Molecularly Imprinted Drug Carrier for Lamotrigine-Design, Synthesis, and Characterization of Physicochemical Parameters.

This study presents the initial attempt at introducing a magnetic molecularly imprinted polymer (MIP) designed specifically for lamotrigine with the purpose of functioning as a drug carrier. First, the composition of the magnetic polymer underwent optimization based on bulk polymer adsorption studies and theoretical analyses. The magnetic MIP was synthesized from itaconic acid and ethylene glycol dimethacrylate exhibiting a drug loading capacity of 3.4 ± 0.9 µg g-1. Structural characterization was performed using powder X-ray diffraction analysis, vibrating sample magnetometry, and Fourier transform infrared spectroscopy. The resulting MIP demonstrated controlled drug released characteristics without a burst effect in the phospahe buffer saline at pH 5 and 8. These findings hold promise for the potential nasal administration of lamotrigine in future applications.

The Role of Bacteria in Acute Oak Decline in South-West Poland.

The process of multifactorial oak disease has been of interest to scientists from all over the world for many years. Recently, a new phenomenon has been added to the model related to oak decline, acute oak dieback, which causes oak decline and was first described in the UK. This study presents research on this phenomenon in the area of the largest oak stand complex in Poland, the so-called Krotoszyn Plateau. This work was carried out in two stages. In the first stage, 54 trees were tested for the presence of bacteria using molecular biology (real-time PCR). Subsequently, a tissue fragment was taken for inoculation from the trees in which the presence of B. goodwinii and G. quercinecans bacteria was confirmed. The isolates obtained were used to test Koch's postulates and for biochemical analyses for Polish strains. In addition, the results obtained were also compared with the presence of A. biguttatus, which is considered a bacterial vector, which, in turn, confirmed that the bacteria responsible for the AOD phenomenon can also be present in trees not inhabited by this insect. The results obtained confirmed the presence of bacteria and their potential to cause necrosis in oaks, which fits into the model of the spiral disease that has been causing mass mortality of oaks in this naturally and economically valuable area since the 1980s.

Comparative characteristics of oat doubled haploids and oat × maize addition lines: Anatomical features of the leaves, chlorophyll a fluorescence and yield parameters.

As a result of oat (Avena sativa L.) × maize (Zea mays L.) crossing, maize chromosomes may not be completely eliminated at the early stages of embryogenesis, leading to the oat × maize addition (OMA) lines development. Introgression of maize chromosomes into oat genome can cause morphological and physiological modifications. The aim of the research was to evaluate the leaves' anatomy, chlorophyll a fluorescence, and yield parameter of oat doubled haploid (DH) and OMA lines obtained by oat × maize crossing. The present study examined two DH and two disomic OMA lines and revealed that they differ significantly in the majority of studied traits, apart from: the number of cells of the outer bundle sheath; light energy absorption; excitation energy trapped in PSII reaction centers; and energy dissipated from PSII. The OMA II line was characterized by larger size of single cells in the outer bundle sheath and greater number of seeds per plant among tested lines.

Antagonistic interactions safeguard mitotic propagation of genetic and epigenetic information in zebrafish.

The stability of cellular phenotypes in developing organisms depends on error-free transmission of epigenetic and genetic information during mitosis. Methylation of cytosine residues in genomic DNA is a key epigenetic mark that modulates gene expression and prevents genome instability. Here, we report on a genetic test of the relationship between DNA replication and methylation in the context of the developing vertebrate organism instead of cell lines. Our analysis is based on the identification of hypomorphic alleles of dnmt1, encoding the DNA maintenance methylase Dnmt1, and pole1, encoding the catalytic subunit of leading-strand DNA polymerase epsilon holoenzyme (Pole). Homozygous dnmt1 mutants exhibit genome-wide DNA hypomethylation, whereas the pole1 mutation is associated with increased DNA methylation levels. In dnmt1/pole1 double-mutant zebrafish larvae, DNA methylation levels are restored to near normal values, associated with partial rescue of mutant-associated transcriptional changes and phenotypes. Hence, a balancing antagonism between DNA replication and maintenance methylation buffers against replicative errors contributing to the robustness of vertebrate development.

Tnpo3 controls splicing of the pre-mRNA encoding the canonical TCR α chain of iNKT cells.

Unconventional T cells, such as innate natural killer T cells (iNKT) cells, are an important part of vertebrate immune defences. iNKT recognise glycolipids through a T cell receptor (TCR) that is composed of a semi-invariant TCR α chain, paired with a restricted set of TCR ß chains. Here, we show that splicing of the cognate Trav11-Traj18-Trac pre-mRNA encoding the characteristic Vα14Jα18 variable region of this semi-invariant TCR depends on the presence of Tnpo3. The Tnpo3 gene encodes a nuclear transporter of the ß-karyopherin family whose cargo includes various splice regulators. The block of iNKT cell development in the absence of Tnpo3 can be overcome by transgenic provision of a rearranged Trav11-Traj18-Trac cDNA, indicating that Tnpo3 deficiency does not interfere with the development of iNKT cells per se. Our study thus identifies a role for Tnpo3 in regulating the splicing of the pre-mRNA encoding the cognate TCRα chain of iNKT cells.

Hyaluronic acid-GPRC5C signalling promotes dormancy in haematopoietic stem cells.

Bone marrow haematopoietic stem cells (HSCs) are vital for lifelong maintenance of healthy haematopoiesis. In inbred mice housed in gnotobiotic facilities, the top of the haematopoietic hierarchy is occupied by dormant HSCs, which reversibly exit quiescence during stress. Whether HSC dormancy exists in humans remains debatable. Here, using single-cell RNA sequencing, we show a continuous landscape of highly purified human bone marrow HSCs displaying varying degrees of dormancy. We identify the orphan receptor GPRC5C, which enriches for dormant human HSCs. GPRC5C is also essential for HSC function, as demonstrated by genetic loss- and gain-of-function analyses. Through structural modelling and biochemical assays, we show that hyaluronic acid, a bone marrow extracellular matrix component, preserves dormancy through GPRC5C. We identify the hyaluronic acid-GPRC5C signalling axis controlling the state of dormancy in mouse and human HSCs.

Disentangling phylogenetic relations and biogeographic history within the Cucujus haematodes species group (Coleoptera: Cucujidae).

Recent progress in the taxonomy of flat bark beetles (Cucujidae), specifically, in the genus Cucujus, has revealed great diversity in subtropical Asia, but the seemingly well-known temperate and boreal taxa need further attention because of their conservation status. Here, we used an integrative approach using morphology, DNA, and species distribution modelling to disentangle phylogenetic relations, verify the number of species, and understand the historical biogeography of Palearctic and Nearctic Cucujus beetles, particularly the C. haematodes species group. Species distinctiveness was supported for C. cinnaberinus, but present-day C. haematodes turned out to be a species complex made up of separate lineages in the western, middle and eastern parts of its Palearctic range. Cucujus muelleri was a member of that complex, being sister to Asian C. haematodes. Moreover, C. haematodes caucasicus was found to be phylogenetically closely related to Italian C. tulliae, and both to be sister to European C. haematodes. North American C. clavipes clavipes and C. c. puniceus resulted to be enough divergent to be considered different species. Interestingly, western American C. puniceus turned out to be closely related to the C. haematodes complex, whereas eastern American C. clavipes constituted a separate lineage, being distantly related to both C. puniceus and C. cinnaberinus. These patterns suggest former trans-continental connections among the ancestors of extant flat bark beetle species. Moreover, a divergent lineage of C. cinnaberinus was found in Calabria, which should be regarded at the very least as a subspecies. The ancestor of C. hameatodes group originated in mid-Miocene, and next, ca. 6.2 Mya, a line leading to C. cinnaberinus had split. Speciation of the American lineages occurred during Pliocene (4.4 Mya for C. clavipes and 3.3 Mya for C. puniceus). Species classified as C. haematodes, C. tulliae and C. muelleri, as well as distinct lineages within C. cinnaberinus split during mid Pleistocene (ca. 1.5 Mya). A comparison of species climatic requirements and their present distribution allowed to identify glacial refugia in south-eastern areas of North America (C. clavipes), south-western areas of North America (C. puniceus), and the Mediterranean and Caspian Sea Basins (European Cucujus species), or south-eastern areas of Asia and the foothills of the central Asian mountains (eastern C. haematodes). Subsequent climatic changes in the Holocene forced these beetles to move their ranges northwards along the coasts of the Pacific (C. puniceus) or Atlantic (C. clavipes), north-eastwards to central, northern, and eastern Europe (C. cinnaberinus and European C. haematodes) or Siberia (Asian C. haematodes). The combined use of molecular, morphological and climatic data allows a comprehensive understanding of the phylogenetic relations and past distributions of Cucujus beetles, highlighting the complexity of C. haematodes species group evolution.

Genetic landscape of T cells identifies synthetic lethality for T-ALL.

To capture the global gene network regulating the differentiation of immature T cells in an unbiased manner, large-scale forward genetic screens in zebrafish were conducted and combined with genetic interaction analysis. After ENU mutagenesis, genetic lesions associated with failure of T cell development were identified by meiotic recombination mapping, positional cloning, and whole genome sequencing. Recessive genetic variants in 33 genes were identified and confirmed as causative by additional experiments. The mutations affected T cell development but did not perturb the development of an unrelated cell type, growth hormone-expressing somatotrophs, providing an important measure of cell-type specificity of the genetic variants. The structure of the genetic network encompassing the identified components was established by a subsequent genetic interaction analysis, which identified many instances of positive (alleviating) and negative (synthetic) genetic interactions. Several examples of synthetic lethality were subsequently phenocopied using combinations of small molecule inhibitors. These drugs not only interfered with normal T cell development, but also elicited remission in a model of T cell acute lymphoblastic leukaemia. Our findings illustrate how genetic interaction data obtained in the context of entire organisms can be exploited for targeted interference with specific cell types and their malignant derivatives.

Transgenerational inheritance of impaired larval T cell development in zebrafish.

Evidence for transgenerational inheritance of epigenetic information in vertebrates is scarce. Aberrant patterns of DNA methylation in gametes may set the stage for transmission into future generations. Here, we describe a viable hypomorphic allele of dnmt1 in zebrafish that causes widespread demethylation of CpG dinucleotides in sperm and somatic tissues. We find that homozygous mutants are essentially normal, with the exception of drastically impaired lymphopoiesis, affecting both larval and adult phases of T cell development. The phenotype of impaired larval (but not adult) T cell development is transmitted to subsequent generations by genotypically wildtype fish. We further find that about 200 differentially methylated regions in sperm DNA of transmitting and non-transmitting males, including hypermethylated sites associated with runx3 and rptor genes, whose reduced activities are associated with impaired larval T cell development. Our results indicate a particular sensitivity of larval T cell development to transgenerationally inherited epimutations.

Epigenetic Protection of Vertebrate Lymphoid Progenitor Cells by Dnmt1.

DNA methylation is a universal epigenetic mechanism involved in regulation of gene expression and genome stability. The DNA maintenance methylase DNMT1 ensures that DNA methylation patterns are faithfully transmitted to daughter cells during cell division. Because loss of DNMT1 is lethal, a pan-organismic analysis of DNMT1 function is lacking. We identified new recessive dnmt1 alleles in medaka and zebrafish and, guided by the structures of mutant proteins, generated a recessive variant of mouse Dnmt1. Each of the three missense mutations studied here distorts the catalytic pocket and reduces enzymatic activity. Because all three DNMT1 mutant animals are viable, it was possible to examine their phenotypes throughout life. The consequences of genome-wide hypomethylation of DNA of somatic tissues in the Dnmt1 mutants are surprisingly mild but consistently affect the development of the lymphoid lineage. Our findings indicate that developing lymphocytes in vertebrates are sensitive to perturbations of DNA maintenance methylation.

Pervasive changes of mRNA splicing in upf1-deficient zebrafish identify rpl10a as a regulator of T cell development.

The transcriptome of eukaryotic cells is constantly monitored for errors to avoid the production of undesired protein variants. The evolutionarily conserved nonsense-mediated mRNA decay (NMD) pathway degrades aberrant mRNAs, but also functions in the regulation of transcript abundance in response to changed physiological states. Here, we describe a zebrafish mutant of upf1, encoding the central component of the NMD machinery. Fish homozygous for the upf1t20450 allele (Y163X) survive until day 10 after fertilization, presenting with impaired T cell development as one of the most conspicuous features of the mutant phenotype. Analysis of differentially expressed genes identified dysregulation of the pre-mRNA splicing pathway, accompanied by perturbed autoregulation of canonical splicing activators (SRSF) and repressors (HNRNP). In upf1-deficient mutants, NMD-susceptible transcripts of ribosomal proteins that are known for their role as noncanonical splicing regulators were greatly increased, most notably, rpl10a When the levels of NMD-susceptible rpl10a transcripts were artificially increased in zebrafish larvae, T cell development was significantly impaired, suggesting that perturbed autoregulation of rpl10a splicing contributes to failing T cell development in upf1 deficiency. Our results identify an extraribosomal tissue-specific function to rpl10a in the immune system, and thus exemplify the advantages of the zebrafish model to study the effects of upf1-deficiency in the context of a vertebrate organism.

Development of Neonectria punicea Pathogenic Symptoms in Juvenile Fraxinus excelsior Trees.

When monitoring the state of health of Fraxinus excelsior trees, unusual symptoms were discovered within a F. excelsior plantation in Bosnia and Herzegovina. These symptoms included the appearance of necrosis and cankers in the basal parts of the trees, followed by the formation of fruiting bodies, however, none of these symptoms were found in the crowns. After sampling and isolation of the necrotic parts from the stem base, pathogen Neonectria punicea was isolated and identified from the characteristics of pure cultures, morphology of the fruiting bodies, and from multilocus sequencing. In field conditions, juvenile F. excelsior trees were inoculated with two N. punicea isolates obtained from the necrotic tissues of both juvenile F. excelsior and mature Fagus sylvatica trees. In both isolates, 12 months post inoculation, the lengths and widths of the necroses were significantly larger compared to the control. Necroses of significantly larger lengths, widths and surfaces were found again in both tested isolates 24 months post inoculation. In the case of the F. excelsior isolate, the lengths of the necroses at both the stem base and at breast height increased by 1.6 times, whereas the F. sylvatica isolate increased in size by up to 1.7 and 1.8 times, respectively. Trees inoculated without a previous bark wound showed no symptoms, similar to the control trees. Scanning electron microscopy and X-ray micro-computed tomography imaging revealed that N. punicea hyphae penetrated from the cankers to the woody outermost annual growth ring and that hyphae were present mostly in the large earlywood vessels and rarely in the axial parenchyma cells. Hyphae also spread radially through the pits in vessels. The infected trees responded with the formation of tyloses in the vessels to prevent a rapid fungal spread through the axial vascular transport pathway. The ability of N. punicea to cause necroses in juvenile ash trees was demonstrated for the first time during this study. It poses a serious threat to planted forests and natural regenerations of F. excelsior especially if F. sylvatica is considered as a possible inoculum reservoir for future infections. This pathogen should be integrated within future ash resistance or breeding programs.

Evolutionary transition from degenerate to nonredundant cytokine signaling networks supporting intrathymic T cell development.

In mammals, T cell development critically depends on the IL-7 cytokine signaling pathway. Here we describe the identification of the zebrafish ortholog of mammalian IL-7 based on chromosomal localization, deduced protein sequence, and expression patterns. To examine the biological role of il7 in teleosts, we generated an il7 allele lacking most of its coding exons using CRISPR/Cas9-based mutagenesis. il7-deficient animals are viable and exhibit no obvious signs of immune disorder. With respect to intrathymic T cell development, il7 deficiency is associated with only a mild reduction of thymocyte numbers, contrasting with a more pronounced impairment of T cell development in il7r-deficient fish. Genetic interaction studies between il7 and il7r mutants, and il7 and crlf2(tslpr) mutants suggest the contribution of additional, as-yet unidentified cytokines to intrathymic T cell development. Such activities were also ascertained for other cytokines, such as il2 and il15, collectively indicating that in contrast to the situation in mammals, T cell development in the thymus of teleosts is driven by a degenerate multicomponent network of γc cytokines; this explains why deficiencies of single components have little detrimental effect. In contrast, the dependence on a single cytokine in the mammalian thymus has catastrophic consequences in cases of congenital deficiencies in genes affecting the IL-7 signaling pathway. We speculate that the transition from a degenerate to a nonredundant cytokine network supporting intrathymic T cell development emerged as a consequence of repurposing evolutionarily ancient constitutive cytokine pathways for regulatory functions in the mammalian peripheral immune system.

Systematic Identification of Cell-Cell Communication Networks in the Developing Brain.

Since the generation of cell-type specific knockout models, the importance of inter-cellular communication between neural, vascular, and microglial cells during neural development has been increasingly appreciated. However, the extent of communication between these major cell populations remains to be systematically mapped. Here, we describe EMBRACE (embryonic brain cell extraction using FACS), a method to simultaneously isolate neural, mural, endothelial, and microglial cells to more than 94% purity in ∼4 h. Utilizing EMBRACE we isolate, transcriptionally analyze, and build a cell-cell communication map of the developing mouse brain. We identify 1,710 unique ligand-receptor interactions between neural, endothelial, mural, and microglial cells in silico and experimentally confirm the APOE-LDLR, APOE-LRP1, VTN-KDR, and LAMA4-ITGB1 interactions in the E14.5 brain. We provide our data via the searchable "Brain interactome explorer", available at https://mpi-ie.shinyapps.io/braininteractomeexplorer/. Together, this study provides a comprehensive map that reveals the richness of communication within the developing brain.

snakePipes: facilitating flexible, scalable and integrative epigenomic analysis.

SUMMARY: Due to the rapidly increasing scale and diversity of epigenomic data, modular and scalable analysis workflows are of wide interest. Here we present snakePipes, a workflow package for processing and downstream analysis of data from common epigenomic assays: ChIP-seq, RNA-seq, Bisulfite-seq, ATAC-seq, Hi-C and single-cell RNA-seq. snakePipes enables users to assemble variants of each workflow and to easily install and upgrade the underlying tools, via its simple command-line wrappers and yaml files. AVAILABILITY AND IMPLEMENTATION: snakePipes can be installed via conda: `conda install -c mpi-ie -c bioconda -c conda-forge snakePipes'. Source code (https://github.com/maxplanck-ie/snakepipes) and documentation (https://snakepipes.readthedocs.io/en/latest/) are available online. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Executive control in language production by children with and without language impairment.

BACKGROUND: Accumulating evidence suggests that the updating, inhibiting and shifting abilities underlying executive control are important for spoken language production in adults. However, little is known about this in children. AIMS: To examine whether children with and without language impairment differ in all or only some of these executive abilities, and whether they show corresponding differences when these abilities are engaged in language production. METHODS & PROCEDURES: Thirty-three children with specific language impairment (SLI) and 41 typically developing (TD) children (age matched, aged 8-12 years) completed standard executive control tests that measure the updating, inhibiting and shifting abilities. All children were native speakers of Dutch. Moreover, they performed a noun-phrase production task involving picture description within a picture-word interference paradigm. We measured their production accuracy and speed to assess length, distractor and switch effects, which reflect the updating, inhibiting and shifting abilities underlying executive control. OUTCOMES & RESULTS: Compared with TD children, the children with SLI had lower scores on all executive control tests. Moreover, they were overall slower and made more errors in the noun-phrase production task. Additionally, the magnitude of the distractor and switch effects was larger for the SLI than for the TD group. CONCLUSIONS & IMPLICATIONS: The results suggest that children with SLI have impaired language production and executive control abilities, and that some of the differences in the executive control abilities between SLI and TD groups were reflected in their language production.

Molecular alterations associated with metastases of solid pseudopapillary neoplasms of the pancreas.

Solid pseudopapillary neoplasms (SPN) of the pancreas are rare, low-grade malignant neoplasms that metastasise to the liver or peritoneum in 10-15% of cases. They almost invariably present somatic activating mutations of CTNNB1. No comprehensive molecular characterisation of metastatic disease has been conducted to date. We performed whole-exome sequencing and copy-number variation (CNV) analysis of 10 primary SPN and comparative sequencing of five matched primary/metastatic tumour specimens by high-coverage targeted sequencing of 409 genes. In addition to CTNNB1-activating mutations, we found inactivating mutations of epigenetic regulators (KDM6A, TET1, BAP1) associated with metastatic disease. Most of these alterations were shared between primary and metastatic lesions, suggesting that they occurred before dissemination. Differently from mutations, the majority of CNVs were not shared among lesions from the same patients and affected genes involved in metabolic and pro-proliferative pathways. Immunostaining of 27 SPNs showed that loss or reduction of KDM6A and BAP1 expression was significantly enriched in metastatic SPNs. Consistent with an increased transcriptional response to hypoxia in pancreatic adenocarcinomas bearing KDM6A inactivation, we showed that mutation or reduced KDM6A expression in SPNs is associated with increased expression of the HIF1α-regulated protein GLUT1 at both primary and metastatic sites. Our results suggest that BAP1 and KDM6A function is a barrier to the development of metastasis in a subset of SPNs, which might open novel avenues for the treatment of this disease. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Placebo Groups in Research on the Effectiveness of ABA Therapeutic Techniques.

Behavior analysts have shown that a single-subject experimental design (SSED) is a useful tool for identifying the effectiveness of specific therapeutic techniques, whereas researchers outside applied behavior analysis (ABA) maintain that randomized placebo-controlled trials (RPCT) provide the most definite test of efficacy. In this paper the possible benefits that could result from supporting SSED studies by placebo control groups are discussed. However, the use of placebo groups in psychotherapy research arouses considerable controversy and many researchers argue against it. The main aim of this paper is to clarify theoretical and methodological problems associated with using placebo groups in psychotherapy research and to demonstrate that these problems can be solved if the assumptions on which they are based are reformulated. The article also discusses ethical issues about the use of placebo groups in research on the effectiveness of psychotherapy.