in the genome of Drosophila melanogaster adults. In view of the presence of transposable elements amongst the identified sequences, it has been suggested that DNA methylation is involved in transposon control in the fly genome. On the contrary, a re-analysis of these data can furnish several intriguing elements that could open new questions about the role that DNA methylation plays in the fly genome. The aim of the present paper is to discuss some features that emerge from the analysis of the identified methylated sequences

Advanced microscopic approaches to the study of eukaryotic chromosomes

Since the first discoveries of Schneider and Flemming, several papers have been published on chromosome structure making the understanding of chromatin organization in chromosomes one of the most attractive topics of the current biosciences. Several new discoveries on chromosome structure arose from advanced microscopy techniques that flanked light and electron microscopy. In the present review, the newest microscopy techniques applied to the study of the chromosome structure are presented and discussed in order to evidence the advantages and disadvantages of each approac

Histone methylation and DNA methylation: a missed pas de deux in invertebrates?

Several Authors have reported that histone methylation interacts with DNA methylation creating a self-propagating epigenetic cycle for long-term transcriptional repression of methylated genome compartments. This phenomenon, observed in plant and vertebrate genomes, does not appear to hold true in invertebrates. In particular, both structural and functional evidences suggest that, in invertebrates, DNA methylation and histone methylation do not interact, thus inhibiting the intimate pas de deux observed in other eukaryotes

Identification of a new hobo element in the cabbage moth, Mamestra brassicae (Lepidoptera).

A complete hobo-like element, called Mbhobo, was identified in the cabbage moth, Mamestra brassicae. This element has a high sequence similarity to the HFL1 hobo element of Drosophila melanogaster. Amplification of Mbhobo termini indicated that transposition occurred into a 5’-GTGGGTAC-3’ target sequence that was duplicated upon insertion. This target site conforms to the consensus sequence established for the insertion sites of insect hAT elements. Mbhobo has a single 1935 bp long ORF with significant homology to the D. melanogaster HFL1 hobo transposase. FISH experiments evidenced Mbhobo clusters located in heterochromatic regions of Z and W sex chromosomes and in heterochromatic areas of chromosome pair 10

Rapidly fatal West Nile virus meningoencephalitis in an immunocompetent patient: a case report

Abstract Background West Nile virus (WNV) is a Flaviviridae most often transmitted by mosquitos. Clinical manifestations vary from no symptoms to neuroinvasive disease. Mortality is rare, but patients with neuroinvasive disease have a fatality rate of 4-14%. Diagnosis is made on epidemiological, clinical and serological criteria. Treatment is based on symptomatic and support therapy. WNV neuroinvasive disease seems associated to advancing age and diabetes, but poor prognosis risk factors are still not clearly recognized. During 2017-2018 10 patients affected by WNV infection were admitted to our Hospital (Policlinico of Modena): 3 patients had a rapid fatal outcome and 3 needed intensive care transfer. We report the most representative case. Case Report A 81-yr-old man from Emilia-Romagna was admitted to our unit with a 6 days history of fever (>38℃), fatigue, nausea, vomiting, hiccough and mild cognitive impairment treated with amoxicillin. Past medical history: type 2 diabetes mellitus, arterial hypertension, permanent pacemaker for type 3 atrioventricular block. Referred exposure to farm animals. No recent travels abroad. Chest x-ray showed a retrocardiac opacity, so empiric levofloxacin was started for suspected community acquired pneumonia. After two days the patient began lethargic with a Glasgow Come Score < 8. Neuroinvasive WNV disease was confirmed by electroencephalogram and rachicentesis. Before serologic results acyclovir and dexamethasone were initiated without benefit and patient diedon the fifth day after admission. Conclusions Risk factors for poor prognosis related to WNV Infection are still not clearly identified. Our patient underwent unexpected rapid clinical deterioration before invasive treatment could positively affect prognosis. This underlines the importance of clinical alert to WNV infections during transmission season in endemic areas and the necessity of more data on fatal WNV cases to define criteria to promptly recognize high-risk patients. Keywords encephalitis; meningoencephalitis; neuroinvasive disease; West Nile virus; fatal meningoencephalitis

JLB: a flexible and effective device in critical patients. Review of clinical cases

JLB catheter (Deltamed Inc) is an alternative way to manage difficult venous access; it is placed under US-guidance in large bore veins, with an easy-sterile approach. Internal jugular vein (IJV) is the first choice for cannulation, followed by subclavian or deep upper-arm veins. The catheter is available in different lengths and gauges, it allows high flow rates and can be left in place up to 30 days. From June 2015 to March 2017, JLB has been positioned in 409 patients: in 354 as primary access in IJV, brachial or subclavian vein; in 55 cases JLB became an introducing line for the Seldinger guidewire and further CVC positioning. All clinical cases were reviewed selecting those with greater clinical relevance. We report 8 cases in which JLB resulted determinant for the patient treatment: a 16 years old obese girl born with perinatal distress, a 78 years old obese woman with hemorrhagic shock caused by gastrointestinal bleeding, a 40 years old man with severe hypokalemia, a 30 years old man with severe sepsis, a 40 years old man with Becker’s muscular dystrophy and severe sepsis, a 40 years old man with multiple myeloma who had to carry out cycles of chemotherapy, a 76 years old man with CMV pancolitis and myelofibrosis who needed parenteral nutrition, antiviral therapy and frequent blood and platelets transfusion. Moreover, it has been useful in elderly patients who needed to carry out palliative care for seniority or cancer lasting up to 30 days . In our experience the JLB catheter is safe, easy to place, quick and cost –effective. It is a valid solution either in unstable patients requiring an immediate access in emergency and stable patients with difficult venous access, in which invasive devices can be considered an over-treatment

Conservation of HP1 and methylated H3 histones as heterochromatic epigenetic markers in the holocentric chromosomes of the cabbage moth, Mamestra brassicae (Lepidoptera)

The methylated H3 histone and heterochromatin protein 1 (HP1) are markers of heterochromatin in several eukaryotes possessing monocentric chromosomes. In order to confirm that these epigenetic markers of heterochromatin arc evolutionary conserved, the distribution of methylated H3 histones and HP1 homologues on the holocentric chromosomes of the cabbage moth Mamestra brassicae (Lepidoptera) were studied. In particular, PCR experiments with degenerated primers identified a HP1 homologue (called MbHP1) in the M. brassicae genome. Sequencing showed that the MbHP1 gene is 737 bp long including a 102 bp 5'UTR and a 635 bp coding portion (comprising an 80 bp intron). The MbHP1 peptide consisted of 184 amino acids, had a 20 kDa molecular mass and a net negative charge. At the structural level, it showed an N terminal chromo-domain and a chromo-shadow-domain at the C terminus linked by a short hinge region. At the cytogenetic level, MbHP1 was located exclusively in the heterochromatic regions of the chromosomes. The same heterochromatic regions became labelled after immuno-staining with antibodies against H3 historic methylated at lysine 9, reinforcing the hypothesis that this modified histone is essential for HP1 binding. Our data, as a whole, confirm that heterochromatic components and markers are evolutionary conserved both in mono- and holocentric chromosomes despite the difference in the distribution of heterochromatin on chromosomes

Analysis of the expression pattern of the defensin gene in the lepidopteran Mamestra brassicae.

Southern blotting experiments performed on M. brassicae genomic DNA after digestion with methylation-sensitive restriction enzymes indicated that defensin gene is methylated at CpG targets in the promoter region. However, defensin gene is actively transcribed despite the presence of methylation. Experiments performed by genome demethylation indicated that demethylated defensin gene resulted in altered expression after bacterial induction. In particular, if defensin gene is demethylated it has not possible to observed any increase in gene expression after induction with Gram positive bacteria. The present results are very intriguing since they indicate not only that in M. brassicae DNA methylation is not involved in gene silencing but also that cytosine methylation could be essential to assure the expression of specific genes. Finally, the above reported data apparently argue against a unifying and evolutionary conserved role of cytosine methylation from invertebrates to vertebrates. In fact, it appears that the DNA methylation/gene silencing correlation, which is typically reported in vertebrates, appears not to hold true for insects