Cytosine analogues as DNA methyltransferase substrates.

DNA methyltransferases are drug targets for myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), acute myelogenous leukemia (AML) and possibly ß-hemoglobinopathies. We characterize the interaction of nucleoside analogues in DNA with a prokaryotic CpG-specific DNA methyltransferase (M.MpeI) as a model for mammalian DNMT1 methyltransferases. We tested DNA containing 5-hydroxymethylcytosine (5hmC), 5-hydroxycytosine (5OHC), 5-methyl-2-pyrimidinone (in the ribosylated form known as 5-methylzebularine, 5mZ), 5,6-dihydro-5-azacytosine (dhaC), 5-fluorocytosine (5FC), 5-chlorocytosine (5ClC), 5-bromocytosine (5BrC) and 5-iodocytosine (5IC). Covalent complex formation was by far most efficient for 5FC. Non-covalent complexes were most abundant for dhaC and 5mZ. Surprisingly, we observed methylation of 5IC and 5BrC, and to a lesser extent 5ClC and 5FC, in the presence, but not the absence of small molecule thiol nucleophiles. For 5IC and 5BrC, we demonstrated by mass spectrometry that the reactions were due to methyltransferase driven dehalogenation, followed by methylation. Crystal structures of M.MpeI-DNA complexes capture the 'in' conformation of the active site loop for analogues with small or rotatable (5mZ) 5-substituents and its 'out' form for bulky 5-substituents. Since very similar 'in' and 'out' loop conformations were also observed for DNMT1, it is likely that our conclusions generalize to other DNA methyltransferases.

Structural analysis of the BisI family of modification dependent restriction endonucleases.

The BisI family of restriction endonucleases is unique in requiring multiple methylated or hydroxymethylated cytosine residues within a short recognition sequence (GCNGC), and in cleaving directly within this sequence, rather than at a distance. Here, we report that the number of modified cytosines that are required for cleavage can be tuned by the salt concentration. We present crystal structures of two members of the BisI family, NhoI and Eco15I_Ntd (N-terminal domain of Eco15I), in the absence of DNA and in specific complexes with tetra-methylated GCNGC target DNA. The structures show that NhoI and Eco15I_Ntd sense modified cytosine bases in the context of double-stranded DNA (dsDNA) without base flipping. In the co-crystal structures of NhoI and Eco15I_Ntd with DNA, the internal methyl groups (G5mCNGC) interact with the side chains of an (H/R)(V/I/T/M) di-amino acid motif near the C-terminus of the distal enzyme subunit and arginine residue from the proximal subunit. The external methyl groups (GCNG5mC) interact with the proximal enzyme subunit, mostly through main chain contacts. Surface plasmon resonance analysis for Eco15I_Ntd shows that the internal and external methyl binding pockets contribute about equally to sensing of cytosine methyl groups.

Evaluation of Linkers' Influence on Peptide-Based Piezoelectric Biosensors' Sensitivity to Aldehydes in the Gas Phase.

Recent findings qualified aldehydes as potential biomarkers for disease diagnosis. One of the possibilities is to use electrochemical biosensors in point-of-care (PoC), but these need further development to overcome some limitations. Currently, the primary goal is to enhance their metrological parameters in terms of sensitivity and selectivity. Previous findings indicate that peptide OBPP4 (KLLFDSLTDLKKKMSEC-NH2) is a promising candidate for further development of aldehyde-sensitive biosensors. To increase the affinity of a receptor layer to long-chain aldehydes, a structure stabilization of the peptide active site via the incorporation of different linkers was studied. Indeed, the incorporation of linkers improved sensitivity to and binding of aldehydes in comparison to that of the original peptide-based biosensor. The tendency to adopt disordered structures was diminished owing to the implementation of suitable linkers. Therefore, to improve the metrological characteristics of peptide-based piezoelectric biosensors, linkers were added at the C-terminus of OBPP4 peptide (KLLFDSLTDLKKKMSE-linker-C-NH2). Those linkers consist of proteinogenic amino acids from group one: glycine, L-proline, L-serine, and non proteinogenic amino acids from group two: ß-alanine, 4-aminobutyric acid, and 6-aminohexanoic acid. Linkers were evaluated with in silico studies, followed by experimental verification. All studied linkers enhanced the detection of aldehydes in the gas phase. The highest difference in frequency (60 Hz, nonanal) was observed between original peptide-based biosensors and ones based on peptides modified with the GSGSGS linker. It allowed evaluation of the limit of detection for nonanal at the level of 2 ppm, which is nine times lower than that of the original peptide. The highest sensitivity values were also obtained for the GSGSGS linker: 0.3312, 0.4281, and 0.4676 Hz/ppm for pentanal, octanal, and nonanal, respectively. An order of magnitude increase in sensitivity was observed for the six linkers used. Generally, the linker's rigidity and the number of amino acid residues are much more essential for biosensors' metrological characteristics than the amino acid sequence itself. It was found that the longer the linkers, the better the effect on docking efficiency.

Chromatin accessibility-based characterisation of brain gene regulatory networks in three distinct honey bee polyphenisms.

The honey bee genome has the capacity to produce three phenotypically distinct organisms (two diploid female castes: queen and worker, and a haploid male drone). Previous studies have implicated metabolic flux acting via epigenetic regulation in directing nutrition-driven phenotypic plasticity in the honey bee. However, the cis-acting DNA regulatory elements that establish tissue and polyphenism -specific epigenomes and gene expression programmes, remain unclear. Using a high resolution multiomic approach including assay for transposase-accessible chromatin by sequencing (ATAC-seq), RNA-seq and ChIP-seq, we produce the first genome-wide maps of the regulatory landscape across all three adult honey bee phenotypes identifying > 5000 regulatory regions in queen, 7500 in worker and 6500 in drone, with the vast majority of these sites located within intronic regions. These regions are defined by positive enrichment of H3K27ac and depletion of H3K4me3 and show a positive correlation with gene expression. Using ATAC-seq footprinting we determine queen, worker and drone -specific transcription factor occupancy and uncover novel phenotype-specific regulatory networks identifying two key nuclear receptors that have previously been implicated in caste-determination and adult behavioural maturation in honey bees; ecdysone receptor and ultraspiracle. Collectively, this study provides novel insights into key gene regulatory networks that are associated with these distinct polyphenisms in the honey bee.

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.

Antifungal Effect of Penicillamine Due to the Selective Targeting of L-Homoserine O-Acetyltransferase.

Due to the apparent similarity of fungal and mammalian metabolic pathways, the number of established antifungal targets is low, and the identification of novel ones is highly desirable. The results of our studies, presented in this work, indicate that the fungal biosynthetic pathway of L-methionine, an amino acid essential for humans, seems to be an attractive perspective. The MET2 gene from Candida albicans encoding L-homoserine O-acetyltransferase (CaMet2p), an enzyme catalyzing the first step in that pathway, was cloned and expressed as the native or the oligo-His-tagged fusion protein in Escherichia coli. The recombinant enzymes were purified and characterized for their basic molecular properties and substrate specificities. The purified MET2 gene product revealed the appropriate activity, catalyzed the conversion of L-homoserine (L-Hom) to O-acetyl-L-homoserine (OALH), and exhibited differential sensitivity to several L-Hom or OALH analogues, including penicillamine. Surprisingly, both penicillamine enantiomers (L- and D-Pen) displayed comparable inhibitory effects. The results of the docking of L- and D-Pen to the model of CaMet2p confirmed that both enantiomeric forms of the inhibitor are able to bind to the catalytic site of the enzyme with similar affinities and a similar binding mode. The sensitivity of some fungal cells to L-Pen, depending on the presence or absence of L-Met in the medium, clearly indicate Met2p targeting. Moreover, C. glabrata clinical strains that are resistant to fluconazole displayed a similar susceptibility to L-Pen as the wild-type strains. Our results prove the potential usefulness of Met2p as a molecular target for antifungal chemotherapy.

Inhibitors of glucosamine-6-phosphate synthase as potential antimicrobials or antidiabetics - synthesis and properties.

Glucosamine-6-phosphate synthase (GlcN-6-P synthase) is known as a promising target for antimicrobial agents and antidiabetics. Several compounds of natural or synthetic origin have been identified as inhibitors of this enzyme. This set comprises highly selective l-glutamine, amino sugar phosphate or transition state intermediate cis-enolamine analogues. Relatively low antimicrobial activity of these inhibitors, poorly penetrating microbial cell membranes, has been improved using the pro-drug approach. On the other hand, a number of heterocyclic and polycyclic compounds demonstrating antimicrobial activity have been presented as putative inhibitors of the enzyme, based on the results of molecular docking to GlcN-6-P synthase matrix. The most active compounds of this group could be considered promising leads for development of novel antimicrobial drugs or antidiabetics, provided their selective toxicity is confirmed.

Dataset for random uniform distributions of 2D circles and 3D spheres.

This dataset contains random uniform distributions for a large number of 2D and 3D balls, along with the description files. It provides the possibility for fast pick up of random, but repeatable, sets of smaller samples, with the guaranteed statistical properties such as random uniform distribution of balls, the predefined expected volume ratio of balls, and also the minimum distance between them. Samples are uniquely identified by the position coordinates in the provided large kernels. The sets of samples can be used in performing numerical predictions of different types for uniform ball distributions while keeping the numerical effort at a reasonable level. Specifically, this can be useful in computational homogenization of fiber and spherical particle reinforced composites, where the provided kernels can be viewed as representative volumes and the samples as the realizations of statistical volume elements. Some secondary results, like the numbers of samples of a given size assuring the required accuracy in expected ball volume ratio representation, are also provided. Data was created by means of the pseudo-random number generator using python scripting and can be loaded and used also in other programming environments.

Novel structure in the nuclei of honey bee brain neurons revealed by immunostaining.

In the course of a screen designed to produce antibodies (ABs) with affinity to proteins in the honey bee brain we found an interesting AB that detects a highly specific epitope predominantly in the nuclei of Kenyon cells (KCs). The observed staining pattern is unique, and its unfamiliarity indicates a novel previously unseen nuclear structure that does not colocalize with the cytoskeletal protein f-actin. A single rod-like assembly, 3.7-4.1 µm long, is present in each nucleus of KCs in adult brains of worker bees and drones with the strongest immuno-labelling found in foraging bees. In brains of young queens, the labelling is more sporadic, and the rod-like structure appears to be shorter (~ 2.1 µm). No immunostaining is detectable in worker larvae. In pupal stage 5 during a peak of brain development only some occasional staining was identified. Although the cellular function of this unexpected structure has not been determined, the unusual distinctiveness of the revealed pattern suggests an unknown and potentially important protein assembly. One possibility is that this nuclear assembly is part of the KCs plasticity underlying the brain maturation in adult honey bees. Because no labelling with this AB is detectable in brains of the fly Drosophila melanogaster and the ant Camponotus floridanus, we tentatively named this antibody AmBNSab (Apis mellifera Brain Neurons Specific antibody). Here we report our results to make them accessible to a broader community and invite further research to unravel the biological role of this curious nuclear structure in the honey bee central brain.

A Pseudomonas monteilii unilateral inguinofemoral granulomatous lymphadenitis in a two-year-old girl. A case report.

Introduction: Pseudomonas monteilii is an environmental contaminant and is considered as an emerging human pathogen. We report the case of a Pseudomonas monteilii granulomatous lymphadenitis in a two-year-old girl.Clinical Presentation and Intervention: A two-year-old, previously healthy, Caucasian girl developed a unilateral inguinofemoral granulomatous lymphadenitis with Pseudomonas monteilii. The protracted course, the violaceous discoloration of the overlying skin, the mild tenderness without constitutional signs, the reactive tuberculin skin test with a negative interferon gamma release assay (IGRA) and the negative Bartonella henselae serology ranked non-tuberculous mycobacterial lymphadenitis high in our differential diagnosis. The ultrasonography showed signs of abcedation. We decided for surgical excision of the nodes. A P.monteilii granulomatous lymphadenitis was revealed. Treatment with an oral course of 2 weeks ciprofloxacin was prescribed. The course after treatment was uneventful and after one year of follow-up, the child is still doing well.Conclusions: Unusual clinical presentation should raise suspicion of uncommon pathogens and uncommon pathogens should raise suspicion of an underlying problem such as immunodeficiency, which was not the case in our patient.

The prevalence of lower airway anomalies in children with Down syndrome compared to controls.

INTRODUCTION: Children with Down syndrome (DS) often present with chronic respiratory symptoms. Congenital airway anomalies have been described but data about prevalence is scarce and a comparison to controls is lacking. We aim to compare the endoscopic and clinical data of children with DS to controls without significant medical history. METHODS: All endoscopic procedures under general anesthesia (broncho- and/or direct laryngoscopy) in patients with DS were reviewed. We compared clinical and endoscopic data to a cohort of children with respiratory symptoms but without any other relevant medical history. RESULTS: Endoscopic data were available for 65 patients with DS. The median age was 2.9 years (range: 0.2-17), 63% were boys. The most common clinical presentation was recurrent respiratory infections (37%). Other major symptoms were chronic cough and/or noisy breathing (23%) and stridor (20%). Endoscopy was normal in 29% of patients. The largest group of patients (44%) had some form of airway malacia. Tracheal bronchus and subglottic stenosis were each isolated findings in 3.1% of patients. Twenty percent presented with combined airway anomalies. The control group consisted of 150 children (matched for age and sex) without significant underlying disease. The most common presentations were chronic cough and/or noisy breathing (29%), persistent radiographic abnormalities (20%), and suspicion of aspiration of a foreign body (15%). In the majority of controls (68%), no airway anomaly was found. Other findings were malacia (22%), tracheal bronchus (1%), and subglottic stenosis (1%). A combined anomaly was found in 5%. CONCLUSION: Congenital airway anomalies were seen in 71% of patients with DS, compared with 32% of controls. Combined anomalies are more frequent in DS. Complete lower airway endoscopy is recommended in patients with DS as it may influence therapeutic decision-making.

A Highly Selective Biosensor Based on Peptide Directly Derived from the HarmOBP7 Aldehyde Binding Site.

This paper presents the results of research on determining the optimal length of a peptide chain to effectively bind octanal molecules. Peptides that map the aldehyde binding site in HarmOBP7 were immobilized on piezoelectric transducers. Based on computational studies, four Odorant Binding Protein-derived Peptides (OBPPs) with different sequences were selected. Molecular modelling results of ligand docking with selected peptides were correlated with experimental results. The use of low-molecular synthetic peptides, instead of the whole protein, enabled the construction OBPPs-based biosensors. This work aims at developing a biomimetic piezoelectric OBPPs sensor for selective detection of octanal. Moreover, the research is concerned with the ligand binding affinity depending on different peptides' chain lengths. The authors believe that the chain length can have a substantial influence on the type and effectiveness of peptide-ligand interaction. A confirmation of in silico investigation results is the correlation with the experimental results, which shows that the highest affinity to octanal is exhibited by the longest peptide (OBPP4 - KLLFDSLTDLKKKMSEC-NH2). We hypothesized that the binding of long chain aldehydes to the peptide, mimicking the binding site of HarmOBP7, induced a conformational change in the peptide deposited on a selected transducer. The constructed OBPP4-based biosensors were able to selectively bind octanal in the gas phase. It was also shown that the sensors were characterized by high selectivity with respect to octanal, as well as to acetaldehyde and benzaldehyde. The results indicate that the OBPP4 peptide, mimicking the binding domain in the Odorant Binding Protein, can provide new opportunities for the development of biomimicking materials in the field of odor biosensors.

Low adherence with national travel medicine recommendations in Belgian expatriate children: A retrospective analysis.

BACKGROUND: Expatriates (expats) from European countries regularly migrate to low-income countries where infectious diseases are more prevalent. Little evidence exists however on pediatric expatriates' compliance with preventive measures related to infectious diseases. This study aims to evaluate compliance in Belgian expat-children. METHODS: Data of 135 Belgian expat-children, visiting the Institute of Tropical Medicine (Antwerp, Belgium), were collected from clinical notes, laboratory results and from a web-based immunization-register. Information on routine vaccinations, yellow fever, hepatitis A, rabies, typhoid fever, meningococcal ACW135Y, Japanese encephalitis, BCG vaccine and anti-malaria chemoprophylaxis was collected. RESULTS: Overall, 87% of expat-children were up-to-date with their routine vaccinations. Although all children were eligible for hepatitis A, typhoid and rabies vaccination, only 8-21% were fully vaccinated. Only 29 and 61% of eligible children were vaccinated against meningococcal (ACW135Y) or yellow fever respectively. Finally, only 10% of children who lived in malaria-endemic-areas, reported chemoprophylaxis-use. CONCLUSION: Although routine vaccination coverage in expat-children seems adequate, additional preventive measures are often needed. Whether this is due to lack of high-quality health care-access, fear of side-effects or insufficient knowledge about the risks/available preventive measures, remains elusive. Nevertheless, expats seem to constitute a separate risk-group for infectious diseases and destination-related health issues.

Modification of quaternary structure of Candida albicans GlcN-6-P synthase and its desensitization to inhibition by UDP-GlcNAc by site-directed mutagenesis.

Site-directed mutagenesis of the CaGFA1 gene encoding glucosamine-6-phosphate synthase from Candida albicans was performed. Desensitization of the enzyme to inhibition by UDPGlcNAc was achieved upon T487I and H492F substitutions at the UDP-GlcNAc binding site, exchange of D524, S525 and S527 for Ala at the dimer:dimer interface and construction of the tail-lock array (L434R and L460A) at the C-tail region. The first two sets if mutageneses but not the last one resulted in conversion of the tetrameric enzyme into its dimeric form. Evidence for links and communication between the UDP-GlcNAc binding site and the dimer-dimer contact areas are presented. The CaGfa1-T487IH492F and CaGfa1-KHSH-D524AS525AS527A muteins are the first examples of the successful conversion of eukaryotic GlcN-6-P synthase into its prokaryotic-like version upon rational site-directed mutagenesis.

Plaque psoriasis following Kawasaki disease and varicella.

We describe the case of a 15-month-old boy with Kawasaki disease who developed varicella 7 days after the beginning of the disease and diffuse plaque psoriasis after 43 days. Associations between Kawasaki disease and psoriasis, between Kawasaki disease and varicella and between varicella and psoriasis have all been reported in the literature. The triple association of Kawasaki disease, varicella and psoriasis is very rare. Neither the double nor the triple associations are well known among a diverse group of practitioners.

Phenotypically distinct female castes in honey bees are defined by alternative chromatin states during larval development.

The capacity of the honey bee to produce three phenotypically distinct organisms (two female castes; queens and sterile workers, and haploid male drones) from one genotype represents one of the most remarkable examples of developmental plasticity in any phylum. The queen-worker morphological and reproductive divide is environmentally controlled during post-embryonic development by differential feeding. Previous studies implicated metabolic flux acting via epigenetic regulation, in particular DNA methylation and microRNAs, in establishing distinct patterns of gene expression underlying caste-specific developmental trajectories. We produce the first genome-wide maps of chromatin structure in the honey bee at a key larval stage in which developmental canalization into queen or worker is virtually irreversible. We find extensive genome-wide differences in H3K4me3, H3K27ac, and H3K36me3, many of which correlate with caste-specific transcription. Furthermore, we identify H3K27ac as a key chromatin modification, with caste-specific regions of intronic H3K27ac directing the worker caste. These regions may harbor the first examples of caste-specific enhancer elements in the honey bee. Our results demonstrate a key role for chromatin modifications in the establishment and maintenance of caste-specific transcriptional programs in the honey bee. We show that at 96 h of larval growth, the queen-specific chromatin pattern is already established, whereas the worker determination is not, thus providing experimental support for the perceived timing of this critical point in developmental heterochrony in two types of honey bee females. In a broader context, our study provides novel data on environmentally regulated organismal plasticity and the molecular foundation of the evolutionary origins of eusociality.

The Role of Functional Respiratory Imaging in Treatment Selection of Children With Obstructive Sleep Apnea and Down Syndrome.

STUDY OBJECTIVES: The complexity of the pathogenesis of obstructive sleep apnea (OSA) in children with Down syndrome (DS) is illustrated by a prevalence of residual OSA after adenotonsillectomy. The aim of this study was to investigate whether upper airway imaging combined with computation fluid dynamics could characterize treatment outcome after adenotonsillectomy in these children. METHODS: Children with DS and OSA were prospectively included. All children underwent an evaluation of the upper airway and an ultra-low dose computed tomography scan of the upper airway before adenotonsillectomy. The upper airway tract was extracted from the scan and combined with computational fluid dynamics. Results were evaluated using control polysomnography after adenotonsillectomy. RESULTS: Thirty-three children were included: 18 boys, age 4.3 ± 2.3 years, median body mass index z-score 0.6 (-2.9 to 3.0), and median obstructive apnea-hypopnea index was 15.7 (3-70) events/h. The minimal upper airway cross-sectional area was significantly smaller in children with more severe OSA (P = .03). Nineteen children underwent a second polysomnography after adenotonsillectomy. Seventy-nine percent had persistent OSA (obstructive apneahypopnea index > 2 events/h). A greater than 50% decrease in obstructive apnea-hypopnea index was observed in 79% and these children had a significantly higher volume of the regions below the tonsils. CONCLUSIONS: This is the first study to characterize treatment outcome in children with DS and OSA using computed tomography upper airway imaging. At baseline, children with more severe OSA had a smaller upper airway. Children with a less favorable response to adenotonsillectomy had a smaller volume of regions below the tonsils, which could be due to enlargement of the lingual tonsils, glossoptosis, or macroglossia. COMMENTARY: A commentary on this article appears in this issue on page 501.