A Screen for Drosophila Genes Relevant to the Nervous System

Understanding the molecular and genetic control of many brain functions remains one of the most challenging problems of modern biology. Although a number of brain structural and functional mutants have been isolated, their number and diversity is small compared to the complexity of the Drosophila nervous system. Using various differential screening methods in conjunction with a method of reverse genetics developed in the laboratory, the aim of the project was to gain molecular and eventually genetic access to this missing diversity. Following an assessment of differential and subtraction screening methods a differential screen was performed to isolate a set of about twenty cDNA clones representing genes expressed strongly in the head and weakly in the body. These clones were sifted further using partial sequence and approximate pattern of expression. Several clones were putatively identified as being derived from genes with homologues in other organisms including one previously known head specific gene, ninaE. Another clone from the screen, pST 123, was shown to be a cloned fragment of a message predicted to encode an unknown homologue of synaptobrevin, a family of molecules known to be involved in synaptic transmission. A third clone class, represented by the pST51 cDNA, was derived from a Na/K ATPase beta-subunit gene family member, a family of molecules involved in establishing membrane potentials in the nervous system, as well as in other processes such as cell-cell recognition. These two cDNAs were selected for further study, together with two other cDNA clones, pST162 and pST170, which defined genes with interesting patterns of transcription. A more detailed molecular characterisation of the four cDNA clones was performed to confirm the predicted gene expression pattern and to further investigate the molecular biology. For example, the beta-subunit cDNA insert from pST51, was sequenced and a full length predicted amino acid sequence identified. Phylogenetic analysis revealed that the encoded polypeptide was most closely related to the only other arthropod Na/K beta-subunit known, that of the brine shrimp. Together they define a new class of beta-subunit, clearly distinct from the three mammalian types. Expression analysis included head/body northerns and in situ hybridisation to head and head/body frozen sections using cDNA derived probes. This showed an expression pattern consistant with a gene expressed exclusively in the nervous system. A set of related cDNA clones identified using the pST51 cDNA insert reveal the likely presence of multiple transcripts generated with a variety of 5' and 3' untranslated regions. The analysis of the related cDNAs also identified the likely presence of a message encoding a second form of the beta-subunit. Genomic Southerns and in situ localisation to polytene chromosomes indicate a single copy gene, although lower stringency hybridisation experiments identify the presence of several related loci in the Drosophila genome. A similar analysis has been performed on the other selected cDNAs. Of these, the study of the pST 123 cDNA derived from a novel Drosophila synaptobrevin gene has proved to be the most informative. Interestingly, synaptobrevins have recently been identified as the target for tetanus toxin cleavage. From the predicted amino acid sequence it appears that the gene product should be cleaved by this toxin. A Drosophila synaptobrevin locus had previously been identified, but it appears that this locus produces proteins unlikely to cleave with the toxin, and has recently been reported as having a predominantly non-neuronal expression profile. In situ hybridisation experiments presented in this thesis suggest a neuronal role for the subunit isolated in this screen

Flavonoids from engineered tomatoes inhibit gut barrier pro-inflammatory cytokines and chemokines, via SAPK/JNK and p38 MAPK pathways

Flavonoids are a diverse group of plant secondary metabolites, known to reduce inflammatory bowel disease symptoms. How they achieve this is largely unknown. Our study focuses on the gut epithelium as it receives high topological doses of dietary constituents, maintains gut homeostasis, and orchestrates gut immunity. Dysregulation leads to chronic gut inflammation, via dendritic cell (DC)-driven immune responses. Tomatoes engineered for enriched sets of flavonoids (anthocyanins or flavonols) provided a unique and complex naturally consumed food matrix to study the effect of diet on chronic inflammation. Primary murine colonic epithelial cell-based inflammation assays consist of chemokine induction, apoptosis and proliferation, and effects on kinase pathways. Primary murine leukocytes and DCs were used to assay effects on transmigration. A murine intestinal cell line was used to assay wound healing. Engineered tomato extracts (enriched in anthocyanins or flavonols) showed strong and specific inhibitory effects on a set of key epithelial pro-inflammatory cytokines and chemokines. Chemotaxis assays showed a resulting reduction in the migration of primary leukocytes and DCs. Activation of epithelial cell SAPK/JNK and p38 MAPK signaling pathways were specifically inhibited. The epithelial wound healing-associated STAT3 pathway was unaffected. Cellular migration, proliferation, and apoptosis assays confirmed that wound healing processes were not affected by flavonoids. We show flavonoids target epithelial pro-inflammatory kinase pathways, inhibiting chemotactic signals resulting in reduced leukocyte and DC chemotaxis. Thus, both anthocyanins and flavonols modulate epithelial cells to become hyporesponsive to bacterial stimulation. Our results identify a viable mechanism to explain the in vivo anti-inflammatory effects of flavonoids

B1 SINE-binding ZFP266 impedes mouse iPSC generation through suppression of chromatin opening mediated by reprogramming factors

Induced pluripotent stem cell (iPSC) reprogramming is inefficient and understanding the molecular mechanisms underlying this inefficiency holds the key to successfully control cellular identity. Here, we report 24 reprogramming roadblock genes identified by CRISPR/Cas9-mediated genome-wide knockout (KO) screening. Of these, depletion of the predicted KRAB zinc finger protein (KRAB-ZFP) Zfp266 strongly and consistently enhances murine iPSC generation in several reprogramming settings, emerging as the most robust roadblock. We show that ZFP266 binds Short Interspersed Nuclear Elements (SINEs) adjacent to binding sites of pioneering factors, OCT4 (POU5F1), SOX2, and KLF4, and impedes chromatin opening. Replacing the KRAB co-suppressor with co-activator domains converts ZFP266 from an inhibitor to a potent facilitator of iPSC reprogramming. We propose that the SINE-KRAB-ZFP interaction is a critical regulator of chromatin accessibility at regulatory elements required for efficient cellular identity changes. In addition, this work serves as a resource to further illuminate molecular mechanisms hindering reprogramming.Induced pluripotent stem cell (iPSC) reprogramming is inherently inefficient. Here the authors identify 24 reprogramming roadblock genes through a CRISPR/Cas9-mediated genome-wide knockout screen including a KRAB-ZFP Zfp266, knockout of which consistently enhances murine iPSC generation.Peer reviewe

Urban Heat Island and Vulnerable Population. The Case of Madrid

The Urban Heat Island effect shows the differences among temperatures in urban areas and the surrounding rural ones. Previous studies have demonstrated that temperature differences could be up to 8 °C during the hottest periods of summer in Madrid , and that it varies according to the urban structure. Associated to this effect, the impact of temperature increase over dwelling indoor thermal comfort seems to double cooling energy demand . In Madrid, fuel poor households already suffering from inadequate indoor temperatures can face important overheating problems and, as a consequence, relevant health problems could become more frequent and stronger. This poses an increment in mortality rates in risk groups that should be evaluated. This research is aimed at establishing the geospatial connection between the urban heat island and the most vulnerable population living in the city of Madrid. Hence, those areas most in need for an urban intervention can be detected and prioritized

A database of microRNA expression patterns in Xenopus laevis

MicroRNAs (miRNAs) are short, non-coding RNAs around 22 nucleotides long. They inhibit gene expression either by translational repression or by causing the degradation of the mRNAs they bind to. Many are highly conserved amongst diverse organisms and have restricted spatio-temporal expression patterns during embryonic development where they are thought to be involved in generating accuracy of developmental timing and in supporting cell fate decisions and tissue identity. We determined the expression patterns of 180 miRNAs in Xenopus laevis embryos using LNA oligonucleotides. In addition we carried out small RNA-seq on different stages of early Xenopus development, identified 44 miRNAs belonging to 29 new families and characterized the expression of 5 of these. Our analyses identified miRNA expression in many organs of the developing embryo. In particular a large number were expressed in neural tissue and in the somites. Surprisingly none of the miRNAs we have looked at show expression in the heart. Our results have been made freely available as a resource in both XenMARK and Xenbase

Thymic epithelial cell fate and potency in early organogenesis assessed by single cell transcriptional and functional analysis

During development, cortical (c) and medullary (m) thymic epithelial cells (TEC) arise from the third pharyngeal pouch endoderm. Current models suggest that within the thymic primordium most TEC exist in a bipotent/common thymic epithelial progenitor cell (TEPC) state able to generate both cTEC and mTEC, at least until embryonic day 12.5 (E12.5) in the mouse. This view, however, is challenged by recent transcriptomics and genetic evidence. We therefore set out to investigate the fate and potency of TEC in the early thymus. Here using single cell (sc) RNAseq we identify a candidate mTEC progenitor population at E12.5, consistent with recent reports. Via lineage-tracing we demonstrate this population as mTEC fate-restricted, validating our bioinformatics prediction. Using potency analyses we also establish that most E11.5 and E12.5 progenitor TEC are cTEC-fated. Finally we show that overnight culture causes most if not all E12.5 cTEC-fated TEPC to acquire functional bipotency, and provide a likely molecular mechanism for this changed differentiation potential. Collectively, our data overturn the widely held view that a common TEPC predominates in the E12.5 thymus, showing instead that sublineage-primed progenitors are present from the earliest stages of thymus organogenesis but that these early fetal TEPC exhibit cell-fate plasticity in response to extrinsic factors. Our data provide a significant advance in the understanding of fetal thymic epithelial development and thus have implications for thymus-related clinical research, in particular research focussed on generating TEC from pluripotent stem cells