516 research outputs found
PhOTO Zebrafish: A Transgenic Resource for In Vivo Lineage Tracing during Development and Regeneration
Background: Elucidating the complex cell dynamics (divisions, movement, morphological changes, etc.) underlying embryonic development and adult tissue regeneration requires an efficient means to track cells with high fidelity in space and time. To satisfy this criterion, we developed a transgenic zebrafish line, called PhOTO, that allows photoconvertible optical tracking of nuclear and membrane dynamics in vivo.
Methodology: PhOTO zebrafish ubiquitously express targeted blue fluorescent protein (FP) Cerulean and photoconvertible FP Dendra2 fusions, allowing for instantaneous, precise targeting and tracking of any number of cells using Dendra2 photoconversion while simultaneously monitoring global cell behavior and morphology. Expression persists through adulthood, making the PhOTO zebrafish an excellent tool for studying tissue regeneration: after tail fin amputation and photoconversion of a ~100µm stripe along the cut area, marked differences seen in how cells contribute to the new tissue give detailed insight into the dynamic process of regeneration. Photoconverted cells that contributed to the regenerate were separated into three distinct populations corresponding to the extent of cell division 7 days after amputation, and a subset of cells that divided the least were organized into an evenly spaced, linear orientation along the length of the newly regenerating fin.
Conclusions/Significance: PhOTO zebrafish have wide applicability for lineage tracing at the systems-level in the early embryo as well as in the adult, making them ideal candidate tools for future research in development, traumatic injury and regeneration, cancer progression, and stem cell behavior
Bi-cultural dynamics for risk and protective factors for cardiometabolic health in an Alaska Native (Yup\u27ik) population
Alaska Native people experience disparities in mortality from heart disease and stroke. This work attempts to better understand the relationships between socioeconomic, behavioral, and cardiometabolic risk factors among Yup\u27ik people of southwestern Alaska, with a focus on the role of the socioeconomic, and cultural components. Using a cross-sectional sample of 486 Yup\u27ik adults, we fitted a Partial Least Squares Path Model (PLS-PM) to assess the associations between components, including demographic factors [age and gender], socioeconomic factors [education, economic status, Yup\u27ik culture, and Western culture], behavioral factors [diet, cigarette smoking and smokeless tobacco use, and physical activity], and cardiometabolic risk factors [adiposity, triglyceride-HDL and LDL lipids, glycemia, and blood pressure]. We found relatively mild associations of education and economic status with cardiometabolic risk factors, in contrast with studies in other populations. The socioeconomic factor and participation in Yup\u27ik culture had potentially protective associations with adiposity, triglyceride-HDL lipids, and blood pressure, whereas participation in Western culture had a protective association with blood pressure. We also found a moderating effect of participation in Western culture on the relationships between Yup\u27ik culture participation and both blood pressure and LDL lipids, indicating a potentially beneficial additional effect of biculturalism. Our results suggest that reinforcing protective effects of both Yup\u27ik and Western cultures could be useful for interventions aimed at reducing cardiometabolic health disparities
Control of intestinal stem cell function and proliferation by mitochondrial pyruvate metabolism.
Most differentiated cells convert glucose to pyruvate in the cytosol through glycolysis, followed by pyruvate oxidation in the mitochondria. These processes are linked by the mitochondrial pyruvate carrier (MPC), which is required for efficient mitochondrial pyruvate uptake. In contrast, proliferative cells, including many cancer and stem cells, perform glycolysis robustly but limit fractional mitochondrial pyruvate oxidation. We sought to understand the role this transition from glycolysis to pyruvate oxidation plays in stem cell maintenance and differentiation. Loss of the MPC in Lgr5-EGFP-positive stem cells, or treatment of intestinal organoids with an MPC inhibitor, increases proliferation and expands the stem cell compartment. Similarly, genetic deletion of the MPC in Drosophila intestinal stem cells also increases proliferation, whereas MPC overexpression suppresses stem cell proliferation. These data demonstrate that limiting mitochondrial pyruvate metabolism is necessary and sufficient to maintain the proliferation of intestinal stem cells
An experimental and theoretical study of the enantioselective deprotonation of cyclohexene oxide with isopinocampheyl-based chiral lithium amides
The mechanism of the enantioselective deprotonation of cyclohexene oxide with isopinocampheyl-based chiral lithium amide was studied by quantum chemical calculations. The transition states of eight molecules were fully optimized at the ab initio HF/3-21G and density functional B3LYP/3-21G levels with Gaussian 98. The activation energies were calculated at the B3LYP/6-31+G(3df,2p)//B3LYP/3-21G level. We found the theoretical evaluation to be consistent with the experimental data. At the best case, an enantiomeric excess of up to 95% for (R)-2-scyclohexen-1-ol was achieved with (−)-N, N-diisopinocampheyl lithium amide
Drosophila SPF45: A Bifunctional Protein with Roles in Both Splicing and DNA Repair
The sequence of the SPF45 protein is significantly conserved, yet functional studies have identified it as a splicing factor in animal cells and as a DNA-repair protein in plants. Using a combined genetic and biochemical approach to investigate this apparent functional discrepancy, we unify and validate both of these studies by demonstrating that the Drosophila melanogaster protein is bifunctional, with independent functions in DNA repair and splicing. We find that SPF45 associates with the U2 snRNP and that mutations that remove the C-terminal end of the protein disrupt this interaction. Although animals carrying this mutation are viable, they are nevertheless compromised in their ability to regulate Sex-lethal splicing, demonstrating that Sex-lethal is an important physiological target of SPF45. Furthermore, these mutant animals exhibit phenotypes diagnostic of difficulties in recovering from exogenously induced DNA damage. The conclusion that SPF45 functions in the DNA-repair pathway is strengthened by finding both genetic and physical interactions between SPF45 and RAD201, a previously uncharacterized member of the RecA/Rad51 protein family. Together with our finding that the fly SPF45 protein increases the survival rate of mutagen-treated bacteria lacking the RecG helicase, these studies provide the tantalizing suggestion that SPF45 has an ancient and evolutionarily conserved role in DNA repair
Temporally-Controlled Site-Specific Recombination in Zebrafish
Conventional use of the site-specific recombinase Cre is a powerful technology in mouse, but almost absent in other vertebrate model organisms. In zebrafish, Cre-mediated recombination efficiency was previously very low. Here we show that using transposon-mediated transgenesis, Cre is in fact highly efficient in this organism. Furthermore, temporal control of recombination can be achieved by using the ligand-inducible CreERT2. Site-specific recombination only occurs upon administration of the drug tamoxifen (TAM) or its active metabolite, 4-hydroxy-tamoxifen (4-OHT). Cre-mediated recombination is detectable already 4 or 2 hours after administration of TAM or 4-OHT, demonstrating fast recombination kinetics. In addition, low doses of TAM allow mosaic labeling of single cells. Combined, our results show that conditional Cre/lox will be a valuable tool for both, embryonic and adult zebrafish studies. Furthermore, single copy insertion transgenesis of Cre/lox constructs suggest a strategy suitable also for other organisms
Involvement of promoter methylation in the regulation of Pregnane X receptor in colon cancer cells
<p>Abstract</p> <p>Background</p> <p>Pregnane X receptor (PXR) is a key transcription factor that regulates drug metabolizing enzymes such as cytochrome P450 (CYP) 3A4, and plays important roles in intestinal first-pass metabolism. Although there is a large inter-individual heterogeneity with intestinal CYP3A4 expression and activity, the mechanism driving these differences is not sufficiently explained by genetic variability of PXR or CYP3A4. We examined whether epigenetic mechanisms are involved in the regulation of PXR/CYP3A4 pathways in colon cancer cells.</p> <p>Methods</p> <p>mRNA levels of PXR, CYP3A4 and vitamin D receptor (VDR) were evaluated by quantitative real-time PCR on 6 colon cancer cell lines (Caco-2, HT29, HCT116, SW48, LS180, and LoVo). DNA methylation status was also examined by bisulfite sequencing of the 6 cell lines and 18 colorectal cancer tissue samples. DNA methylation was reversed by the treatment of these cell lines with 5-aza-2'-deoxycytidine (5-aza-dC).</p> <p>Results</p> <p>The 6 colon cancer cell lines were classified into two groups (high or low expression cells) based on the basal level of PXR/CYP3A4 mRNA. DNA methylation of the CpG-rich sequence of the <it>PXR </it>promoter was more densely detected in the low expression cells (Caco-2, HT29, HCT116, and SW48) than in the high expression cells (LS180 and LoVo). This methylation was reversed by treatment with 5-aza-dC, in association with re-expression of PXR and CYP3A4 mRNA, but not VDR mRNA. Therefore, PXR transcription was silenced by promoter methylation in the low expression cells, which most likely led to downregulation of CYP3A4 transactivation. Moreover, a lower level of <it>PXR </it>promoter methylation was observed in colorectal cancer tissues compared with adjacent normal mucosa, suggesting upregulation of the PXR/CYP3A4 mRNAs during carcinogenesis.</p> <p>Conclusions</p> <p><it>PXR </it>promoter methylation is involved in the regulation of intestinal PXR and CYP3A4 mRNA expression and might be associated with the inter-individual variability of the drug responses of colon cancer cells.</p
Expression and regulation of caudal in the lower cyclorrhaphan fly Megaselia
The homeobox gene caudal (cad) regulates posterior development in Drosophila. In early embryos, the cad protein (CAD) is expressed in a posterior-to-anterior concentration gradient, which contributes polarity to the developing embryo. The CAD gradient is complementary to and dependent on the anterior pattern organizer Bicoid (BCD), which represses the translation of ubiquitous maternal cad transcripts in the anterior embryo through a direct interaction with the cad 3′ untranslated region (UTR). Here, we show that early embryos of the lower cyclorrhaphan fly Megaselia express the putative cad orthologue Mab-cad throughout the posterior three quarters of the blastoderm but lack maternal transcripts. In transgenic blastoderm embryos of Drosophila, Mab-cad cis-regulatory DNA drives the expression of a reporter gene in a similar pattern, while Mab-cad 3′ UTR fails to mediate translational repression of a ubiquitously transcribed reporter. For another lower cyclorrhaphan fly (Lonchoptera) and two related outgroup taxa of Cyclorrhapha (Empis, Haematopota), we report maternal cad expression in ovarian follicles. Together, our results suggest that BCD is not required for the translational repression of Mab-cad, and that maternal cad expression was lost in the Megaselia lineage
The nuclear receptors of Biomphalaria glabrata and Lottia gigantea: Implications for developing new model organisms
© 2015 Kaur et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedNuclear receptors (NRs) are transcription regulators involved in an array of diverse physiological functions including key roles in endocrine and metabolic function. The aim of this study was to identify nuclear receptors in the fully sequenced genome of the gastropod snail, Biomphalaria glabrata, intermediate host for Schistosoma mansoni and compare these to known vertebrate NRs, with a view to assessing the snail's potential as a invertebrate model organism for endocrine function, both as a prospective new test organism and to elucidate the fundamental genetic and mechanistic causes of disease. For comparative purposes, the genome of a second gastropod, the owl limpet, Lottia gigantea was also investigated for nuclear receptors. Thirty-nine and thirty-three putative NRs were identified from the B. glabrata and L. gigantea genomes respectively, based on the presence of a conserved DNA-binding domain and/or ligand-binding domain. Nuclear receptor transcript expression was confirmed and sequences were subjected to a comparative phylogenetic analysis, which demonstrated that these molluscs have representatives of all the major NR subfamilies (1-6). Many of the identified NRs are conserved between vertebrates and invertebrates, however differences exist, most notably, the absence of receptors of Group 3C, which includes some of the vertebrate endocrine hormone targets. The mollusc genomes also contain NR homologues that are present in insects and nematodes but not in vertebrates, such as Group 1J (HR48/DAF12/HR96). The identification of many shared receptors between humans and molluscs indicates the potential for molluscs as model organisms; however the absence of several steroid hormone receptors indicates snail endocrine systems are fundamentally different.The National Centre for the Replacement, Refinement and Reduction of Animals in Research, Grant Ref:G0900802 to CSJ, LRN, SJ & EJR [www.nc3rs.org.uk]
Large introns in relation to alternative splicing and gene evolution: a case study of Drosophila bruno-3
Background:
Alternative splicing (AS) of maturing mRNA can generate structurally and functionally distinct transcripts from the same gene. Recent bioinformatic analyses of available genome databases inferred a positive correlation between intron length and AS. To study the interplay between intron length and AS empirically and in more detail, we analyzed the diversity of alternatively spliced transcripts (ASTs) in the Drosophila RNA-binding Bruno-3 (Bru-3) gene. This gene was known to encode thirteen exons separated by introns of diverse sizes, ranging from 71 to 41,973 nucleotides in D. melanogaster. Although Bru-3's structure is expected to be conducive to AS, only two ASTs of this gene were previously described.
Results:
Cloning of RT-PCR products of the entire ORF from four species representing three diverged Drosophila lineages provided an evolutionary perspective, high sensitivity, and long-range contiguity of splice choices currently unattainable by high-throughput methods. Consequently, we identified three new exons, a new exon fragment and thirty-three previously unknown ASTs of Bru-3. All exon-skipping events in the gene were mapped to the exons surrounded by introns of at least 800 nucleotides, whereas exons split by introns of less than 250 nucleotides were always spliced contiguously in mRNA. Cases of exon loss and creation during Bru-3 evolution in Drosophila were also localized within large introns. Notably, we identified a true de novo exon gain: exon 8 was created along the lineage of the obscura group from intronic sequence between cryptic splice sites conserved among all Drosophila species surveyed. Exon 8 was included in mature mRNA by the species representing all the major branches of the obscura group. To our knowledge, the origin of exon 8 is the first documented case of exonization of intronic sequence outside vertebrates.
Conclusion:
We found that large introns can promote AS via exon-skipping and exon turnover during evolution likely due to frequent errors in their removal from maturing mRNA. Large introns could be a reservoir of genetic diversity, because they have a greater number of mutable sites than short introns. Taken together, gene structure can constrain and/or promote gene evolution
- …