1,721,097 research outputs found
Nucleic acid purification from plants, animals and microbes in under 30 seconds
No full textNucleic acid amplification is a powerful molecular biology tool, although its use outside the modern laboratory environment is limited due to the relatively cumbersome methods required to extract nucleic acids from biological samples. To address this issue, we investigated a variety of materials for their suitability for nucleic acid capture and purification. We report here that untreated cellulose-based paper can rapidly capture nucleic acids within seconds and retain them during a single washing step, while contaminants present in complex biological samples are quickly removed. Building on this knowledge, we have successfully created an equipment-free nucleic acid extraction dipstick methodology that can obtain amplification-ready DNA and RNA from plants, animals, and microbes from difficult biological samples such as blood and leaves from adult trees in less than 30 seconds. The simplicity and speed of this method as well as the low cost and availability of suitable materials (e.g., common paper towelling), means that nucleic acid extraction is now more accessible and affordable for researchers and the broader community. Furthermore, when combined with recent advancements in isothermal amplification and naked eye DNA visualization techniques, the dipstick extraction technology makes performing molecular diagnostic assays achievable in limited resource settings including university and high school classrooms, field-based environments, and developing countries
The in vivo kinetics of RNA polymerase II elongation during co-transcriptional splicing.
Get PDFRNA processing events that take place on the transcribed pre-mRNA include capping, splicing, editing, 3' processing, and polyadenylation. Most of these processes occur co-transcriptionally while the RNA polymerase II (Pol II) enzyme is engaged in transcriptional elongation. How Pol II elongation rates are influenced by splicing is not well understood. We generated a family of inducible gene constructs containing increasing numbers of introns and exons, which were stably integrated in human cells to serve as actively transcribing gene loci. By monitoring the association of the transcription and splicing machineries on these genes in vivo, we showed that only U1 snRNP localized to the intronless gene, consistent with a splicing-independent role for U1 snRNP in transcription. In contrast, all snRNPs accumulated on intron-containing genes, and increasing the number of introns increased the amount of spliceosome components recruited. This indicates that nascent RNA can assemble multiple spliceosomes simultaneously. Kinetic measurements of Pol II elongation in vivo, Pol II ChIP, as well as use of Spliceostatin and Meayamycin splicing inhibitors showed that polymerase elongation rates were uncoupled from ongoing splicing. This study shows that transcription elongation kinetics proceed independently of splicing at the model genes studied here. Surprisingly, retention of polyadenylated mRNA was detected at the transcription site after transcription termination. This suggests that the polymerase is released from chromatin prior to the completion of splicing, and the pre-mRNA is post-transcriptionally processed while still tethered to chromatin near the gene end
Lamin B1 and lamin B2 are long-lived proteins with distinct functions in retinal development
Get PDFLamin B1 and lamin B2 are essential building blocks of the nuclear lamina, a filamentous meshwork lining the nucleoplasmic side of the inner nuclear membrane. Deficiencies in lamin B1 and lamin B2 impair neurodevelopment, but distinct functions for the two proteins in the development and homeostasis of the CNS have been elusive. Here we show that embryonic depletion of lamin B1 in retinal progenitors and postmitotic neurons affects nuclear integrity, leads to the collapse of the laminB2 meshwork, impairs neuronal survival, and markedly reduces the cellularity of adult retinas. In stark contrast, a deficiency of lamin B2 in the embryonic retina has no obvious effect on lamin B1 localization or nuclear integrity in embryonic retinas, suggesting that lamin B1, but not lamin B2, is strictly required for nucleokinesis during embryonic neurogenesis. However, the absence of lamin B2 prevents proper lamination of adult retinal neurons, impairs synaptogenesis, and reduces cone photoreceptor survival. We also show that lamin B1 and lamin B2 are extremely long-lived proteins in rod and cone photoreceptors. OF interest, a complete absence of both proteins during postnatal life has little or no effect on the survival and function of cone photoreceptors.Version of Recor
An unbiased cell morphology-based screen for new, biologically active small molecules.
Get PDFWe have implemented an unbiased cell morphology-based screen to identify small-molecule modulators of cellular processes using the Cytometrix (TM) automated imaging and analysis system. This assay format provides unbiased analysis of morphological effects induced by small molecules by capturing phenotypic readouts of most known classes of pharmacological agents and has the potential to read out pathways for which little is known. Four human-cancer cell lines and one noncancerous primary cell type were treated with 107 small molecules comprising four different protein kinase-inhibitor scaffolds. Cellular phenotypes induced by each compound were quantified by multivariate statistical analysis of the morphology, staining intensity, and spatial attributes of the cellular nuclei, microtubules, and Golgi compartments. Principal component analysis was used to identify inhibitors of cellular components not targeted by known protein kinase inhibitors. Here we focus on a hydroxyl-substituted analog (hydroxy-PP) of the known Src-family kinase inhibitor PP2 because it induced cell-specific morphological features distinct from all known kinase inhibitors in the collection. We used affinity purification to identify a target of hydroxy-PP, carbonyl reductase 1 (CBR1), a short-chain dehydrogenase-reductase. We solved the X-ray crystal structure of the CBR1/hydroxy-PP complex to 1.24 A resolution. Structure-based design of more potent and selective CBR1 inhibitors provided probes for analyzing the biological function of CBR1 in A549 cells. These studies revealed a previously unknown function for CBR1 in serum-withdrawal-induced apoptosis. Further studies indicate CBR1 inhibitors may enhance the effectiveness of anticancer anthracyclines. Morphology-based screening of diverse cancer cell types has provided a method for discovering potent new small-molecule probes for cell biological studies and anticancer drug candidates
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NAD+ consumption by PARP1 in response to DNA damage triggers metabolic shift critical for damaged cell survival
Get PDFDNA damage signaling is critical for the maintenance of genome integrity and cell fate decision. Poly(ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor rapidly activated in a damage dose- and complexity-dependent manner playing a critical role in the initial chromatin organization and DNA repair pathway choice at damage sites. However, our understanding of a cell-wide consequence of its activation in damaged cells is still limited. Using the phasor approach to fluorescence lifetime imaging microscopy and fluorescence-based biosensors in combination with laser microirradiation, we found a rapid cell-wide increase of the bound NADH fraction in response to nuclear DNA damage, which is triggered by PARP-dependent NAD+ depletion. This change is linked to the metabolic balance shift to oxidative phosphorylation (oxphos) over glycolysis. Inhibition of oxphos, but not glycolysis, resulted in parthanatos due to rapid PARP-dependent ATP deprivation, indicating that oxphos becomes critical for damaged cell survival. The results reveal the novel prosurvival response to PARP activation through a change in cellular metabolism and demonstrate how unique applications of advanced fluorescence imaging and laser microirradiation-induced DNA damage can be a powerful tool to interrogate damage-induced metabolic changes at high spatiotemporal resolution in a live cell
p16(INK4a) prevents centrosome dysfunction and genomic instability in primary cells.
Get PDFAneuploidy, frequently observed in premalignant lesions, disrupts gene dosage and contributes to neoplastic progression. Theodor Boveri hypothesized nearly 100 years ago that aneuploidy was due to an increase in centrosome number (multipolar mitoses) and the resultant abnormal segregation of chromosomes. We performed immunocytochemistry, quantitative immunofluorescence, karyotypic analysis, and time-lapse microscopy on primary human diploid epithelial cells and fibroblasts to better understand the mechanism involved in the production of supernumerary centrosomes (more than two microtubule nucleating bodies) to directly demonstrate that the presence of supernumerary centrosomes in genomically intact cells generates aneuploid daughter cells. We show that loss of p16(INK4a) generates supernumerary centrosomes through centriole pair splitting. Generation of supernumerary centrosomes in human diploid epithelial cells was shown to nucleate multipolar spindles and directly drive production of aneuploid daughter cells as a result of unequal segregation of the genomic material during mitosis. Finally, we demonstrate that p16(INK4a) cooperates with p21 through regulation of cyclin-dependent kinase activity to prevent centriole pair splitting. Cells with loss of p16(INK4a) activity have been found in vivo in histologically normal mammary tissue from a substantial fraction of healthy, disease-free women. Demonstration of centrosome dysfunction in cells due to loss of p16(INK4a) suggests that, under the appropriate conditions, these cells can become aneuploid. Gain or loss of genomic material (aneuploidy) may provide the necessary proproliferation and antiapoptotic mechanisms needed for the earliest stages of tumorigenesis
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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