Psf2 plays important roles in normal eye development in Xenopus laevis

PubMed Central

Walter, Brian E.; Perry, Kimberly J.; Fukui, Lisa; Malloch, Erica L.; Wever, Jason

2008-01-01

Purpose Psf2 (partner of Sld5 2) represents a member of the GINS (go, ichi, ni, san) heterotetramer [1] and functions in DNA replication as a “sliding clamp.” Previous in situ hybridization analyses revealed that Psf2 is expressed during embryonic development in a tissue-specific manner, including the optic cup (retina) and the lens [2]. This article provides an analysis of Psf2 function during eye development in Xenopus laevis. Methods A morpholino targeted to Psf2 mRNA was designed to knockdown Psf2 translation and was injected into specific embryonic cells during early cleavage stages in the frog, Xenopus laevis. Injected embryos were assayed for specific defects in morphology, cell proliferation, and apoptosis. Synthetic Psf2 RNA was also co-injected with the morpholino to rescue morpholino-mediated developmental defects. It is well known that reciprocal inductive interactions control the development of the optic cup and lens. Therefore, control- and morpholino-injected embryos were used for reciprocal transplantation experiments to distinguish the intrinsic role of Psf2 in the development of the optic cup (retina) versus the lens. Results Morpholino-mediated knockdown of Psf2 expression resulted in dosage-dependent phenotypes, which included microphthalmia, incomplete closure of the ventral retinal fissure, and retinal and lens dysgenesis. Defects were also observed in other embryonic tissues that normally express Psf2 including the pharyngeal arches and the otic vesicle, although other tissues that express Psf2 were not found to be grossly defective. Eye defects could be rescued by co-injection of synthetic Psf2 RNA. Examination of cell proliferation via an antibody against phospho-histone H3 S10P revealed no significant differences in the retina and lens following Psf2 knockdown. However, there was a significant increase in the level of apoptosis in retinal as well as forebrain tissues, as revealed by TUNEL (terminal deoxynucleotide transferase dUTP nick end labeling) assay. Conclusions The results demonstrate intrinsic roles for Psf2 in both retinal and to a lesser extent, lens tissues. Observed lens defects can mainly be attributed to deficiencies in retinal development and consequently the late phase of lens induction, which involves instructive cues from the optic cup. Developmental defects were not observed in all tissues that express Psf2, which could be related to differences in the translation of Psf2 or redundant effects of related factors such as proliferating cell nuclear antigen (PCNA). PMID:18509549

The structure of human SFPQ reveals a coiled-coil mediated polymer essential for functional aggregation in gene regulation

PubMed Central

Lee, Mihwa; Sadowska, Agata; Bekere, Indra; Ho, Diwei; Gully, Benjamin S.; Lu, Yanling; Iyer, K. Swaminathan; Trewhella, Jill; Fox, Archa H.; Bond, Charles S.

2015-01-01

SFPQ, (a.k.a. PSF), is a human tumor suppressor protein that regulates many important functions in the cell nucleus including coordination of long non-coding RNA molecules into nuclear bodies. Here we describe the first crystal structures of Splicing Factor Proline and Glutamine Rich (SFPQ), revealing structural similarity to the related PSPC1/NONO heterodimer and a strikingly extended structure (over 265 Å long) formed by an unusual anti-parallel coiled-coil that results in an infinite linear polymer of SFPQ dimers within the crystals. Small-angle X-ray scattering and transmission electron microscopy experiments show that polymerization is reversible in solution and can be templated by DNA. We demonstrate that the ability to polymerize is essential for the cellular functions of SFPQ: disruptive mutation of the coiled-coil interaction motif results in SFPQ mislocalization, reduced formation of nuclear bodies, abrogated molecular interactions and deficient transcriptional regulation. The coiled-coil interaction motif thus provides a molecular explanation for the functional aggregation of SFPQ that directs its role in regulating many aspects of cellular nucleic acid metabolism. PMID:25765647

Aberrant alternative splicing is another hallmark of cancer.

PubMed

Ladomery, Michael

2013-01-01

The vast majority of human genes are alternatively spliced. Not surprisingly, aberrant alternative splicing is increasingly linked to cancer. Splice isoforms often encode proteins that have distinct and even antagonistic properties. The abnormal expression of splice factors and splice factor kinases in cancer changes the alternative splicing of critically important pre-mRNAs. Aberrant alternative splicing should be added to the growing list of cancer hallmarks.

PSF reconstruction for Compton-based prompt gamma imaging

NASA Astrophysics Data System (ADS)

Jan, Meei-Ling; Lee, Ming-Wei; Huang, Hsuan-Ming

2018-02-01

Compton-based prompt gamma (PG) imaging has been proposed for in vivo range verification in proton therapy. However, several factors degrade the image quality of PG images, some of which are due to inherent properties of a Compton camera such as spatial resolution and energy resolution. Moreover, Compton-based PG imaging has a spatially variant resolution loss. In this study, we investigate the performance of the list-mode ordered subset expectation maximization algorithm with a shift-variant point spread function (LM-OSEM-SV-PSF) model. We also evaluate how well the PG images reconstructed using an SV-PSF model reproduce the distal falloff of the proton beam. The SV-PSF parameters were estimated from simulation data of point sources at various positions. Simulated PGs were produced in a water phantom irradiated with a proton beam. Compared to the LM-OSEM algorithm, the LM-OSEM-SV-PSF algorithm improved the quality of the reconstructed PG images and the estimation of PG falloff positions. In addition, the 4.44 and 5.25 MeV PG emissions can be accurately reconstructed using the LM-OSEM-SV-PSF algorithm. However, for the 2.31 and 6.13 MeV PG emissions, the LM-OSEM-SV-PSF reconstruction provides limited improvement. We also found that the LM-OSEM algorithm followed by a shift-variant Richardson-Lucy deconvolution could reconstruct images with quality visually similar to the LM-OSEM-SV-PSF-reconstructed images, while requiring shorter computation time.

Hypoxia leads to significant changes in alternative splicing and elevated expression of CLK splice factor kinases in PC3 prostate cancer cells.

PubMed

Bowler, Elizabeth; Porazinski, Sean; Uzor, Simon; Thibault, Philippe; Durand, Mathieu; Lapointe, Elvy; Rouschop, Kasper M A; Hancock, John; Wilson, Ian; Ladomery, Michael

2018-04-02

Mounting evidence suggests that one of the ways that cells adapt to hypoxia is through alternative splicing. The aim of this study was firstly to examine the effect of hypoxia on the alternative splicing of cancer associated genes using the prostate cancer cell line PC3 as a model. Secondly, the effect of hypoxia on the expression of several regulators of splicing was examined. PC3 cells were grown in 1% oxygen in a hypoxic chamber for 48 h, RNA extracted and sent for high throughput PCR analysis at the RNomics platform at the University of Sherbrooke, Canada. Genes whose exon inclusion rate PSI (ψ) changed significantly were identified, and their altered exon inclusion rates verified by RT-PCR in three cell lines. The expression of splice factors and splice factor kinases in response to hypoxia was examined by qPCR and western blotting. The splice factor kinase CLK1 was inhibited with the benzothiazole TG003. In PC3 cells the exon inclusion rate PSI (ψ) was seen to change by > 25% in 12 cancer-associated genes; MBP, APAF1, PUF60, SYNE2, CDC42BPA, FGFR10P, BTN2A2, UTRN, RAP1GDS1, PTPN13, TTC23 and CASP9 (caspase 9). The expression of the splice factors SRSF1, SRSF2, SRSF3, SAM68, HuR, hnRNPA1, and of the splice factor kinases SRPK1 and CLK1 increased significantly in hypoxia. We also observed that the splice factor kinase CLK3, but not CLK2 and CLK4, was also induced in hypoxic DU145 prostate, HT29 colon and MCF7 breast cancer cell lines. Lastly, we show that the inhibition of CLK1 in PC3 cells with the benzothiazole TG003 increased expression of the anti-apoptotic isoform caspase 9b. Significant changes in alternative splicing of cancer associated genes occur in prostate cancer cells in hypoxic conditions. The expression of several splice factors and splice factor kinases increases during hypoxia, in particular the Cdc-like splice factor kinases CLK1 and CLK3. We suggest that in hypoxia the elevated expression of these regulators of splicing helps cells adapt through alternative splicing of key cancer-associated genes. We suggest that the CLK splice factor kinases could be targeted in cancers in which hypoxia contributes to resistance to therapy.

MYCN controls an alternative RNA splicing program in high-risk metastatic neuroblastoma

PubMed Central

Zhang, Shile; Wei, Jun S.; Li, Samuel Q.; Badgett, Tom C.; Song, Young K.; Agarwal, Saurabh; Coarfa, Cristian; Tolman, Catherine; Hurd, Laura; Liao, Hongling; He, Jianbin; Wen, Xinyu; Liu, Zhihui; Thiele, Carol J.; Westermann, Frank; Asgharzadeh, Shahab; Seeger, Robert C.; Maris, John M.; Auvil, Jamie M Guidry; Smith, Malcolm A; Kolaczyk, Eric D; Shohet, Jason; Khan, Javed

2016-01-01

The molecular mechanisms underlying the aggressive behavior of MYCN driven neuroblastoma (NBL) is under intense investigation; however, little is known about the impact of this family of transcription factors on the splicing program. Here we used high-throughput RNA sequencing to systematically study the expression of RNA isoforms in stage 4 MYCN-amplified NBL, an aggressive subtype of metastatic NBL. We show that MYCN-amplified NBL tumors display a distinct gene splicing pattern affecting multiple cancer hallmark functions. Six splicing factors displayed unique differential expression patterns in MYCN-amplified tumors and cell lines, and the binding motifs for some of these splicing factors are significantly enriched in differentially-spliced genes. Direct binding of MYCN to promoter regions of the splicing factors PTBP1 and HNRNPA1 detected by ChIP-seq demonstrates MYCN controls the splicing pattern by direct regulation of the expression of these key splicing factors. Furthermore, high expression of PTBP1 and HNRNPA1 was significantly associated with poor overall survival of stage4 NBL patients (p≤0.05). Knocking down PTBP1, HNRNPA1 and their downstream target PKM2, an isoform of pro-tumor-growth, result in repressed growth of NBL cells. Therefore, our study reveals a novel role of MYCN in controlling global splicing program through regulation of splicing factors in addition to its well-known role in the transcription program. These findings suggest a therapeutically potential to target the key splicing factors or gene isoforms in high-risk NBL with MYCN-amplification. PMID:26683771

[Deregulation of pre-messenger RNA splicing and rare diseases].

PubMed

de la Grange, Pierre

2016-12-01

Most of protein-coding human genes are subjected to alternative pre-mRNA splicing. This mechanism is highly regulated to precisely modulate detection of specific splice sites. This regulation is under control of the spliceosome and several splicing factors are also required to modulate the alternative usage of splice sites. Splicing factors and spliceosome components recognize splicing signals and regulatory sequences of the pre-mRNAs. These splicing sequences make splicing susceptible to polymorphisms and mutations. Examples of associations between human rare diseases and defects in pre-messenger RNA splicing are accumulating. Although many alterations are caused by mutations in splicing sequence (i.e., cis acting mutations), recent studies described the disruptive impact of mutations within spliceosome components or splicing factors (i.e., trans acting mutations). Following growing of knowledge regarding splicing regulation, several approaches have been developed to compensate for the effect of deleterious mutations and to restore sufficient amounts of functional protein. © 2016 médecine/sciences – Inserm.

The Human Splicing Factor ASF/SF2 can Specifically Recognize Pre-mRNA 5' Splice Sites

NASA Astrophysics Data System (ADS)

Zuo, Ping; Manley, James L.

1994-04-01

ASF/SF2 is a human protein previously shown to function in in vitro pre-mRNA splicing as an essential factor necessary for all splices and also as an alternative splicing factor, capable of switching selection of 5' splice sites. To begin to study the protein's mechanism of action, we have investigated the RNA binding properties of purified recombinant ASF/SF2. Using UV crosslinking and gel shift assays, we demonstrate that the RNA binding region of ASF/SF2 can interact with RNA in a sequence-specific manner, recognizing the 5' splice site in each of two different pre-mRNAs. Point mutations in the 5' splice site consensus can reduce binding by as much as a factor of 100, with the largest effects observed in competition assays. These findings support a model in which ASF/SF2 aids in the recognition of pre-mRNA 5' splice sites.

Competition between pre-mRNAs for the splicing machinery drives global regulation of splicing

PubMed Central

Munding, Elizabeth M.; Shiue, Lily; Katzman, Sol; Donohue, John Paul; Ares, Manuel

2013-01-01

Summary During meiosis in yeast, global splicing efficiency increases and then decreases. Here we provide evidence that splicing improves due to reduced competition for the splicing machinery. The timing of this regulation corresponds to repression and reactivation of ribosomal protein genes (RPGs) during meiosis. In vegetative cells RPG repression by rapamycin treatment also increases splicing efficiency. Down-regulation of the RPG-dedicated transcription factor gene IFH1 genetically suppresses two spliceosome mutations prp11-1 and prp4-1, and globally restores splicing efficiency in prp4-1 cells. We conclude that the splicing apparatus is limiting and pre-mRNAs compete. Splicing efficiency of a pre-mRNA therefore depends not just on its own concentration and affinity for limiting splicing factor(s) but also on those of competing pre-mRNAs. Competition between RNAs for limiting RNA processing factors appears to be a general condition in eukaryotic cells important for function of a variety of post-transcriptional control mechanisms including miRNA repression, polyadenylation and splicing. PMID:23891561

[Filamentous and phosphate solubilizing fungi relationships with some edaphic parameters and coffee plantations management].

PubMed

Posada, Raúl Hernando; Sánchez de Prager, Marina; Sieverding, Ewald; Aguilar Dorantes, Karla; Heredia-Abarca, Gabriela Patricia

2012-09-01

Soil properties and the environment have multiple outcomes on fungal communities. Although, the interaction effects between management intensity, pH, available phosphorus, organic carbon, soil texture and different fractions of water stable macro-aggregates on the communities of microscopic filamentous fungi (MFF), iron phosphate solubilizing fungi (PSF-Fe), and iron and calcium phosphate solubilizing fungi (PSF-(Fe+Ca)), have been previously evaluated in field conditions, this has never been performed in terms of their combined effects, neither with phosphate solubilizing fungi. To assess this, we collected 40 composite soil samples from eight Mexican and Colombian coffee plantations, with different management intensities and physico-chemical edaphic parameters, during 2008-2009. We isolated different communities of MFF, PSF-Fe and PSF-(Fe+Ca), by wet sieving and soil particles culture in Potato-Dextrose-Agar from soil samples, and we classified isolates in terms of their phosphate solubilizing ability. Following the principal component analysis results, we decided to analyze fungal communities and abiotic factors interactions for each country separately. Structural Equation Models revealed that organic carbon was positively associated to MFF richness and number of isolates (lambda>0.58), but its relationship with PSF-Fe and PSF-(Fe+Ca) were variable; while the available phosphorus, pH and water stable macro-aggregate fractions did not show a clear pattern. Management intensity was negatively related to PSF-Fe (lambda < or = -0.21) morphotype richness and the number of isolates in Colombian coffee plantations. We found that the relationships of clay and organic carbon content, and available phosphorus and soil pH, with the species richness and number of isolates of MFF, PSF-Fe and PSF-(Fe+Ca) were highly variable; this made impossible to generalize the responses between saprotrophic fungal groups and geographic zones. The management intensity was not related to species richness and number of isolates of MFF in any coffee areas, while for PSF the relationship could not be defined. The different water stable macro-aggregates fractions did not show a defined pattern in relation to the species richness and the number of isolates of saprophytic and phosphate solubilizing fungi (PSF). This study highlights the need to take into account edaphic and geographic context in order to reach a better understanding of the intensity management effects on MFF and PSF function in agroecosystems.

MYCN controls an alternative RNA splicing program in high-risk metastatic neuroblastoma.

PubMed

Zhang, Shile; Wei, Jun S; Li, Samuel Q; Badgett, Tom C; Song, Young K; Agarwal, Saurabh; Coarfa, Cristian; Tolman, Catherine; Hurd, Laura; Liao, Hongling; He, Jianbin; Wen, Xinyu; Liu, Zhihui; Thiele, Carol J; Westermann, Frank; Asgharzadeh, Shahab; Seeger, Robert C; Maris, John M; Guidry Auvil, Jamie M; Smith, Malcolm A; Kolaczyk, Eric D; Shohet, Jason; Khan, Javed

2016-02-28

The molecular mechanisms underlying the aggressive behavior of MYCN driven neuroblastoma (NBL) is under intense investigation; however, little is known about the impact of this family of transcription factors on the splicing program. Here we used high-throughput RNA sequencing to systematically study the expression of RNA isoforms in stage 4 MYCN-amplified NBL, an aggressive subtype of metastatic NBL. We show that MYCN-amplified NBL tumors display a distinct gene splicing pattern affecting multiple cancer hallmark functions. Six splicing factors displayed unique differential expression patterns in MYCN-amplified tumors and cell lines, and the binding motifs for some of these splicing factors are significantly enriched in differentially-spliced genes. Direct binding of MYCN to promoter regions of the splicing factors PTBP1 and HNRNPA1 detected by ChIP-seq demonstrates that MYCN controls the splicing pattern by direct regulation of the expression of these key splicing factors. Furthermore, high expression of PTBP1 and HNRNPA1 was significantly associated with poor overall survival of stage4 NBL patients (p ≤ 0.05). Knocking down PTBP1, HNRNPA1 and their downstream target PKM2, an isoform of pro-tumor-growth, result in repressed growth of NBL cells. Therefore, our study reveals a novel role of MYCN in controlling global splicing program through regulation of splicing factors in addition to its well-known role in the transcription program. These findings suggest a therapeutically potential to target the key splicing factors or gene isoforms in high-risk NBL with MYCN-amplification. Published by Elsevier Ireland Ltd.

Impact of spliceosome mutations on RNA splicing in myelodysplasia: dysregulated genes/pathways and clinical associations.

PubMed

Pellagatti, Andrea; Armstrong, Richard N; Steeples, Violetta; Sharma, Eshita; Repapi, Emmanouela; Singh, Shalini; Sanchi, Andrea; Radujkovic, Aleksandar; Horn, Patrick; Dolatshad, Hamid; Roy, Swagata; Broxholme, John; Lockstone, Helen; Taylor, Stephen; Giagounidis, Aristoteles; Vyas, Paresh; Schuh, Anna; Hamblin, Angela; Papaemmanuil, Elli; Killick, Sally; Malcovati, Luca; Hennrich, Marco L; Gavin, Anne-Claude; Ho, Anthony D; Luft, Thomas; Hellström-Lindberg, Eva; Cazzola, Mario; Smith, Christopher W J; Smith, Stephen; Boultwood, Jacqueline

2018-06-21

SF3B1, SRSF2 and U2AF1 are the most frequently mutated splicing factor genes in the myelodysplastic syndromes (MDS). We have performed a comprehensive and systematic analysis to determine the impact of these commonly mutated splicing factors on pre-mRNA splicing in the bone marrow stem/progenitor cells and in the erythroid and myeloid precursors in splicing factor mutant MDS. Using RNA-seq, we determined the aberrantly spliced genes and dysregulated pathways in CD34 + cells of 84 MDS patients. Splicing factor mutations result in different alterations in splicing and largely affect different genes, but these converge in common dysregulated pathways and cellular processes, focused on RNA splicing, protein synthesis and mitochondrial dysfunction, suggesting common mechanisms of action in MDS. Many of these dysregulated pathways and cellular processes can be linked to the known disease pathophysiology associated with splicing factor mutations in MDS, whilst several others have not been previously associated with MDS, such as sirtuin signaling. We identified aberrantly spliced events associated with clinical variables, and isoforms which independently predict survival in MDS and implicate dysregulation of focal adhesion and extracellular exosomes as drivers of poor survival. Aberrantly spliced genes and dysregulated pathways were identified in the MDS-affected lineages in splicing factor mutant MDS. Functional studies demonstrated that knockdown of the mitosis regulators SEPT2 and AKAP8, aberrantly spliced target genes of SF3B1 and SRSF2 mutations respectively, led to impaired erythroid cell growth and differentiation. This study illuminates the impact of the common spliceosome mutations on the MDS phenotype and provides novel insights into disease pathophysiology. Copyright © 2018 American Society of Hematology.

Splicing factor gene mutations in the myelodysplastic syndromes: impact on disease phenotype and therapeutic applications.

PubMed

Pellagatti, Andrea; Boultwood, Jacqueline

2017-01-01

Splicing factor gene mutations are the most frequent mutations found in patients with the myeloid malignancy myelodysplastic syndrome (MDS), suggesting that spliceosomal dysfunction plays a major role in disease pathogenesis. The aberrantly spliced target genes and deregulated cellular pathways associated with the commonly mutated splicing factor genes in MDS (SF3B1, SRSF2 and U2AF1) are being identified, illuminating the molecular mechanisms underlying MDS. Emerging data from mouse modeling studies indicate that the presence of splicing factor gene mutations can lead to bone marrow hematopoietic stem/myeloid progenitor cell expansion, impaired hematopoiesis and dysplastic differentiation that are hallmarks of MDS. Importantly, recent evidence suggests that spliceosome inhibitors and splicing modulators may have therapeutic value in the treatment of splicing factor mutant myeloid malignancies. Copyright © 2016 Elsevier Ltd. All rights reserved.

SRSF2 mutations drive oncogenesis by activating a global program of aberrant alternative splicing in hematopoietic cells.

PubMed

Liang, Yang; Tebaldi, Toma; Rejeski, Kai; Joshi, Poorval; Stefani, Giovanni; Taylor, Ashley; Song, Yuanbin; Vasic, Radovan; Maziarz, Jamie; Balasubramanian, Kunthavai; Ardasheva, Anastasia; Ding, Alicia; Quattrone, Alessandro; Halene, Stephanie

2018-06-01

Recurrent mutations in the splicing factor SRSF2 are associated with poor clinical outcomes in myelodysplastic syndromes (MDS). Their high frequency suggests these mutations drive oncogenesis, yet the molecular explanation for this process is unclear. SRSF2 mutations could directly affect pre-mRNA splicing of a vital gene product; alternatively, a whole network of gene products could be affected. Here we determine how SRSF2 mutations globally affect RNA binding and splicing in vivo using HITS-CLIP. Remarkably, the majority of differential binding events do not translate into alternative splicing of exons with SRSF2 P95H binding sites. Alternative splice alterations appear to be dominated by indirect effects. Importantly, SRSF2 P95H targets are enriched in RNA processing and splicing genes, including several members of the hnRNP and SR families of proteins, suggesting a "splicing-cascade" phenotype wherein mutation of a single splicing factor leads to widespread modifications in multiple RNA processing and splicing proteins. We show that splice alteration of HNRNPA2B1, a splicing factor differentially bound and spliced by SRSF2 P95H , impairs hematopoietic differentiation in vivo. Our data suggests a model whereby the recurrent mutations in splicing factors set off a cascade of gene regulatory events that together affect hematopoiesis and drive cancer.

The RNA Splicing Response to DNA Damage.

PubMed

Shkreta, Lulzim; Chabot, Benoit

2015-10-29

The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging.

The RNA Splicing Response to DNA Damage

PubMed Central

Shkreta, Lulzim; Chabot, Benoit

2015-01-01

The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging. PMID:26529031

Perturbation of Chromatin Structure Globally Affects Localization and Recruitment of Splicing Factors

PubMed Central

Risso, Guillermo J.; Pawellek, Andrea; Ule, Jernej; Lamond, Angus I.; Kornblihtt, Alberto R.

2012-01-01

Chromatin structure is an important factor in the functional coupling between transcription and mRNA processing, not only by regulating alternative splicing events, but also by contributing to exon recognition during constitutive splicing. We observed that depolarization of neuroblastoma cell membrane potential, which triggers general histone acetylation and regulates alternative splicing, causes a concentration of SR proteins in nuclear speckles. This prompted us to analyze the effect of chromatin structure on splicing factor distribution and dynamics. Here, we show that induction of histone hyper-acetylation results in the accumulation in speckles of multiple splicing factors in different cell types. In addition, a similar effect is observed after depletion of the heterochromatic protein HP1α, associated with repressive chromatin. We used advanced imaging approaches to analyze in detail both the structural organization of the speckle compartment and nuclear distribution of splicing factors, as well as studying direct interactions between splicing factors and their association with chromatin in vivo. The results support a model where perturbation of normal chromatin structure decreases the recruitment efficiency of splicing factors to nascent RNAs, thus causing their accumulation in speckles, which buffer the amount of free molecules in the nucleoplasm. To test this, we analyzed the recruitment of the general splicing factor U2AF65 to nascent RNAs by iCLIP technique, as a way to monitor early spliceosome assembly. We demonstrate that indeed histone hyper-acetylation decreases recruitment of U2AF65 to bulk 3′ splice sites, coincident with the change in its localization. In addition, prior to the maximum accumulation in speckles, ∼20% of genes already show a tendency to decreased binding, while U2AF65 seems to increase its binding to the speckle-located ncRNA MALAT1. All together, the combined imaging and biochemical approaches support a model where chromatin structure is essential for efficient co-transcriptional recruitment of general and regulatory splicing factors to pre-mRNA. PMID:23152763

Development of a scale to measure symptoms of anxiety and depression in the general UK population: the psychiatric symptom frequency scale.

PubMed Central

Lindelow, M; Hardy, R; Rodgers, B

1997-01-01

OBJECTIVES: The psychiatric symptom frequency (PSF) scale was developed to assess symptoms of anxiety and depression (i.e. affective symptoms) experienced over the past year in the general population. This study aimed to examine the distribution of PSF scores, internal consistency, and factor structure and to investigate relationships between total scores for this scale and other indicators of poor mental health. PARTICIPANTS: The Medical Research Council national survey of health and development, a class stratified cohort study of men and women followed up from birth in 1946, with the most recent interview at age 43 when the PSF scale was administered. MAIN RESULTS: The PSF scale showed high internal consistency between the 18 items (Cronbach's alpha = 0.88). Ratings on items of the scale reflected one predominant factor, incorporating both depression and anxiety, and two additional factors of less statistical importance, one reflecting sleep problems and the other panic and situational anxiety. Total scores were calculated by adding 18 items of the scale, and high total scores were found to be strongly associated with reports of contact with a doctor or other health professional and use of prescribed medication for "nervous or emotional trouble or depression," and with suicidal ideas. CONCLUSIONS: The PSF is a useful and valid scale for evaluating affective symptoms in the general population. It is appropriate for administration by lay interviewers with minimal training, is relatively brief, and generates few missing data. The total score is a flexible measure which can be used in continuous or binary form to suit the purposes of individual investigations, and provides discrimination at lower as well as upper levels of symptom severity. PMID:9425466

Public speaking fears and their correlates among 17,615 Japanese adolescents.

PubMed

Furukawa, Toshi A; Watanabe, Norio; Kinoshita, Yoshihiro; Kinoshita, Kuni; Sasaki, Tsukasa; Nishida, Atsushi; Okazaki, Yuji; Shimodera, Shinji

2014-03-01

Public speaking fears (PSF) are highly prevalent in the general population. They are also among the most common symptoms of social anxiety disorder, which typically has an adolescent onset and has recently been increasingly recognized as a persistent and impairing disorder across various cultures in the world. This study examined PSF and their associated factors among 17,615 adolescents, aged 12-18, in a large-scale school-based community survey in Japan. PSF was noted in 7.3% of the students. The prevalence was higher among girls than among boys, and it had a tendency to decrease with age. Across the gender and age groups, PSF was associated with psychopathology, including depression and anxiety, suicidal thoughts, deliberate self-harm, violence towards objects and people, and increased difficulties at school. We should no longer make light of public speaking fears among adolescents as transient, common-sense phenomena. Copyright © 2012 Blackwell Publishing Asia Pty Ltd.

Functional domains of the human splicing factor ASF/SF2.

PubMed Central

Zuo, P; Manley, J L

1993-01-01

The human splicing factor ASF/SF2 displays two predominant activities in in vitro splicing assays: (i) it is an essential factor apparently required for all splices and (ii) it is able to switch utilization of alternative 5' splice sites in a concentration-dependent manner. ASF/SF2 is the prototype of a family of proteins typified by the presence of one or two RNP-type RNA binding domains (RBDs) and a region highly enriched in repeating arginine-serine dipeptides (RS regions). Here we describe a functional analysis of ASF/SF2, which defines several regions essential for one, or both, of its two principal activities, and provides insights into how this type of protein functions in splicing. Two isoforms of the protein, which arise from alternative splicing, are by themselves inactive, but each can block the activity of ASF/SF2, thereby functioning as splicing repressors. Some, but not all, mutations in the RS region prevent ASF/SF2 from functioning as an essential splicing factor. However, the entire RS region can be deleted without reducing splice site switching activity, indicating that it is not absolutely required for interaction with other splicing factors. Experiments with deletion and substitution mutants reveal that the protein contains two related, but highly diverged, RBDs, and that both are essential for activity. Each RBD by itself retains the ability to bind RNA, although optimal binding requires both domains. Images PMID:8223481

Pre-mRNA Splicing in Plants: In Vivo Functions of RNA-Binding Proteins Implicated in the Splicing Process

PubMed Central

Meyer, Katja; Koester, Tino; Staiger, Dorothee

2015-01-01

Alternative pre-messenger RNA splicing in higher plants emerges as an important layer of regulation upon exposure to exogenous and endogenous cues. Accordingly, mutants defective in RNA-binding proteins predicted to function in the splicing process show severe phenotypic alterations. Among those are developmental defects, impaired responses to pathogen threat or abiotic stress factors, and misregulation of the circadian timing system. A suite of splicing factors has been identified in the model plant Arabidopsis thaliana. Here we summarize recent insights on how defects in these splicing factors impair plant performance. PMID:26213982

Mutations of RNA splicing factors in hematological malignancies.

PubMed

Shukla, Girish C; Singh, Jagjit

2017-11-28

Systematic large-scale cancer genomic studies have produced numerous significant findings. These studies have not only revealed new cancer-promoting genes, but they also have identified cancer-promoting functions of previously known "housekeeping" genes. These studies have identified numerous mutations in genes which play a fundamental role in nuclear precursor mRNA splicing. Somatic mutations and copy number variation in many of the splicing factors which participate in the formation of multiple spliceosomal complexes appear to play a role in many cancers and in particular in myelodysplastic syndromes (MDS). Mutated proteins seem to interfere with the recognition of the authentic splice sites (SS) leading to utilization of suboptimal alternative splicing sites generating aberrantly spliced mRNA isoforms. This short review is focusing on the function of the splice factors involved in the formation of splicing complexes and potential mechanisms which affect usage of the authentic splice site recognition. Copyright © 2017 Elsevier B.V. All rights reserved.

Matching rendered and real world images by digital image processing

NASA Astrophysics Data System (ADS)

Mitjà, Carles; Bover, Toni; Bigas, Miquel; Escofet, Jaume

2010-05-01

Recent advances in computer-generated images (CGI) have been used in commercial and industrial photography providing a broad scope in product advertising. Mixing real world images with those rendered from virtual space software shows a more or less visible mismatching between corresponding image quality performance. Rendered images are produced by software which quality performance is only limited by the resolution output. Real world images are taken with cameras with some amount of image degradation factors as lens residual aberrations, diffraction, sensor low pass anti aliasing filters, color pattern demosaicing, etc. The effect of all those image quality degradation factors can be characterized by the system Point Spread Function (PSF). Because the image is the convolution of the object by the system PSF, its characterization shows the amount of image degradation added to any taken picture. This work explores the use of image processing to degrade the rendered images following the parameters indicated by the real system PSF, attempting to match both virtual and real world image qualities. The system MTF is determined by the slanted edge method both in laboratory conditions and in the real picture environment in order to compare the influence of the working conditions on the device performance; an approximation to the system PSF is derived from the two measurements. The rendered images are filtered through a Gaussian filter obtained from the taking system PSF. Results with and without filtering are shown and compared measuring the contrast achieved in different final image regions.

Evolution of a tissue-specific splicing network

PubMed Central

Taliaferro, J. Matthew; Alvarez, Nehemiah; Green, Richard E.; Blanchette, Marco; Rio, Donald C.

2011-01-01

Alternative splicing of precursor mRNA (pre-mRNA) is a strategy employed by most eukaryotes to increase transcript and proteomic diversity. Many metazoan splicing factors are members of multigene families, with each member having different functions. How these highly related proteins evolve unique properties has been unclear. Here we characterize the evolution and function of a new Drosophila splicing factor, termed LS2 (Large Subunit 2), that arose from a gene duplication event of dU2AF50, the large subunit of the highly conserved heterodimeric general splicing factor U2AF (U2-associated factor). The quickly evolving LS2 gene has diverged from the splicing-promoting, ubiquitously expressed dU2AF50 such that it binds a markedly different RNA sequence, acts as a splicing repressor, and is preferentially expressed in testes. Target transcripts of LS2 are also enriched for performing testes-related functions. We therefore propose a path for the evolution of a new splicing factor in Drosophila that regulates specific pre-mRNAs and contributes to transcript diversity in a tissue-specific manner. PMID:21406555

Alterations in expression pattern of splicing factors in epithelial ovarian cancer and its clinical impact.

PubMed

Iborra, Severine; Hirschfeld, Marc; Jaeger, Markus; Zur Hausen, Axel; Braicu, Iona; Sehouli, Jalid; Gitsch, Gerald; Stickeler, Elmar

2013-07-01

Alternative splicing represents an important nuclear mechanism in the posttranscriptional regulation of gene expression, which is frequently altered during tumorigenesis. Previously, we described marked changes in alternative splicing of the CD44 gene in ovarian and breast cancer as well as specific induction of distinct splicing factors during tumor development. The present study was focused on the expression profiles of different splicing factors, including classical serine-arginine (SR) proteins including ASF/SF2, hTra2β1, hTra2α, and Y-box-binding protein (YB-1) in physiological and malignant epithelial ovarian tissue to evaluate their expression pattern with regard to tumor development and disease progression. Expression levels of the different splicing factors were analyzed in physiological epithelial ovarian tissue samples, primary tumors, and metastatic samples of patients with a diagnosis of epithelial ovarian cancer using quantified reverse transcription polymerase chain reaction analysis. We examined more closely the splicing factor hTra2β1 using Western blot analysis and immunohistochemistry. The analysis revealed a marked and specific induction of ASF/SF2, SRp20, hTra2β1, and YB-1 in primary tumors as well as in their metastatic sites. However, in our patient cohort, no induction was seen for the other investigated splicing factors SRp55, SRp40, and hTra2α. Our results suggest a specific induction of distinct splicing factors in ovarian cancer tumorigenesis. The involvement of hTra2β1, YB-1, SRp20, and ASF/SF2 in exon recognition and alternative splicing may be important for gene regulation of alternatively spliced genes like CD44 with potential functional consequences in this tumor type leading to progression and metastasis.

Adaptive recovery of motion blur point spread function from differently exposed images

NASA Astrophysics Data System (ADS)

Albu, Felix; Florea, Corneliu; Drîmbarean, Alexandru; Zamfir, Adrian

2010-01-01

Motion due to digital camera movement during the image capture process is a major factor that degrades the quality of images and many methods for camera motion removal have been developed. Central to all techniques is the correct recovery of what is known as the Point Spread Function (PSF). A very popular technique to estimate the PSF relies on using a pair of gyroscopic sensors to measure the hand motion. However, the errors caused either by the loss of the translational component of the movement or due to the lack of precision in gyro-sensors measurements impede the achievement of a good quality restored image. In order to compensate for this, we propose a method that begins with an estimation of the PSF obtained from 2 gyro sensors and uses a pair of under-exposed image together with the blurred image to adaptively improve it. The luminance of the under-exposed image is equalized with that of the blurred image. An initial estimation of the PSF is generated from the output signal of 2 gyro sensors. The PSF coefficients are updated using 2D-Least Mean Square (LMS) algorithms with a coarse-to-fine approach on a grid of points selected from both images. This refined PSF is used to process the blurred image using known deblurring methods. Our results show that the proposed method leads to superior PSF support and coefficient estimation. Also the quality of the restored image is improved compared to 2 gyro only approach or to blind image de-convolution results.

A novel protein factor is required for use of distal alternative 5' splice sites in vitro.

PubMed Central

Harper, J E; Manley, J L

1991-01-01

Adenovirus E1A pre-mRNA was used as a model to examine alternative 5' splice site selection during in vitro splicing reactions. Strong preference for the downstream 13S 5' splice site over the upstream 12S or 9S 5' splice sites was observed. However, the 12S 5' splice site was used efficiently when a mutant pre-mRNA lacking the 13S 5' splice site was processed, and 12S splicing from this substrate was not reduced by 13S splicing from a separate pre-mRNA, demonstrating that 13S splicing reduced 12S 5' splice site selection through a bona fide cis-competition. DEAE-cellulose chromatography of nuclear extract yielded two fractions with different splicing activities. The bound fraction contained all components required for efficient splicing of simple substrates but was unable to utilize alternative 5' splice sites. In contrast, the flow-through fraction, which by itself was inactive, contained an activity required for alternative splicing and was shown to stimulate 12S and 9S splicing, while reducing 13S splicing, when added to reactions carried out by the bound fraction. Furthermore, the activity, which we have called distal splicing factor (DSF), enhanced utilization of an upstream 5' splice site on a simian virus 40 early pre-mRNA, suggesting that the factor acts in a position-dependent, substrate-independent fashion. Several lines of evidence are presented suggesting that DSF is a non-small nuclear ribonucleoprotein protein. Finally, we describe a functional interaction between DSF and ASF, a protein that enhances use of downstream 5' splice sites. Images PMID:1658620

Muscle-Specific Mis-Splicing and Heart Disease Exemplified by RBM20.

PubMed

Rexiati, Maimaiti; Sun, Mingming; Guo, Wei

2018-01-05

Alternative splicing is an essential post-transcriptional process to generate multiple functional RNAs or proteins from a single transcript. Progress in RNA biology has led to a better understanding of muscle-specific RNA splicing in heart disease. The recent discovery of the muscle-specific splicing factor RNA-binding motif 20 (RBM20) not only provided great insights into the general alternative splicing mechanism but also demonstrated molecular mechanism of how this splicing factor is associated with dilated cardiomyopathy. Here, we review our current knowledge of muscle-specific splicing factors and heart disease, with an emphasis on RBM20 and its targets, RBM20-dependent alternative splicing mechanism, RBM20 disease origin in induced Pluripotent Stem Cells (iPSCs), and RBM20 mutations in dilated cardiomyopathy. In the end, we will discuss the multifunctional role of RBM20 and manipulation of RBM20 as a potential therapeutic target for heart disease.

A serine–arginine-rich (SR) splicing factor modulates alternative splicing of over a thousand genes in Toxoplasma gondii

PubMed Central

Yeoh, Lee M.; Goodman, Christopher D.; Hall, Nathan E.; van Dooren, Giel G.; McFadden, Geoffrey I.; Ralph, Stuart A.

2015-01-01

Single genes are often subject to alternative splicing, which generates alternative mature mRNAs. This phenomenon is widespread in animals, and observed in over 90% of human genes. Recent data suggest it may also be common in Apicomplexa. These parasites have small genomes, and economy of DNA is evolutionarily favoured in this phylum. We investigated the mechanism of alternative splicing in Toxoplasma gondii, and have identified and localized TgSR3, a homologue of ASF/SF2 (alternative-splicing factor/splicing factor 2, a serine-arginine–rich, or SR protein) to a subnuclear compartment. In addition, we conditionally overexpressed this protein, which was deleterious to growth. qRT-PCR was used to confirm perturbation of splicing in a known alternatively-spliced gene. We performed high-throughput RNA-seq to determine the extent of splicing modulated by this protein. Current RNA-seq algorithms are poorly suited to compact parasite genomes, and hence we complemented existing tools by writing a new program, GeneGuillotine, that addresses this deficiency by segregating overlapping reads into distinct genes. In order to identify the extent of alternative splicing, we released another program, JunctionJuror, that detects changes in intron junctions. Using this program, we identified about 2000 genes that were constitutively alternatively spliced in T. gondii. Overexpressing the splice regulator TgSR3 perturbed alternative splicing in over 1000 genes. PMID:25870410

Characterization of polysulfone and polysulfone/vanillin microcapsules by 1H NMR spectroscopy, solid-state 13C CP/MAS-NMR spectroscopy, and N2 adsorption-desorption analyses.

PubMed

Peña, Brisa; de Ménorval, Louis-Charles; Garcia-Valls, Ricard; Gumí, Tània

2011-11-01

Textile detergent and softener industries have incorporated perfume microencapsulation technology to improve their products. Perfume encapsulation allows perfume protection until use and provides a long-lasting fragrance release. But, certain industrial microcapsules show low encapsulation capacity and low material stability. Polysulfone capsules have been already proposed to solve these drawbacks. Among them, PSf/Vanillin capsules were considered as a desirable system. They present both good material stability and high encapsulation capacity. However, several factors such as the final location of the perfume in the polymeric matrix, the aggregation state that it has in the capsule and its interaction with the capsule components have not been studied yet. These factors can provide vast information about the capsule performance and its improvement. With the aim to characterize these parameters, the physical and chemical properties of PSf/Vanillin capsules have been investigated by nuclear magnetic resonance (NMR) spectroscopy, scanning electron microscopy (SEM), atomic force microscopy (AFM), and N(2) adsorption-desorption measurements. AFM micrograph and N(2) isotherms confirm that the presence of vanillin modify the physical structure of PSf/Vanillin microcapsules as it is trapped in the capsule porosity. NMR results show that vanillin is present in solid state in PSf/Vanillin microcapsules.

Optimal Matched Filter in the Low-number Count Poisson Noise Regime and Implications for X-Ray Source Detection

NASA Astrophysics Data System (ADS)

Ofek, Eran O.; Zackay, Barak

2018-04-01

Detection of templates (e.g., sources) embedded in low-number count Poisson noise is a common problem in astrophysics. Examples include source detection in X-ray images, γ-rays, UV, neutrinos, and search for clusters of galaxies and stellar streams. However, the solutions in the X-ray-related literature are sub-optimal in some cases by considerable factors. Using the lemma of Neyman–Pearson, we derive the optimal statistics for template detection in the presence of Poisson noise. We demonstrate that, for known template shape (e.g., point sources), this method provides higher completeness, for a fixed false-alarm probability value, compared with filtering the image with the point-spread function (PSF). In turn, we find that filtering by the PSF is better than filtering the image using the Mexican-hat wavelet (used by wavdetect). For some background levels, our method improves the sensitivity of source detection by more than a factor of two over the popular Mexican-hat wavelet filtering. This filtering technique can also be used for fast PSF photometry and flare detection; it is efficient and straightforward to implement. We provide an implementation in MATLAB. The development of a complete code that works on real data, including the complexities of background subtraction and PSF variations, is deferred for future publication.

Molecularly imprinted membranes (MIMs) for selective removal of polychlorinated biphenyls (PCBs) in environmental waters: fabrication and characterization.

PubMed

Mkhize, Dennis S; Nyoni, Hlengilizwe; Quinn, Laura P; Mamba, Bhekie B; Msagati, Titus A M

2017-04-01

Molecularly imprinted membranes (MIMs) with selective removal properties for polychlorinated biphenyls (PCBs) were prepared through the phase inversion technique. The MIMs were obtained from casting the viscous solutions of molecularly imprinted polymers (MIPs), polysulfone (PSf), and N-methyl-2-pyrrolidone (NMP) as the casting solvent. Different membranes were prepared at different concentration of MIPs and PSf. The resulting MIMs were characterized by atomic force microscope (AFM), scanning electron microscope (SEM), and Fourier transform infrared spectroscopy (FTIR). Moreover, the performance of the membranes was evaluated by determining and interpreting the rejection (%), flux (F), permeability coefficient (P), permselectivity factor ( α ' PCB/DDT or anthracene ), and enrichment factors of PCBs, dichlorodiphenyltrichloroethane (p,p'-DDT), and anthracene from model contaminated water using the dead-end filtration cell. Molecularly imprinted membrane prepared with 18 wt% PSf and 20 wt% MIP 4 exhibited a well-defined porous structure, which was accompanied by enhanced PCB enrichment. Furthermore, molecularly imprinted membrane showed good enrichment factors for PCBs even from spiked natural water samples of Hartbeespoort dam.

Serine/Arginine-Rich Splicing Factor 3 and Heterogeneous Nuclear Ribonucleoprotein A1 Regulate Alternative RNA Splicing and Gene Expression of Human Papillomavirus 18 through Two Functionally Distinguishable cis Elements.

PubMed

Ajiro, Masahiko; Tang, Shuang; Doorbar, John; Zheng, Zhi-Ming

2016-10-15

Human papillomavirus 18 (HPV18) is the second most common oncogenic HPV type associated with cervical, anogenital, and oropharyngeal cancers. Like other oncogenic HPVs, HPV18 encodes two major (one early and one late) polycistronic pre-mRNAs that are regulated by alternative RNA splicing to produce a repertoire of viral transcripts for the expression of individual viral genes. However, RNA cis-regulatory elements and trans-acting factors contributing to HPV18 alternative RNA splicing remain unknown. In this study, an exonic splicing enhancer (ESE) in the nucleotide (nt) 3520 to 3550 region in the HPV18 genome was identified and characterized for promotion of HPV18 929^3434 splicing and E1^E4 production through interaction with SRSF3, a host oncogenic splicing factor differentially expressed in epithelial cells and keratinocytes. Introduction of point mutations in the SRSF3-binding site or knockdown of SRSF3 expression in cells reduces 929^3434 splicing and E1^E4 production but activates other, minor 929^3465 and 929^3506 splicing. Knockdown of SRSF3 expression also enhances the expression of E2 and L1 mRNAs. An exonic splicing silencer (ESS) in the HPV18 nt 612 to 639 region was identified as being inhibitory to the 233^416 splicing of HPV18 E6E7 pre-mRNAs via binding to hnRNP A1, a well-characterized, abundantly and ubiquitously expressed RNA-binding protein. Introduction of point mutations into the hnRNP A1-binding site or knockdown of hnRNP A1 expression promoted 233^416 splicing and reduced E6 expression. These data provide the first evidence that the alternative RNA splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host trans-acting splicing factors. Expression of HPV18 genes is regulated by alternative RNA splicing of viral polycistronic pre-mRNAs to produce a repertoire of viral early and late transcripts. RNA cis elements and trans-acting factors contributing to HPV18 alternative RNA splicing have been discovered in this study for the first time. The identified ESS at the E7 open reading frame (ORF) prevents HPV18 233^416 splicing in the E6 ORF through interaction with a host splicing factor, hnRNP A1, and regulates E6 and E7 expression of the early E6E7 polycistronic pre-mRNA. The identified ESE at the E1^E4 ORF promotes HPV18 929^3434 splicing of both viral early and late pre-mRNAs and E1^E4 production through interaction with SRSF3. This study provides important observations on how alternative RNA splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host splicing factors and offers potential therapeutic targets to overcome HPV-related cancer. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Serine/Arginine-Rich Splicing Factor 3 and Heterogeneous Nuclear Ribonucleoprotein A1 Regulate Alternative RNA Splicing and Gene Expression of Human Papillomavirus 18 through Two Functionally Distinguishable cis Elements

PubMed Central

Ajiro, Masahiko; Tang, Shuang; Doorbar, John

2016-01-01

ABSTRACT Human papillomavirus 18 (HPV18) is the second most common oncogenic HPV type associated with cervical, anogenital, and oropharyngeal cancers. Like other oncogenic HPVs, HPV18 encodes two major (one early and one late) polycistronic pre-mRNAs that are regulated by alternative RNA splicing to produce a repertoire of viral transcripts for the expression of individual viral genes. However, RNA cis-regulatory elements and trans-acting factors contributing to HPV18 alternative RNA splicing remain unknown. In this study, an exonic splicing enhancer (ESE) in the nucleotide (nt) 3520 to 3550 region in the HPV18 genome was identified and characterized for promotion of HPV18 929^3434 splicing and E1^E4 production through interaction with SRSF3, a host oncogenic splicing factor differentially expressed in epithelial cells and keratinocytes. Introduction of point mutations in the SRSF3-binding site or knockdown of SRSF3 expression in cells reduces 929^3434 splicing and E1^E4 production but activates other, minor 929^3465 and 929^3506 splicing. Knockdown of SRSF3 expression also enhances the expression of E2 and L1 mRNAs. An exonic splicing silencer (ESS) in the HPV18 nt 612 to 639 region was identified as being inhibitory to the 233^416 splicing of HPV18 E6E7 pre-mRNAs via binding to hnRNP A1, a well-characterized, abundantly and ubiquitously expressed RNA-binding protein. Introduction of point mutations into the hnRNP A1-binding site or knockdown of hnRNP A1 expression promoted 233^416 splicing and reduced E6 expression. These data provide the first evidence that the alternative RNA splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host trans-acting splicing factors. IMPORTANCE Expression of HPV18 genes is regulated by alternative RNA splicing of viral polycistronic pre-mRNAs to produce a repertoire of viral early and late transcripts. RNA cis elements and trans-acting factors contributing to HPV18 alternative RNA splicing have been discovered in this study for the first time. The identified ESS at the E7 open reading frame (ORF) prevents HPV18 233^416 splicing in the E6 ORF through interaction with a host splicing factor, hnRNP A1, and regulates E6 and E7 expression of the early E6E7 polycistronic pre-mRNA. The identified ESE at the E1^E4 ORF promotes HPV18 929^3434 splicing of both viral early and late pre-mRNAs and E1^E4 production through interaction with SRSF3. This study provides important observations on how alternative RNA splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host splicing factors and offers potential therapeutic targets to overcome HPV-related cancer. PMID:27489271

Regulation of Alternative Splicing in Vivo by Overexpression of Antagonistic Splicing Factors

NASA Astrophysics Data System (ADS)

Caceres, Javier F.; Stamm, Stefan; Helfman, David M.; Krainer, Adrian R.

1994-09-01

The opposing effects of SF2/ASF and heterogeneous nuclear ribonucleoprotein (hnRNP) A1 influence alternative splicing in vitro. SF2/ASF or hnRNP A1 complementary DNAs were transiently overexpressed in HeLa cells, and the effect on alternative splicing of several cotransfected reporter genes was measured. Increased expression of SF2/ASF activated proximal 5' splice sites, promoted inclusion of a neuron-specific exon, and prevented abnormal exon skipping. Increased expression of hnRNP A1 activated distal 5' splice sites. Therefore, variations in the intracellular levels of antagonistic splicing factors influence different modes of alternative splicing in vivo and may be a natural mechanism for tissue-specific or developmental regulation of gene expression.

SIMULATED HUMAN ERROR PROBABILITY AND ITS APPLICATION TO DYNAMIC HUMAN FAILURE EVENTS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Herberger, Sarah M.; Boring, Ronald L.

Abstract Objectives: Human reliability analysis (HRA) methods typically analyze human failure events (HFEs) at the overall task level. For dynamic HRA, it is important to model human activities at the subtask level. There exists a disconnect between dynamic subtask level and static task level that presents issues when modeling dynamic scenarios. For example, the SPAR-H method is typically used to calculate the human error probability (HEP) at the task level. As demonstrated in this paper, quantification in SPAR-H does not translate to the subtask level. Methods: Two different discrete distributions were generated for each SPAR-H Performance Shaping Factor (PSF) tomore » define the frequency of PSF levels. The first distribution was a uniform, or uninformed distribution that assumed the frequency of each PSF level was equally likely. The second non-continuous distribution took the frequency of PSF level as identified from an assessment of the HERA database. These two different approaches were created to identify the resulting distribution of the HEP. The resulting HEP that appears closer to the known distribution, a log-normal centered on 1E-3, is the more desirable. Each approach then has median, average and maximum HFE calculations applied. To calculate these three values, three events, A, B and C are generated from the PSF level frequencies comprised of subtasks. The median HFE selects the median PSF level from each PSF and calculates HEP. The average HFE takes the mean PSF level, and the maximum takes the maximum PSF level. The same data set of subtask HEPs yields starkly different HEPs when aggregated to the HFE level in SPAR-H. Results: Assuming that each PSF level in each HFE is equally likely creates an unrealistic distribution of the HEP that is centered at 1. Next the observed frequency of PSF levels was applied with the resulting HEP behaving log-normally with a majority of the values under 2.5% HEP. The median, average and maximum HFE calculations did yield different answers for the HFE. The HFE maximum grossly over estimates the HFE, while the HFE distribution occurs less than HFE median, and greater than HFE average. Conclusions: Dynamic task modeling can be perused through the framework of SPAR-H. Identification of distributions associated with each PSF needs to be defined, and may change depending upon the scenario. However it is very unlikely that each PSF level is equally likely as the resulting HEP distribution is strongly centered at 100%, which is unrealistic. Other distributions may need to be identified for PSFs, to facilitate the transition to dynamic task modeling. Additionally discrete distributions need to be exchanged for continuous so that simulations for the HFE can further advance. This paper provides a method to explore dynamic subtask to task translation and provides examples of the process using the SPAR-H method.« less

Connecting the dots: chromatin and alternative splicing in EMT.

PubMed

Warns, Jessica A; Davie, James R; Dhasarathy, Archana

2016-02-01

Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases, and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process.

RNA Splicing: Regulation and Dysregulation in the Heart.

PubMed

van den Hoogenhof, Maarten M G; Pinto, Yigal M; Creemers, Esther E

2016-02-05

RNA splicing represents a post-transcriptional mechanism to generate multiple functional RNAs or proteins from a single transcript. The evolution of RNA splicing is a prime example of the Darwinian function follows form concept. A mutation that leads to a new mRNA (form) that encodes for a new functional protein (function) is likely to be retained, and this way, the genome has gradually evolved to encode for genes with multiple isoforms, thereby creating an enormously diverse transcriptome. Advances in technologies to characterize RNA populations have led to a better understanding of RNA processing in health and disease. In the heart, alternative splicing is increasingly being recognized as an important layer of post-transcriptional gene regulation. Moreover, the recent identification of several cardiac splice factors, such as RNA-binding motif protein 20 and SF3B1, not only provided important insight into the mechanisms underlying alternative splicing but also revealed how these splicing factors impact functional properties of the heart. Here, we review our current knowledge of alternative splicing in the heart, with a particular focus on the major and minor spliceosome, the factors controlling RNA splicing, and the role of alternative splicing in cardiac development and disease. © 2016 American Heart Association, Inc.

The influence of Argonaute proteins on alternative RNA splicing.

PubMed

Batsché, Eric; Ameyar-Zazoua, Maya

2015-01-01

Alternative splicing of precursor RNAs is an important process in multicellular species because it impacts several aspects of gene expression: from the increase of protein repertoire to the level of expression. A large body of evidences demonstrates that factors regulating chromatin and transcription impact the outcomes of alternative splicing. Argonaute (AGO) proteins were known to play key roles in the regulation of gene expression at the post-transcriptional level. More recently, their role in the nucleus of human somatic cells has emerged. Here, we will discuss some of the nuclear functions of AGO, with special emphasis on alternative splicing. The AGO-mediated modulation of alternative splicing is based on several properties of these proteins: their binding to transcripts on chromatin and their interactions with many proteins, especially histone tail-modifying enzymes, HP1γ and splicing factors. AGO proteins may favor a decrease in the RNA-polymerase II kinetics at actively transcribed genes leading to the modulation of alternative splicing decisions. They could also influence alternative splicing through their interaction with core components of the splicing machinery and several splicing factors. We will discuss the modes of AGO recruitment on chromatin at active genes. We suggest that long intragenic antisense transcripts (lincRNA) might be an important feature of genes containing splicing events regulated by AGO. © 2014 John Wiley & Sons, Ltd.

Histone and RNA-binding protein interaction creates crosstalk network for regulation of alternative splicing.

PubMed

Kim, Yong-Eun; Park, Chungoo; Kim, Kyoon Eon; Kim, Kee K

2018-04-30

Alternative splicing is an essential process in eukaryotes, as it increases the complexity of gene expression by generating multiple proteins from a single pre-mRNA. However, information on the regulatory mechanisms for alternative splicing is lacking, because splicing occurs over a short period via the transient interactions of proteins within functional complexes of the spliceosome. Here, we investigated in detail the molecular mechanisms connecting alternative splicing with epigenetic mechanisms. We identified interactions between histone proteins and splicing factors such as Rbfox2, Rbfox3, and splicing factor proline and glutamine rich protein (SFPQ) by in vivo crosslinking and immunoprecipitation. Furthermore, we confirmed that splicing factors were bound to specific modified residues of histone proteins. Additionally, changes in histone methylation due to histone methyltransferase inhibitor treatment notably affected alternative splicing in selected genes. Therefore, we suggested that there may be crosstalk mechanisms connecting histone modifications and RNA-binding proteins that increase the local concentration of RNA-binding proteins in alternative exon loci of nucleosomes by binding specific modified histone proteins, leading to alternative splicing. This crosstalk mechanism may play a major role in epigenetic processes such as histone modification and the regulation of alternative splicing. Copyright © 2018 Elsevier Inc. All rights reserved.

In vivo effects on intron retention and exon skipping by the U2AF large subunit and SF1/BBP in the nematode Caenorhabditis elegans

PubMed Central

Ma, Long; Tan, Zhiping; Teng, Yanling; Hoersch, Sebastian; Horvitz, H. Robert

2011-01-01

The in vivo analysis of the roles of splicing factors in regulating alternative splicing in animals remains a challenge. Using a microarray-based screen, we identified a Caenorhabditis elegans gene, tos-1, that exhibited three of the four major types of alternative splicing: intron retention, exon skipping, and, in the presence of U2AF large subunit mutations, the use of alternative 3′ splice sites. Mutations in the splicing factors U2AF large subunit and SF1/BBP altered the splicing of tos-1. 3′ splice sites of the retained intron or before the skipped exon regulate the splicing pattern of tos-1. Our study provides in vivo evidence that intron retention and exon skipping can be regulated largely by the identities of 3′ splice sites. PMID:22033331

Disturbed expression of type 1 iodothyronine deiodinase splice variants in human renal cancer.

PubMed

Piekielko-Witkowska, Agnieszka; Master, Adam; Wojcicka, Anna; Boguslawska, Joanna; Brozda, Izabela; Tanski, Zbigniew; Nauman, Alicja

2009-10-01

Alternative splicing, one of the sources of protein diversity, is often disturbed in cancer. Type 1 iodothyronine deiodinase (DIO1) catalyzes deiodination of thyroxine generating triiodothyronine, an important regulator of cell proliferation and differentiation. The expression of DIO1 is disturbed in different types of cancer. The aim of the study was to analyze the alternative splicing of DIO1 and its possible disturbance in renal cancer. Using real-time PCR, we analyzed 19 tissue samples (T) of renal cancer and 19 matched control samples (C) of the opposite pole of the kidney, not infiltrated by tumor, and 6 control samples (N) (nonneoplastic kidney abnormalities). Cloning of DIO1 mRNA isoforms revealed 11 different transcripts, among them 7 new splice variants, not previously reported. The expression of all variants of DIO1 was dramatically (>90%) and significantly (p < or = 0.0003) lowered in samples T compared to control samples C. The ratio of mRNA isoforms encoding DIO1 protein variants possessing or lacking the active center was lowered in samples T compared with control samples C, suggesting disturbed alternative splicing of DIO1. The expression of mRNA of splicing factors SF2/ASF (splicing factor-2/alternative-splicing factor) and hnRNPA1 (heterogeneous ribonucleoprotein A1), regulating 5'-splice site selection, was significantly but not proportionally lowered in samples T compared to samples C. The mRNA ratio of splicing factors SF2/ASF and hnRNPA1 correlated with the ratio of mRNA isoforms encoding DIO1 protein variants possessing or lacking the active center in controls C but not in samples T. Our results show that the expression and alternative splicing of DIO1 mRNA is disturbed in renal cancer, possibly due to changes in expression of splicing factors SF2/ASF and hnRNPA1.

Interplay between DMD Point Mutations and Splicing Signals in Dystrophinopathy Phenotypes

PubMed Central

Juan-Mateu, Jonàs; González-Quereda, Lidia; Rodríguez, Maria José; Verdura, Edgard; Lázaro, Kira; Jou, Cristina; Nascimento, Andrés; Jiménez-Mallebrera, Cecilia; Colomer, Jaume; Monges, Soledad; Lubieniecki, Fabiana; Foncuberta, Maria Eugenia; Pascual-Pascual, Samuel Ignacio; Molano, Jesús; Baiget, Montserrat; Gallano, Pia

2013-01-01

DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements. PMID:23536893

Therapeutic targeting of RNA splicing in myelodysplasia.

PubMed

Kim, Young Joon; Abdel-Wahab, Omar

2017-07-01

Genomic analysis of patients with myelodysplastic syndromes (MDS) has identified that mutations within genes encoding RNA splicing factors represent the most common class of genetic alterations in MDS. These mutations primarily affect SF3B1, SRSF2, U2AF1, and ZRSR2. Current data suggest that these mutations perturb RNA splicing catalysis in a manner distinct from loss of function but how exactly the global changes in RNA splicing imparted by these mutations result in MDS is not well delineated. At the same time, cells bearing mutations in RNA splicing factors are exquisitely dependent on the presence of the remaining wild-type (WT) allele to maintain residual normal splicing for cell survival. The high frequency of these mutations in MDS, combined with their mutual exclusivity and noteworthy dependence on the WT allele, make targeting RNA splicing attractive in MDS. To this end, two promising therapeutic approaches targeting RNA splicing are being tested clinically currently. These include molecules targeting core RNA splicing catalysis by interfering with the ability of the SF3b complex to interact with RNA, as well as molecules degrading the auxiliary RNA splicing factor RBM39. The preclinical and clinical evaluation of these compounds are discussed here in addition to their potential as therapies for spliceosomal mutant MDS. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of cis-Acting Elements and Splicing Factors Involved in the Regulation of BIM Pre-mRNA Splicing

PubMed Central

Juan, Wen Chun; Roca, Xavier; Ong, S. Tiong

2014-01-01

Aberrant changes in the expression of the pro-apoptotic protein, BCL-2-like 11 (BIM), can result in either impaired or excessive apoptosis, which can contribute to tumorigenesis and degenerative disorders, respectively. Altering BIM pre-mRNA splicing is an attractive approach to modulate apoptosis because BIM activity is partly determined by the alternative splicing of exons 3 or 4, whereby exon 3-containing transcripts are not apoptotic. Here we identified several cis-acting elements and splicing factors involved in BIM alternative splicing, as a step to better understand the regulation of BIM expression. We analyzed a recently discovered 2,903-bp deletion polymorphism within BIM intron 2 that biased splicing towards exon 3, and which also impaired BIM-dependent apoptosis. We found that this region harbors multiple redundant cis-acting elements that repress exon 3 inclusion. Furthermore, we have isolated a 23-nt intronic splicing silencer at the 3′ end of the deletion that is important for excluding exon 3. We also show that PTBP1 and hnRNP C repress exon 3 inclusion, and that downregulation of PTBP1 inhibited BIM-mediated apoptosis. Collectively, these findings start building our understanding of the cis-acting elements and splicing factors that regulate BIM alternative splicing, and also suggest potential approaches to alter BIM splicing for therapeutic purposes. PMID:24743263

Identification of cis-acting elements and splicing factors involved in the regulation of BIM Pre-mRNA splicing.

PubMed

Juan, Wen Chun; Roca, Xavier; Ong, S Tiong

2014-01-01

Aberrant changes in the expression of the pro-apoptotic protein, BCL-2-like 11 (BIM), can result in either impaired or excessive apoptosis, which can contribute to tumorigenesis and degenerative disorders, respectively. Altering BIM pre-mRNA splicing is an attractive approach to modulate apoptosis because BIM activity is partly determined by the alternative splicing of exons 3 or 4, whereby exon 3-containing transcripts are not apoptotic. Here we identified several cis-acting elements and splicing factors involved in BIM alternative splicing, as a step to better understand the regulation of BIM expression. We analyzed a recently discovered 2,903-bp deletion polymorphism within BIM intron 2 that biased splicing towards exon 3, and which also impaired BIM-dependent apoptosis. We found that this region harbors multiple redundant cis-acting elements that repress exon 3 inclusion. Furthermore, we have isolated a 23-nt intronic splicing silencer at the 3' end of the deletion that is important for excluding exon 3. We also show that PTBP1 and hnRNP C repress exon 3 inclusion, and that downregulation of PTBP1 inhibited BIM-mediated apoptosis. Collectively, these findings start building our understanding of the cis-acting elements and splicing factors that regulate BIM alternative splicing, and also suggest potential approaches to alter BIM splicing for therapeutic purposes.

Generalized PSF modeling for optimized quantitation in PET imaging.

PubMed

Ashrafinia, Saeed; Mohy-Ud-Din, Hassan; Karakatsanis, Nicolas A; Jha, Abhinav K; Casey, Michael E; Kadrmas, Dan J; Rahmim, Arman

2017-06-21

Point-spread function (PSF) modeling offers the ability to account for resolution degrading phenomena within the PET image generation framework. PSF modeling improves resolution and enhances contrast, but at the same time significantly alters image noise properties and induces edge overshoot effect. Thus, studying the effect of PSF modeling on quantitation task performance can be very important. Frameworks explored in the past involved a dichotomy of PSF versus no-PSF modeling. By contrast, the present work focuses on quantitative performance evaluation of standard uptake value (SUV) PET images, while incorporating a wide spectrum of PSF models, including those that under- and over-estimate the true PSF, for the potential of enhanced quantitation of SUVs. The developed framework first analytically models the true PSF, considering a range of resolution degradation phenomena (including photon non-collinearity, inter-crystal penetration and scattering) as present in data acquisitions with modern commercial PET systems. In the context of oncologic liver FDG PET imaging, we generated 200 noisy datasets per image-set (with clinically realistic noise levels) using an XCAT anthropomorphic phantom with liver tumours of varying sizes. These were subsequently reconstructed using the OS-EM algorithm with varying PSF modelled kernels. We focused on quantitation of both SUV mean and SUV max , including assessment of contrast recovery coefficients, as well as noise-bias characteristics (including both image roughness and coefficient of-variability), for different tumours/iterations/PSF kernels. It was observed that overestimated PSF yielded more accurate contrast recovery for a range of tumours, and typically improved quantitative performance. For a clinically reasonable number of iterations, edge enhancement due to PSF modeling (especially due to over-estimated PSF) was in fact seen to lower SUV mean bias in small tumours. Overall, the results indicate that exactly matched PSF modeling does not offer optimized PET quantitation, and that PSF overestimation may provide enhanced SUV quantitation. Furthermore, generalized PSF modeling may provide a valuable approach for quantitative tasks such as treatment-response assessment and prognostication.

RNA splicing factors as oncoproteins and tumor suppressors

PubMed Central

Dvinge, Heidi; Kim, Eunhee; Abdel-Wahab, Omar; Bradley, Robert K.

2016-01-01

Preface The recent genomic characterization of cancers has revealed recurrent somatic point mutations and copy number changes affecting genes encoding RNA splicing factors. Initial studies of these ‘spliceosomal mutations’ suggest that the proteins bearing these mutations exhibit altered splice site and/or exon recognition preferences relative to their wild-type counterparts, resulting in cancer-specific mis-splicing. Such changes in the splicing machinery may create novel vulnerabilities in cancer cells that can be therapeutically exploited using compounds that can influence the splicing process. Further studies to dissect the biochemical, genomic, and biological effects of spliceosomal mutations are critical for the development of cancer therapies targeted to these mutations. PMID:27282250

Connecting the dots: chromatin and alternative splicing in EMT

PubMed Central

Warns, Jessica A.; Davie, James R.; Dhasarathy, Archana

2015-01-01

Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process. PMID:26291837

Functions for fission yeast splicing factors SpSlu7 and SpPrp18 in alternative splice-site choice and stress-specific regulated splicing.

PubMed

Melangath, Geetha; Sen, Titash; Kumar, Rakesh; Bawa, Pushpinder; Srinivasan, Subha; Vijayraghavan, Usha

2017-01-01

Budding yeast spliceosomal factors ScSlu7 and ScPrp18 interact and mediate intron 3'ss choice during second step pre-mRNA splicing. The fission yeast genome with abundant multi-intronic transcripts, degenerate splice signals and SR proteins is an apt unicellular fungal model to deduce roles for core spliceosomal factors in alternative splice-site choice, intron retention and to study the cellular implications of regulated splicing. From our custom microarray data we deduce a stringent reproducible subset of S. pombe alternative events. We examined the role of factors SpSlu7 or SpPrp18 for these splice events and investigated the relationship to growth phase and stress. Wild-type log and stationary phase cells showed ats1+ exon 3 skipped and intron 3 retained transcripts. Interestingly the non-consensus 5'ss in ats1+ intron 3 caused SpSlu7 and SpPrp18 dependent intron retention. We validated the use of an alternative 5'ss in dtd1+ intron 1 and of an upstream alternative 3'ss in DUF3074 intron 1. The dtd1+ intron 1 non-canonical 5'ss yielded an alternative mRNA whose levels increased in stationary phase. Utilization of dtd1+ intron 1 sub-optimal 5' ss required functional SpPrp18 and SpSlu7 while compromise in SpSlu7 function alone hampered the selection of the DUF3074 intron 1 non canonical 3'ss. We analysed the relative abundance of these splice isoforms during mild thermal, oxidative and heavy metal stress and found stress-specific splice patterns for ats1+ and DUF3074 intron 1 some of which were SpSlu7 and SpPrp18 dependent. By studying ats1+ splice isoforms during compromised transcription elongation rates in wild-type, spslu7-2 and spprp18-5 mutant cells we found dynamic and intron context-specific effects in splice-site choice. Our work thus shows the combinatorial effects of splice site strength, core splicing factor functions and transcription elongation kinetics to dictate alternative splice patterns which in turn serve as an additional recourse of gene regulation in fission yeast.

Functions for fission yeast splicing factors SpSlu7 and SpPrp18 in alternative splice-site choice and stress-specific regulated splicing

PubMed Central

Kumar, Rakesh; Bawa, Pushpinder; Srinivasan, Subha

2017-01-01

Budding yeast spliceosomal factors ScSlu7 and ScPrp18 interact and mediate intron 3’ss choice during second step pre-mRNA splicing. The fission yeast genome with abundant multi-intronic transcripts, degenerate splice signals and SR proteins is an apt unicellular fungal model to deduce roles for core spliceosomal factors in alternative splice-site choice, intron retention and to study the cellular implications of regulated splicing. From our custom microarray data we deduce a stringent reproducible subset of S. pombe alternative events. We examined the role of factors SpSlu7 or SpPrp18 for these splice events and investigated the relationship to growth phase and stress. Wild-type log and stationary phase cells showed ats1+ exon 3 skipped and intron 3 retained transcripts. Interestingly the non-consensus 5’ss in ats1+ intron 3 caused SpSlu7 and SpPrp18 dependent intron retention. We validated the use of an alternative 5’ss in dtd1+ intron 1 and of an upstream alternative 3’ss in DUF3074 intron 1. The dtd1+ intron 1 non-canonical 5’ss yielded an alternative mRNA whose levels increased in stationary phase. Utilization of dtd1+ intron 1 sub-optimal 5’ ss required functional SpPrp18 and SpSlu7 while compromise in SpSlu7 function alone hampered the selection of the DUF3074 intron 1 non canonical 3’ss. We analysed the relative abundance of these splice isoforms during mild thermal, oxidative and heavy metal stress and found stress-specific splice patterns for ats1+ and DUF3074 intron 1 some of which were SpSlu7 and SpPrp18 dependent. By studying ats1+ splice isoforms during compromised transcription elongation rates in wild-type, spslu7-2 and spprp18-5 mutant cells we found dynamic and intron context-specific effects in splice-site choice. Our work thus shows the combinatorial effects of splice site strength, core splicing factor functions and transcription elongation kinetics to dictate alternative splice patterns which in turn serve as an additional recourse of gene regulation in fission yeast. PMID:29236736

Analysis of Subcellular RNA Fractions Revealed a Transcription-Independent Effect of Tumor Necrosis Factor Alpha on Splicing, Mediated by Spt5.

PubMed

Diamant, Gil; Eisenbaum, Tal; Leshkowitz, Dena; Dikstein, Rivka

2016-05-01

The proinflammatory cytokine tumor necrosis factor alpha (TNF-α) modulates the expression of many genes, primarily through activation of NF-κB. Here, we examined the global effects of the elongation factor Spt5 on nascent and mature mRNAs of TNF-α-induced cells using chromatin and cytosolic subcellular fractions. We identified several classes of TNF-α-induced genes controlled at the level of transcription, splicing, and chromatin retention. Spt5 was found to facilitate splicing and chromatin release in genes displaying high induction rates. Further analysis revealed striking effects of TNF-α on the splicing of 25% of expressed genes; the vast majority were not transcriptionally induced. Splicing enhancement of noninduced genes by TNF-α was transient and independent of NF-κB. Investigating the underlying basis, we found that Spt5 is required for the splicing facilitation of the noninduced genes. In line with this, Spt5 interacts with Sm core protein splicing factors. Furthermore, following TNF-α treatment, levels of RNA polymerase II (Pol II) but not Spt5 are reduced from the splicing-induced genes, suggesting that these genes become enriched with a Pol II-Spt5 form. Our findings revealed the Pol II-Spt5 complex as a highly competent coordinator of cotranscriptional splicing. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

A deconvolution extraction method for 2D multi-object fibre spectroscopy based on the regularized least-squares QR-factorization algorithm

NASA Astrophysics Data System (ADS)

Yu, Jian; Yin, Qian; Guo, Ping; Luo, A.-li

2014-09-01

This paper presents an efficient method for the extraction of astronomical spectra from two-dimensional (2D) multifibre spectrographs based on the regularized least-squares QR-factorization (LSQR) algorithm. We address two issues: we propose a modified Gaussian point spread function (PSF) for modelling the 2D PSF from multi-emission-line gas-discharge lamp images (arc images), and we develop an efficient deconvolution method to extract spectra in real circumstances. The proposed modified 2D Gaussian PSF model can fit various types of 2D PSFs, including different radial distortion angles and ellipticities. We adopt the regularized LSQR algorithm to solve the sparse linear equations constructed from the sparse convolution matrix, which we designate the deconvolution spectrum extraction method. Furthermore, we implement a parallelized LSQR algorithm based on graphics processing unit programming in the Compute Unified Device Architecture to accelerate the computational processing. Experimental results illustrate that the proposed extraction method can greatly reduce the computational cost and memory use of the deconvolution method and, consequently, increase its efficiency and practicability. In addition, the proposed extraction method has a stronger noise tolerance than other methods, such as the boxcar (aperture) extraction and profile extraction methods. Finally, we present an analysis of the sensitivity of the extraction results to the radius and full width at half-maximum of the 2D PSF.

Coupling between alternative polyadenylation and alternative splicing is limited to terminal introns.

PubMed

Movassat, Maliheh; Crabb, Tara L; Busch, Anke; Yao, Chengguo; Reynolds, Derrick J; Shi, Yongsheng; Hertel, Klemens J

2016-07-02

Alternative polyadenylation has been implicated as an important regulator of gene expression. In some cases, alternative polyadenylation is known to couple with alternative splicing to influence last intron removal. However, it is unknown whether alternative polyadenylation events influence alternative splicing decisions at upstream exons. Knockdown of the polyadenylation factors CFIm25 or CstF64 in HeLa cells was used as an approach in identifying alternative polyadenylation and alternative splicing events on a genome-wide scale. Although hundreds of alternative splicing events were found to be differentially spliced in the knockdown of CstF64, genes associated with alternative polyadenylation did not exhibit an increased incidence of alternative splicing. These results demonstrate that the coupling between alternative polyadenylation and alternative splicing is usually limited to defining the last exon. The striking influence of CstF64 knockdown on alternative splicing can be explained through its effects on UTR selection of known splicing regulators such as hnRNP A2/B1, thereby indirectly influencing splice site selection. We conclude that changes in the expression of the polyadenylation factor CstF64 influences alternative splicing through indirect effects.

The transcription factor FBI-1 inhibits SAM68-mediated BCL-X alternative splicing and apoptosis.

PubMed

Bielli, Pamela; Busà, Roberta; Di Stasi, Savino M; Munoz, Manuel J; Botti, Flavia; Kornblihtt, Alberto R; Sette, Claudio

2014-04-01

Alternative splicing (AS) is tightly coupled to transcription for the majority of human genes. However, how these two processes are linked is not well understood. Here, we unveil a direct role for the transcription factor FBI-1 in the regulation of AS. FBI-1 interacts with the splicing factor SAM68 and reduces its binding to BCL-X mRNA. This, in turn, results in the selection of the proximal 5' splice site in BCL-X exon 2, thereby favoring the anti-apoptotic BCL-XL variant and counteracting SAM68-mediated apoptosis. Conversely, depletion of FBI-1, or expression of a SAM68 mutant lacking the FBI-1 binding region, restores the ability of SAM68 to induce BCL-XS splicing and apoptosis. FBI-1's role in splicing requires the activity of histone deacetylases, whose pharmacological inhibition recapitulates the effects of FBI-1 knockdown. Our study reveals an unexpected function for FBI-1 in splicing modulation with a direct impact on cell survival.

The transcription factor FBI-1 inhibits SAM68-mediated BCL-X alternative splicing and apoptosis

PubMed Central

Bielli, Pamela; Busà, Roberta; Di Stasi, Savino M; Munoz, Manuel J; Botti, Flavia; Kornblihtt, Alberto R; Sette, Claudio

2014-01-01

Alternative splicing (AS) is tightly coupled to transcription for the majority of human genes. However, how these two processes are linked is not well understood. Here, we unveil a direct role for the transcription factor FBI-1 in the regulation of AS. FBI-1 interacts with the splicing factor SAM68 and reduces its binding to BCL-X mRNA. This, in turn, results in the selection of the proximal 5′ splice site in BCL-X exon 2, thereby favoring the anti-apoptotic BCL-XL variant and counteracting SAM68-mediated apoptosis. Conversely, depletion of FBI-1, or expression of a SAM68 mutant lacking the FBI-1 binding region, restores the ability of SAM68 to induce BCL-XS splicing and apoptosis. FBI-1's role in splicing requires the activity of histone deacetylases, whose pharmacological inhibition recapitulates the effects of FBI-1 knockdown. Our study reveals an unexpected function for FBI-1 in splicing modulation with a direct impact on cell survival. PMID:24514149

Incorporating the soil environment and microbial community into plant competition theory

PubMed Central

Ke, Po-Ju; Miki, Takeshi

2015-01-01

Plants affect microbial communities and abiotic properties of nearby soils, which in turn influence plant growth and interspecific interaction, forming a plant-soil feedback (PSF). PSF is a key determinant influencing plant population dynamics, community structure, and ecosystem functions. Despite accumulating evidence for the importance of PSF and development of specific PSF models, different models are not yet fully integrated. Here, we review the theoretical progress in understanding PSF. When first proposed, PSF was integrated with various mathematical frameworks to discuss its influence on plant competition. Recent theoretical models have advanced PSF research at different levels of ecological organizations by considering multiple species, applying spatially explicit simulations to examine how local-scale predictions apply to larger scales, and assessing the effect of PSF on plant temporal dynamics over the course of succession. We then review two foundational models for microbial- and litter-mediated PSF. We present a theoretical framework to illustrate that although the two models are typically presented separately, their behavior can be understood together by invasibility analysis. We conclude with suggestions for future directions in PSF theoretical studies, which include specifically addressing microbial diversity to integrate litter- and microbial-mediated PSF, and apply PSF to general coexistence theory through a trait-based approach. PMID:26500621

Systematic profiling of alternative splicing signature reveals prognostic predictor for ovarian cancer.

PubMed

Zhu, Junyong; Chen, Zuhua; Yong, Lei

2018-02-01

The majority of genes are alternatively spliced and growing evidence suggests that alternative splicing is modified in cancer and is associated with cancer progression. Systematic analysis of alternative splicing signature in ovarian cancer is lacking and greatly needed. We profiled genome-wide alternative splicing events in 408 ovarian serous cystadenocarcinoma (OV) patients in TCGA. Seven types of alternative splicing events were curated and prognostic analyses were performed with predictive models and splicing network built for OV patients. Among 48,049 mRNA splicing events in 10,582 genes, we detected 2,611 alternative splicing events in 2,036 genes which were significant associated with overall survival of OV patients. Exon skip events were the most powerful prognostic factors among the seven types. The area under the curve of the receiver-operator characteristic curve for prognostic predictor, which was built with top significant alternative splicing events, was 0.937 at 2,000 days of overall survival, indicating powerful efficiency in distinguishing patient outcome. Interestingly, splicing correlation network suggested obvious trends in the role of splicing factors in OV. In summary, we built powerful prognostic predictors for OV patients and uncovered interesting splicing networks which could be underlying mechanisms. Copyright © 2017 Elsevier Inc. All rights reserved.

A 5′ Splice Site-Proximal Enhancer Binds SF1 and Activates Exon Bridging of a Microexon

PubMed Central

Carlo, Troy; Sierra, Rebecca; Berget, Susan M.

2000-01-01

Internal exon size in vertebrates occurs over a narrow size range. Experimentally, exons shorter than 50 nucleotides are poorly included in mRNA unless accompanied by strengthened splice sites or accessory sequences that act as splicing enhancers, suggesting steric interference between snRNPs and other splicing factors binding simultaneously to the 3′ and 5′ splice sites of microexons. Despite these problems, very small naturally occurring exons exist. Here we studied the factors and mechanism involved in recognizing a constitutively included six-nucleotide exon from the cardiac troponin T gene. Inclusion of this exon is dependent on an enhancer located downstream of the 5′ splice site. This enhancer contains six copies of the simple sequence GGGGCUG. The enhancer activates heterologous microexons and will work when located either upstream or downstream of the target exon, suggesting an ability to bind factors that bridge splicing units. A single copy of this sequence is sufficient for in vivo exon inclusion and is the binding site for the known bridging mammalian splicing factor 1 (SF1). The enhancer and its bound SF1 act to increase recognition of the upstream exon during exon definition, such that competition of in vitro reactions with RNAs containing the GGGGCUG repeated sequence depress splicing of the upstream intron, assembly of the spliceosome on the 3′ splice site of the exon, and cross-linking of SF1. These results suggest a model in which SF1 bridges the small exon during initial assembly, thereby effectively extending the domain of the exon. PMID:10805741

SU-F-T-367: Using PRIMO, a PENELOPE-Based Software, to Improve the Small Field Dosimetry of Linear Accelerators

DOE Office of Scientific and Technical Information (OSTI.GOV)

Benmakhlouf, H; Andreo, P; Brualla, L

2016-06-15

Purpose: To calculate output correction factors for Varian Clinac 2100iX beams for seven small field detectors and use the values to determine the small field output factors for the linacs at Karolinska university hospital. Methods: Phase space files (psf) for square fields between 0.25cm and 10cm were calculated using the PENELOPE-based PRIMO software. The linac MC-model was tuned by comparing PRIMO-estimated and experimentally determined depth doses and lateral dose-profiles for 40cmx40cm fields. The calculated psf were used as radiation sources to calculate the correction factors of IBA and PTW detectors with the code penEasy/PENELOPE. Results: The optimal tuning parameters ofmore » the MClinac model in PRIMO were 5.4 MeV incident electron energy and zero energy spread, focal spot size and beam divergence. Correction factors obtained for the liquid ion chamber (PTW-T31018) are within 1% down to 0.5 cm fields. For unshielded diodes (IBA-EFD, IBA-SFD, PTW-T60017 and PTW-T60018) the corrections are up to 2% at intermediate fields (>1cm side), becoming down to −11% for fields smaller than 1cm. The shielded diode (IBA-PFD and PTW-T60016) corrections vary with field size from 0 to −4%. Volume averaging effects are found for most detectors in the presence of 0.25cm fields. Conclusion: Good agreement was found between correction factors based on PRIMO-generated psf and those from other publications. The calculated factors will be implemented in output factor measurements (using several detectors) in the clinic. PRIMO is a userfriendly general code capable of generating small field psf and can be used without having to code own linac geometries. It can therefore be used to improve the clinical dosimetry, especially in the commissioning of linear accelerators. Important dosimetry data, such as dose-profiles and output factors can be determined more accurately for a specific machine, geometry and setup by using PRIMO and having a MC-model of the detector used.« less

Identification of a chemical inhibitor for nuclear speckle formation: Implications for the function of nuclear speckles in regulation of alternative pre-mRNA splicing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kurogi, Yutaro; Matsuo, Yota; Mihara, Yuki

2014-03-28

Highlights: • We identified tubercidin as a compound inducing aberrant formation of the speckles. • Tubercidin causes delocalization of poly (A){sup +}RNAs from nuclear speckles. • Tubercidin induces dispersion of splicing factors from nuclear speckles. • Tubercidin affects alternative pre-mRNA splicing. • Nuclear speckles play a role in regulation of alternative pre-mRNA splicing. - Abstract: Nuclear speckles are subnuclear structures enriched with RNA processing factors and poly (A){sup +} RNAs comprising mRNAs and poly (A){sup +} non-coding RNAs (ncRNAs). Nuclear speckles are thought to be involved in post-transcriptional regulation of gene expression, such as pre-mRNA splicing. By screening 3585 culturemore » extracts of actinomycetes with in situ hybridization using an oligo dT probe, we identified tubercidin, an analogue of adenosine, as an inhibitor of speckle formation, which induces the delocalization of poly (A){sup +} RNA and dispersion of splicing factor SRSF1/SF2 from nuclear speckles in HeLa cells. Treatment with tubercidin also decreased steady-state MALAT1 long ncRNA, thought to be involved in the retention of SRSF1/SF2 in nuclear speckles. In addition, we found that tubercidin treatment promoted exon skipping in the alternative splicing of Clk1 pre-mRNA. These results suggest that nuclear speckles play a role in modulating the concentration of splicing factors in the nucleoplasm to regulate alternative pre-mRNA splicing.« less

THE SYSTEMATIC ERROR TEST FOR PSF CORRECTION IN WEAK GRAVITATIONAL LENSING SHEAR MEASUREMENT BY THE ERA METHOD BY IDEALIZING PSF

DOE Office of Scientific and Technical Information (OSTI.GOV)

Okura, Yuki; Futamase, Toshifumi, E-mail: yuki.okura@riken.jp

We improve the ellipticity of re-smeared artificial image (ERA) method of point-spread function (PSF) correction in a weak lensing shear analysis in order to treat the realistic shape of galaxies and the PSF. This is done by re-smearing the PSF and the observed galaxy image using a re-smearing function (RSF) and allows us to use a new PSF with a simple shape and to correct the PSF effect without any approximations or assumptions. We perform a numerical test to show that the method applied for galaxies and PSF with some complicated shapes can correct the PSF effect with a systematicmore » error of less than 0.1%. We also apply the ERA method for real data of the Abell 1689 cluster to confirm that it is able to detect the systematic weak lensing shear pattern. The ERA method requires less than 0.1 or 1 s to correct the PSF for each object in a numerical test and a real data analysis, respectively.« less

In vivo Three-Dimensional Superresolution Fluorescence Tracking using a Double-Helix Point Spread Function

PubMed Central

Lew, Matthew D.; Thompson, Michael A.; Badieirostami, Majid; Moerner, W. E.

2010-01-01

The point spread function (PSF) of a widefield fluorescence microscope is not suitable for three-dimensional super-resolution imaging. We characterize the localization precision of a unique method for 3D superresolution imaging featuring a double-helix point spread function (DH-PSF). The DH-PSF is designed to have two lobes that rotate about their midpoint in any transverse plane as a function of the axial position of the emitter. In effect, the PSF appears as a double helix in three dimensions. By comparing the Cramer-Rao bound of the DH-PSF with the standard PSF as a function of the axial position, we show that the DH-PSF has a higher and more uniform localization precision than the standard PSF throughout a 2 μm depth of field. Comparisons between the DH-PSF and other methods for 3D super-resolution are briefly discussed. We also illustrate the applicability of the DH-PSF for imaging weak emitters in biological systems by tracking the movement of quantum dots in glycerol and in live cells. PMID:20563317

17β-estradiol regulates the RNA-binding protein Nova1, which then regulates the alternative splicing of estrogen receptor β in the aging female rat brain.

PubMed

Shults, Cody L; Dingwall, Caitlin B; Kim, Chun K; Pinceti, Elena; Rao, Yathindar S; Pak, Toni R

2018-01-01

Alternative RNA splicing results in the translation of diverse protein products arising from a common nucleotide sequence. These alternative protein products are often functional and can have widely divergent actions from the canonical protein. Studies in humans and other vertebrate animals have demonstrated that alternative splicing events increase with advanced age, sometimes resulting in pathological consequences. Menopause represents a critical transition for women, where the beneficial effects of estrogens are no longer evident; therefore, factors underlying increased pathological conditions in women are confounded by the dual factors of aging and declining estrogens. Estrogen receptors (ERs) are subject to alternative splicing, the spliced variants increase following menopause, and they fail to efficiently activate estrogen-dependent signaling pathways. However, the factors that regulate the alternative splicing of ERs remain unknown. We demonstrate novel evidence supporting a potential biological feedback loop where 17β-estradiol regulates the RNA-binding protein Nova1, which, in turn, regulates the alternative splicing of ERβ. These data increase our understanding of ER alternative splicing and could have potential implications for women taking hormone replacement therapy after menopause. Copyright © 2017 Elsevier Inc. All rights reserved.

Effects of high pressure freezing (HPF) on denaturation of natural actomyosin extracted from prawn (Metapenaeus ensis).

PubMed

Cheng, Lina; Sun, Da-Wen; Zhu, Zhiwei; Zhang, Zhihang

2017-08-15

Effects of protein denaturation caused by high pressure freezing, involving Pressure-Factors (pressure, time) and Freezing-Factors (temperature, phase transition, recrystallization, ice crystal types), are complicated. In the current study, the conformation and functional changes of natural actomyosin (NAM) under pressure assisted freezing (PAF, 100,150,300,400,500MPa P -20°C/25min ), pressure shift freezing (PSF, 200MPa P -20°C/25min ), and immersion freezing ( 0.1MPa P -20°C/5min ) after pressure was released to 0.1MPa, as compared to normal immersion freezing process (IF, 0.1MPa P -20°C/30min ). Results indicated that PSF ( 200MPa P -20°C/30min ) could reduce the denaturation of frozen NAM and a pressure of 300MPa was the critical point to induce such a denaturation. During the periods of B→D in PSF or B→C→D in PAF, the generation and growth of ice crystals played an important role on changing the secondary and tertiary structure of the treated NAM. Copyright © 2017 Elsevier Ltd. All rights reserved.

Serum protein adsorption and platelet adhesion on aspartic-acid-immobilized polysulfone membranes.

PubMed

Higuchi, Akon; Hashiba, Hirokazu; Hayashi, Rika; Yoon, Boo Ok; Sakurai, Masaru; Hara, Mariko

2004-01-01

Polysulfone (PSf) membranes that covalently conjugated with aspartic acid (ASP-PSf) were prepared and analyzed for hemocompatability. Compared to PSf or other types of surface-modified PSf membranes, the ASP-PSf membranes had a reduced ability to adsorb protein from either a plasma solution or a mixed solution of albumin, globulin and fibrinogen. This appears to be due to the creation of a hydrophilic surface by the aspartic acid zwitterion immobilized on the ASP-PSf membranes. Furthermore, the analyses of membrane protein adsorption showed that a mixed protein solution recapitulates the cooperative adsorption of proteins that occurs in plasma. We also found that the number of adhering platelets was the lowest on the ASP-PSf membranes and, in general, that platelet adhesion decreased in parallel with fibrinogen adsorption. In summary, aspartic acid immobilized on the ASP-PSf membranes, which have zwitterions with a net zero charge, effectively contributes to the hydrophilic and hemocompatible sites on the surface of the hydrophobic PSf membranes.

Incorporating significant amino acid pairs and protein domains to predict RNA splicing-related proteins with functional roles

NASA Astrophysics Data System (ADS)

Hsu, Justin Bo-Kai; Huang, Kai-Yao; Weng, Tzu-Ya; Huang, Chien-Hsun; Lee, Tzong-Yi

2014-01-01

Machinery of pre-mRNA splicing is carried out through the interaction of RNA sequence elements and a variety of RNA splicing-related proteins (SRPs) (e.g. spliceosome and splicing factors). Alternative splicing, which is an important post-transcriptional regulation in eukaryotes, gives rise to multiple mature mRNA isoforms, which encodes proteins with functional diversities. However, the regulation of RNA splicing is not yet fully elucidated, partly because SRPs have not yet been exhaustively identified and the experimental identification is labor-intensive. Therefore, we are motivated to design a new method for identifying SRPs with their functional roles in the regulation of RNA splicing. The experimentally verified SRPs were manually curated from research articles. According to the functional annotation of Splicing Related Gene Database, the collected SRPs were further categorized into four functional groups including small nuclear Ribonucleoprotein, Splicing Factor, Splicing Regulation Factor and Novel Spliceosome Protein. The composition of amino acid pairs indicates that there are remarkable differences among four functional groups of SRPs. Then, support vector machines (SVMs) were utilized to learn the predictive models for identifying SRPs as well as their functional roles. The cross-validation evaluation presents that the SVM models trained with significant amino acid pairs and functional domains could provide a better predictive performance. In addition, the independent testing demonstrates that the proposed method could accurately identify SRPs in mammals/plants as well as effectively distinguish between SRPs and RNA-binding proteins. This investigation provides a practical means to identifying potential SRPs and a perspective for exploring the regulation of RNA splicing.

Incorporating significant amino acid pairs and protein domains to predict RNA splicing-related proteins with functional roles.

PubMed

Hsu, Justin Bo-Kai; Huang, Kai-Yao; Weng, Tzu-Ya; Huang, Chien-Hsun; Lee, Tzong-Yi

2014-01-01

Machinery of pre-mRNA splicing is carried out through the interaction of RNA sequence elements and a variety of RNA splicing-related proteins (SRPs) (e.g. spliceosome and splicing factors). Alternative splicing, which is an important post-transcriptional regulation in eukaryotes, gives rise to multiple mature mRNA isoforms, which encodes proteins with functional diversities. However, the regulation of RNA splicing is not yet fully elucidated, partly because SRPs have not yet been exhaustively identified and the experimental identification is labor-intensive. Therefore, we are motivated to design a new method for identifying SRPs with their functional roles in the regulation of RNA splicing. The experimentally verified SRPs were manually curated from research articles. According to the functional annotation of Splicing Related Gene Database, the collected SRPs were further categorized into four functional groups including small nuclear Ribonucleoprotein, Splicing Factor, Splicing Regulation Factor and Novel Spliceosome Protein. The composition of amino acid pairs indicates that there are remarkable differences among four functional groups of SRPs. Then, support vector machines (SVMs) were utilized to learn the predictive models for identifying SRPs as well as their functional roles. The cross-validation evaluation presents that the SVM models trained with significant amino acid pairs and functional domains could provide a better predictive performance. In addition, the independent testing demonstrates that the proposed method could accurately identify SRPs in mammals/plants as well as effectively distinguish between SRPs and RNA-binding proteins. This investigation provides a practical means to identifying potential SRPs and a perspective for exploring the regulation of RNA splicing.

Biochemical and proteomic analysis of spliceosome factors interacting with intron-1 of human papillomavirus type-16.

PubMed

Martínez-Salazar, Martha; López-Urrutia, Eduardo; Arechaga-Ocampo, Elena; Bonilla-Moreno, Raul; Martínez-Castillo, Macario; Díaz-Hernández, Job; Del Moral-Hernández, Oscar; Cedillo-Barrón, Leticia; Martines-Juarez, Víctor; De Nova-Ocampo, Monica; Valdes, Jesús; Berumen, Jaime; Villegas-Sepúlveda, Nicolás

2014-12-05

The human papillomavirus type 16 (HPV-16) E6/E7 spliced transcripts are heterogeneously expressed in cervical carcinoma. The heterogeneity of the E6/E7 splicing profile might be in part due to the intrinsic variation of splicing factors in tumor cells. However, the splicing factors that bind the E6/E7 intron 1 (In-1) have not been defined. Therefore, we aimed to identify these factors; we used HeLa nuclear extracts (NE) for in vitro spliceosome assembly. The proteins were allowed to bind to an RNA/DNA hybrid formed by the In-1 transcript and a 5'-biotinylated DNA oligonucleotide complementary to the upstream exon sequence, which prevented interference in protein binding to the intron. The hybrid probes bound with the nuclear proteins were coupled to streptavidin magnetic beads for chromatography affinity purification. Proteins were eluted and identified by mass spectrometry (MS). Approximately 170 proteins were identified by MS, 80% of which were RNA binding proteins, including canonical spliceosome core components, helicases and regulatory splicing factors. The canonical factors were identified as components of the spliceosomal B-complex. Although 35-40 of the identified factors were cognate splicing factors or helicases, they have not been previously detected in spliceosome complexes that were assembled using in vivo or in vitro models. Copyright © 2014 Elsevier B.V. All rights reserved.

Factors influencing alternative splice site utilization in vivo.

PubMed Central

Fu, X Y; Manley, J L

1987-01-01

To study factors that influence the choice of alternative pre-mRNA splicing pathways, we introduced plasmids expressing either wild-type or mutated simian virus 40 (SV40) early regions into tissue culture cells and then measured the quantities of small-t and large-T RNAs produced. One important element controlling splice site selection was found to be the size of the intron removed in the production of small-t mRNA; expansion of this intron (from 66 to 77 or more nucleotides) resulted in a substantial increase in the amount of small-t mRNA produced relative to large-T mRNA. This suggests that in the normal course of SV40 early pre-mRNA processing, large-T splicing is at a competitive advantage relative to small-t splicing because of the small size of the latter intron. Several additional features of the pre-mRNA that can influence splice site selection were also identified by analyzing the effects of mutations containing splice site duplications. These include the strengths of competing 5' splice sites and the relative positions of splice sites in the pre-mRNA. Finally, we showed that the ratio of small-t to large-T mRNA was 10 to 15-fold greater in human 293 cells than in HeLa cells or other mammalian cell types. These results suggest the existence of cell-specific trans-acting factors that can dramatically alter the pattern of splice site selection in a pre-mRNA. Images PMID:3029566

Splicing Factor 1 Modulates Dietary Restriction and TORC1 Pathway Longevity in C. elegans

PubMed Central

Heintz, Caroline; Escoubas, Caroline; Zhang, Yue; Weir, Heather J.; Dutta, Sneha; Silva-García, Carlos Giovanni; Bruun, Gitte Hoffmann; Morantte, Ianessa; Hoxhaj, Gerta; Manning, Brendan D.; Andresen, Brage S.; Mair, William B.

2016-01-01

Ageing is driven by a loss of transcriptional and protein homeostasis1–3 and is the key risk factor for multiple chronic diseases. Interventions that attenuate or reverse systemic dysfunction seen with age therefore have potential to reduce overall disease risk in the elderly. Pre-mRNA splicing is a fundamental link between gene expression and the proteome, and deregulation of the splicing machinery is linked to multiple age-related chronic diseases4,5. However, the role of splicing homeostasis in healthy ageing remains unclear. Here we demonstrate that pre-mRNA splicing homeostasis is a biomarker and predictor of life expectancy in Caenorhabditis elegans. Using transcriptomics and in-depth splicing analysis in young and old animals fed ad libitum or on dietary restriction (DR), we find defects in global pre-mRNA splicing with age that are reduced by DR via the branch point binding protein (BBP)/splicing factor 1 (SFA-1). We show that SFA-1 is specifically required for lifespan extension both by DR, and modulation of TORC1 pathway components AMPK, RAGA-1 and RSKS-1/S6 Kinase. Lastly, we demonstrate that overexpression of SFA-1 is sufficient to extend lifespan. Together, these data demonstrate a role for RNA splicing homeostasis in DR longevity and suggest modulation of specific spliceosome components can prolong healthy ageing. PMID:27919065

Pre-mRNA splicing in cancer: the relevance in oncogenesis, treatment and drug resistance.

PubMed

Wojtuszkiewicz, Anna; Assaraf, Yehuda G; Maas, Marielle J P; Kaspers, Gertjan J L; Jansen, Gerrit; Cloos, Jacqueline

2015-05-01

Aberrant pre-mRNA splicing in cancer is emerging as an important determinant of oncogenesis, response to treatment and anticancer drug resistance. At the same time, the spliceosome has become a target for a novel class of pre-clinical chemotherapeutics with a potential future application in cancer treatment. Taken together, these findings offer novel opportunities for the enhancement of the efficacy of cancer therapy. This review presents a comprehensive overview of the molecular mechanisms involved in splicing and current developments regarding splicing aberrations in relation to several aspects of cancer formation and therapy. Identified mutations in the various components of the spliceosome and their implications for cancer prognosis are delineated. Moreover, the contribution of abnormal splicing patterns as well as deregulated splicing factors to chemoresistance is discussed, along with novel splicing-based therapeutic approaches. Significant progress has been made in deciphering the role of splicing factors in cancer including carcinogenesis and drug resistance. Splicing-based prognostic tools as well as therapeutic options hold great potential towards improvements in cancer therapy. However, gaining more in-depth molecular insight into the consequences of mutations in various components of the splicing machinery as well as of cellular effects of spliceosome inhibition is a prerequisite to establish the role of splicing in tumor progression and treatment options, respectively.

Effect of time-of-flight and point spread function modeling on detectability of myocardial defects in PET

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schaefferkoetter, Joshua, E-mail: dnrjds@nus.edu.sg; Ouyang, Jinsong; Rakvongthai, Yothin

2014-06-15

Purpose: A study was designed to investigate the impact of time-of-flight (TOF) and point spread function (PSF) modeling on the detectability of myocardial defects. Methods: Clinical FDG-PET data were used to generate populations of defect-present and defect-absent images. Defects were incorporated at three contrast levels, and images were reconstructed by ordered subset expectation maximization (OSEM) iterative methods including ordinary Poisson, alone and with PSF, TOF, and PSF+TOF. Channelized Hotelling observer signal-to-noise ratio (SNR) was the surrogate for human observer performance. Results: For three iterations, 12 subsets, and no postreconstruction smoothing, TOF improved overall defect detection SNR by 8.6% as comparedmore » to its non-TOF counterpart for all the defect contrasts. Due to the slow convergence of PSF reconstruction, PSF yielded 4.4% less SNR than non-PSF. For reconstruction parameters (iteration number and postreconstruction smoothing kernel size) optimizing observer SNR, PSF showed larger improvement for faint defects. The combination of TOF and PSF improved mean detection SNR as compared to non-TOF and non-PSF counterparts by 3.0% and 3.2%, respectively. Conclusions: For typical reconstruction protocol used in clinical practice, i.e., less than five iterations, TOF improved defect detectability. In contrast, PSF generally yielded less detectability. For large number of iterations, TOF+PSF yields the best observer performance.« less

Post-stroke fatigue and its dimensions within first 3 months after stroke.

PubMed

Delva, Iryna I; Lytvynenko, Nataliya V; Delva, Mykhaylo Y

Post-stroke fatigue (PSF) is a common stroke complication with long-term negative consequences. Assess the qualitative and quantitative PSF characteristics during 3 month post-stroke period. There were examined 141 patients with acute ischemic or hemorrhagic strokes at hospital stay, in 1, 2 and 3 months after stroke. PSF was measured by fatigue assessment scale (FAS), multidimensional fatigue inventory-20 (MFI-20) and fatigue severity scale (FSS). 116 (82.3%) patients had ischemic strokes, 25 (17.7%) had hemorrhagic strokes. According to FAS and MFI-20 "global fatigue" sub-scale, PSF was present, respectively, in 22.0% and 25.5% cases at hospital stay, in 38.3% and 35.5% cases in 3 month after stroke. The growing prevalence of PSF was based on significantly increasing the rates of PSD physical domain (from 28.4% to 41.1%) and the rates of PSF mental domain (from 19.1% to 31.9%). On the other hand, the rates of PSF activity-related component had been significantly reduced from 36.2% to 17.0% within observation period. Moreover, according to MFI-20, it had been revealed significant increasing of PSF intensities in global, physical and mental domains during first 3 post-stroke months. According to FSS value ranks, proportions of patients with "no PSF" had been decreased in 1.5 times due to simultaneously rising rates of "moderate" as well as "severe" PSF impacts on daily life. The PSF spreading is significantly increased during the first 3 post-stroke months due to increasing of rates and intensities of physical and mental PSF domains.

Accurately estimating PSF with straight lines detected by Hough transform

NASA Astrophysics Data System (ADS)

Wang, Ruichen; Xu, Liangpeng; Fan, Chunxiao; Li, Yong

2018-04-01

This paper presents an approach to estimating point spread function (PSF) from low resolution (LR) images. Existing techniques usually rely on accurate detection of ending points of the profile normal to edges. In practice however, it is often a great challenge to accurately localize profiles of edges from a LR image, which hence leads to a poor PSF estimation of the lens taking the LR image. For precisely estimating the PSF, this paper proposes firstly estimating a 1-D PSF kernel with straight lines, and then robustly obtaining the 2-D PSF from the 1-D kernel by least squares techniques and random sample consensus. Canny operator is applied to the LR image for obtaining edges and then Hough transform is utilized to extract straight lines of all orientations. Estimating 1-D PSF kernel with straight lines effectively alleviates the influence of the inaccurate edge detection on PSF estimation. The proposed method is investigated on both natural and synthetic images for estimating PSF. Experimental results show that the proposed method outperforms the state-ofthe- art and does not rely on accurate edge detection.

Urban area change detection procedures with remote sensing data

NASA Technical Reports Server (NTRS)

Maxwell, E. L. (Principal Investigator); Riordan, C. J.

1980-01-01

The underlying factors affecting the detection and identification of nonurban to urban land cover change using satellite data were studied. Computer programs were developed to create a digital scene and to simulate the effect of the sensor point spread function (PSF) on the transfer of modulation from the scene to an image of the scene. The theory behind the development of a digital filter representing the PSF is given as well as an example of its application. Atmospheric effects on modulation transfer are also discussed. A user's guide and program listings are given.

Facile preparation of heparinized polysulfone membrane assisted by polydopamine/polyethyleneimine co-deposition for simultaneous LDL selectivity and biocompatibility

NASA Astrophysics Data System (ADS)

Wang, Liwei; Fang, Fei; Liu, Yang; Li, Jing; Huang, Xiaojun

2016-11-01

Low-density lipoprotein (LDL) gains worldwide attention for decades as the key risk factor to atherosclerosis that progressively deteriorating into cardiovascular diseases. Until recent years, LDL-apheresis comes to be extensively used as a direct and efficient LDL removal method, with LDL adsorption materials particularly important. In this paper, a new strategy based on the co-deposition of polydopamine (PDA) with polyethylenimine (PEI) onto polysulfone (PSf) membranes, then subsequent heparinization by amino-carbonyl reactions, to achieve LDL selectivity and simultaneous biocompatibility, is proposed. Surface properties of modified PSf membranes are characterized by ATR-FTIR, XPS, FESEM, Zeta potential and WCA measurements. LDL adsorption ability is investigated by ELISA, while blood biocompatibility is evaluated by platelet adhesion experiments. Results suggest that heparin-modified PSf membranes show high selectivity for LDL removal and fine biocompatibility in contact with plasma, as excellent potential materials for LDL-apheresis.

The Chandra X-ray Observatory PSF Library

NASA Astrophysics Data System (ADS)

Karovska, M.; Beikman, S. J.; Elvis, M. S.; Flanagan, J. M.; Gaetz, T.; Glotfelty, K. J.; Jerius, D.; McDowell, J. C.; Rots, A. H.

Pre-flight and on-orbit calibration of the Chandra X-Ray Observatory provided a unique base for developing detailed models of the optics and detectors. Using these models we have produced a set of simulations of the Chandra point spread function (PSF) which is available to the users via PSF library files. We describe here how the PSF models are generated and the design and content of the Chandra PSF library files.

Characteristic investigation of Golay9 multiple mirror telescope with a spherical primary mirror

NASA Astrophysics Data System (ADS)

Wu, Feng; Wu, Quanying; Zhu, Xifang; Xiang, Ruxi; Qian, Lin

2017-10-01

The sparse aperture provides a novel solution to the manufacturing difficulties of modern super large telescopes. Golay configurations are optimal in the sparse aperture family. Characteristics of the Golay9 multiple mirror telescope having a spherical primary mirror are investigated. The arrangement of the nine sub-mirrors is discussed after the planar Golay9 configuration is analyzed. The characteristics of the entrance pupil are derived by analyzing the sub-aperture shapes with different relative apertures and sub-mirror sizes. Formulas about the fill factor and the overlay factor are deduced. Their maximal values are presented based on the derived tangency condition. Formulas for the point spread function (PSF) and the modulation transfer function (MTF) of the Golay9 MMT are also deduced. Two Golay9 MMT have been developed by Zemax simulation. Their PSF, MTF, fill factors, and overlay factors prove that our theoretical results are consistent with the practical simulation ones.

Modelling reveals kinetic advantages of co-transcriptional splicing.

PubMed

Aitken, Stuart; Alexander, Ross D; Beggs, Jean D

2011-10-01

Messenger RNA splicing is an essential and complex process for the removal of intron sequences. Whereas the composition of the splicing machinery is mostly known, the kinetics of splicing, the catalytic activity of splicing factors and the interdependency of transcription, splicing and mRNA 3' end formation are less well understood. We propose a stochastic model of splicing kinetics that explains data obtained from high-resolution kinetic analyses of transcription, splicing and 3' end formation during induction of an intron-containing reporter gene in budding yeast. Modelling reveals co-transcriptional splicing to be the most probable and most efficient splicing pathway for the reporter transcripts, due in part to a positive feedback mechanism for co-transcriptional second step splicing. Model comparison is used to assess the alternative representations of reactions. Modelling also indicates the functional coupling of transcription and splicing, because both the rate of initiation of transcription and the probability that step one of splicing occurs co-transcriptionally are reduced, when the second step of splicing is abolished in a mutant reporter.

MeCP2 regulates Tet1-catalyzed demethylation, CTCF binding, and learning-dependent alternative splicing of the BDNF gene in Turtle

PubMed Central

Zheng, Zhaoqing; Ambigapathy, Ganesh; Keifer, Joyce

2017-01-01

MECP2 mutations underlying Rett syndrome cause widespread misregulation of gene expression. Functions for MeCP2 other than transcriptional are not well understood. In an ex vivo brain preparation from the pond turtle Trachemys scripta elegans, an intraexonic splicing event in the brain-derived neurotrophic factor (BDNF) gene generates a truncated mRNA transcript in naïve brain that is suppressed upon classical conditioning. MeCP2 and its partners, splicing factor Y-box binding protein 1 (YB-1) and methylcytosine dioxygenase 1 (Tet1), bind to BDNF chromatin in naïve but dissociate during conditioning; the dissociation correlating with decreased DNA methylation. Surprisingly, conditioning results in new occupancy of BDNF chromatin by DNA insulator protein CCCTC-binding factor (CTCF), which is associated with suppression of splicing in conditioning. Knockdown of MeCP2 shows it is instrumental for splicing and inhibits Tet1 and CTCF binding thereby negatively impacting DNA methylation and conditioning-dependent splicing regulation. Thus, mutations in MECP2 can have secondary effects on DNA methylation and alternative splicing. DOI: http://dx.doi.org/10.7554/eLife.25384.001 PMID:28594324

Identifying RNA splicing factors using IFT genes in Chlamydomonas reinhardtii.

PubMed

Lin, Huawen; Zhang, Zhengyan; Iomini, Carlo; Dutcher, Susan K

2018-03-01

Intraflagellar transport moves proteins in and out of flagella/cilia and it is essential for the assembly of these organelles. Using whole-genome sequencing, we identified splice site mutations in two IFT genes, IFT81 ( fla9 ) and IFT121 ( ift121-2 ), which lead to flagellar assembly defects in the unicellular green alga Chlamydomonas reinhardtii The splicing defects in these ift mutants are partially corrected by mutations in two conserved spliceosome proteins, DGR14 and FRA10. We identified a dgr14 deletion mutant, which suppresses the 3' splice site mutation in IFT81 , and a frameshift mutant of FRA10 , which suppresses the 5' splice site mutation in IFT121 Surprisingly, we found dgr14-1 and fra10 mutations suppress both splice site mutations. We suggest these two proteins are involved in facilitating splice site recognition/interaction; in their absence some splice site mutations are tolerated. Nonsense mutations in SMG1 , which is involved in nonsense-mediated decay, lead to accumulation of aberrant transcripts and partial restoration of flagellar assembly in the ift mutants. The high density of introns and the conservation of noncore splicing factors, together with the ease of scoring the ift mutant phenotype, make Chlamydomonas an attractive organism to identify new proteins involved in splicing through suppressor screening. © 2018 The Authors.

Interconnections Between RNA-Processing Pathways Revealed by a Sequencing-Based Genetic Screen for Pre-mRNA Splicing Mutants in Fission Yeast.

PubMed

Larson, Amy; Fair, Benjamin Jung; Pleiss, Jeffrey A

2016-06-01

Pre-mRNA splicing is an essential component of eukaryotic gene expression and is highly conserved from unicellular yeasts to humans. Here, we present the development and implementation of a sequencing-based reverse genetic screen designed to identify nonessential genes that impact pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe, an organism that shares many of the complex features of splicing in higher eukaryotes. Using a custom-designed barcoding scheme, we simultaneously queried ∼3000 mutant strains for their impact on the splicing efficiency of two endogenous pre-mRNAs. A total of 61 nonessential genes were identified whose deletions resulted in defects in pre-mRNA splicing; enriched among these were factors encoding known or predicted components of the spliceosome. Included among the candidates identified here are genes with well-characterized roles in other RNA-processing pathways, including heterochromatic silencing and 3' end processing. Splicing-sensitive microarrays confirm broad splicing defects for many of these factors, revealing novel functional connections between these pathways. Copyright © 2016 Larson et al.

SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy

PubMed Central

Pagliarini, Vittoria; Pelosi, Laura; Bustamante, Maria Blaire; Nobili, Annalisa; Berardinelli, Maria Grazia; D’Amelio, Marcello; Musarò, Antonio

2015-01-01

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss of motor neurons in patients with null mutations in the SMN1 gene. The almost identical SMN2 gene is unable to compensate for this deficiency because of the skipping of exon 7 during pre–messenger RNA (mRNA) processing. Although several splicing factors can modulate SMN2 splicing in vitro, the physiological regulators of this disease-causing event are unknown. We found that knockout of the splicing factor SAM68 partially rescued body weight and viability of SMAΔ7 mice. Ablation of SAM68 function promoted SMN2 splicing and expression in SMAΔ7 mice, correlating with amelioration of SMA-related defects in motor neurons and skeletal muscles. Mechanistically, SAM68 binds to SMN2 pre-mRNA, favoring recruitment of the splicing repressor hnRNP A1 and interfering with that of U2AF65 at the 3′ splice site of exon 7. These findings identify SAM68 as the first physiological regulator of SMN2 splicing in an SMA mouse model. PMID:26438828

Interconnections Between RNA-Processing Pathways Revealed by a Sequencing-Based Genetic Screen for Pre-mRNA Splicing Mutants in Fission Yeast

PubMed Central

Larson, Amy; Fair, Benjamin Jung; Pleiss, Jeffrey A.

2016-01-01

Pre-mRNA splicing is an essential component of eukaryotic gene expression and is highly conserved from unicellular yeasts to humans. Here, we present the development and implementation of a sequencing-based reverse genetic screen designed to identify nonessential genes that impact pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe, an organism that shares many of the complex features of splicing in higher eukaryotes. Using a custom-designed barcoding scheme, we simultaneously queried ∼3000 mutant strains for their impact on the splicing efficiency of two endogenous pre-mRNAs. A total of 61 nonessential genes were identified whose deletions resulted in defects in pre-mRNA splicing; enriched among these were factors encoding known or predicted components of the spliceosome. Included among the candidates identified here are genes with well-characterized roles in other RNA-processing pathways, including heterochromatic silencing and 3ʹ end processing. Splicing-sensitive microarrays confirm broad splicing defects for many of these factors, revealing novel functional connections between these pathways. PMID:27172183

Serine/Arginine-rich Splicing Factor 2 Modulates Herpes Simplex Virus Type 1 Replication via Regulating Viral Gene Transcriptional Activity and Pre-mRNA Splicing.

PubMed

Wang, Ziqiang; Liu, Qing; Lu, Jinhua; Fan, Ping; Xie, Weidong; Qiu, Wei; Wang, Fan; Hu, Guangnan; Zhang, Yaou

2016-12-16

Once it enters the host cell, herpes simplex virus type 1 (HSV-1) recruits a series of host cell factors to facilitate its life cycle. Here, we demonstrate that serine/arginine-rich splicing factor 2 (SRSF2), which is an important component of the splicing speckle, mediates HSV-1 replication by regulating viral gene expression at the transcriptional and posttranscriptional levels. Our results indicate that SRSF2 functions as a transcriptional activator by directly binding to infected cell polypeptide 0 (ICP0), infected cell polypeptide 27 (ICP27), and thymidine kinase promoters. Moreover, SRSF2 participates in ICP0 pre-mRNA splicing by recognizing binding sites in ICP0 exon 3. These findings provide insight into the functions of SRSF2 in HSV-1 replication and gene expression. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Pre-mRNA mis-splicing of sarcomeric genes in heart failure.

PubMed

Zhu, Chaoqun; Chen, Zhilong; Guo, Wei

2017-08-01

Pre-mRNA splicing is an important biological process that allows production of multiple proteins from a single gene in the genome, and mainly contributes to protein diversity in eukaryotic organisms. Alternative splicing is commonly governed by RNA binding proteins to meet the ever-changing demands of the cell. However, the mis-splicing may lead to human diseases. In the heart of human, mis-regulation of alternative splicing has been associated with heart failure. In this short review, we focus on alternative splicing of sarcomeric genes and review mis-splicing related heart failure with relatively well studied Sarcomeric genes and splicing mechanisms with identified regulatory factors. The perspective of alternative splicing based therapeutic strategies in heart failure has also been discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

Alternative splicing in cancers: From aberrant regulation to new therapeutics.

PubMed

Song, Xiaowei; Zeng, Zhenyu; Wei, Huanhuan; Wang, Zefeng

2018-03-01

Alternative splicing is one of the most common mechanisms for gene regulation in humans, and plays a vital role to increase the complexity of functional proteins. In this article, we seek to provide a general review on the relationships between alternative splicing and tumorigenesis. We briefly introduce the basic rules for regulation of alternative splicing, and discuss recent advances on dynamic regulation of alternative splicing in cancers by highlighting the roles of a variety of RNA splicing factors in tumorigenesis. We further discuss several important questions regarding the splicing of long noncoding RNAs and back-splicing of circular RNAs in cancers. Finally, we discuss the current technologies that can be used to manipulate alternative splicing and serve as potential cancer treatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Majidpour, Mostafa; Qiu, Charlie; Chu, Peter

Three algorithms for the forecasting of energy consumption at individual EV charging outlets have been applied to real world data from the UCLA campus. Out of these three algorithms, namely k-Nearest Neighbor (kNN), ARIMA, and Pattern Sequence Forecasting (PSF), kNN with k=1, was the best and PSF was the worst performing algorithm with respect to the SMAPE measure. The advantage of PSF is its increased robustness to noise by substituting the real valued time series with an integer valued one, and the advantage of NN is having the least SMAPE for our data. We propose a Modified PSF algorithm (MPSF)more » which is a combination of PSF and NN; it could be interpreted as NN on integer valued data or as PSF with considering only the most recent neighbor to produce the output. Some other shortcomings of PSF are also addressed in the MPSF. Results show that MPSF has improved the forecast performance.« less

Resveratrol, by modulating RNA processing factor levels, can influence the alternative splicing of pre-mRNAs.

PubMed

Markus, M Andrea; Marques, Francine Z; Morris, Brian J

2011-01-01

Alternative pre-mRNA splicing defects can contribute to, or result from, various diseases, including cancer. Aberrant mRNAs, splicing factors and other RNA processing factors have therefore become targets for new therapeutic interventions. Here we report that the natural polyphenol resveratrol can modulate alternative splicing in a target-specific manner. We transfected minigenes of several alternatively spliceable primary mRNAs into HEK293 cells in the presence or absence of 1, 5, 20 and 50 µM resveratrol and measured exon levels by semi-quantitative PCR after separation by agarose gel electrophoresis. We found that 20 µg/ml and 50 µg/ml of resveratrol affected exon inclusion of SRp20 and SMN2 pre-mRNAs, but not CD44v5 or tau pre-mRNAs. By Western blotting and immunofluorescence we showed that this effect may be due to the ability of resveratrol to change the protein level but not the localization of several RNA processing factors. The processing factors that increased significantly were ASF/SF2, hnRNPA1 and HuR, but resveratrol did not change the levels of RBM4, PTBP1 and U2AF35. By means of siRNA-mediated knockdown we depleted cells of SIRT1, regarded as a major target of resveratrol, and showed that the effect on splicing was not dependent on SIRT1. Our results suggest that resveratrol might be an attractive small molecule to treat diseases in which aberrant splicing has been implicated, and justify more extensive research on the effects of resveratrol on the splicing machinery.

A few nucleotide polymorphisms are sufficient to recruit nuclear factors differentially to the intron 1 of HPV-16 intratypic variants.

PubMed

López-Urrutia, Eduardo; Valdés, Jesús; Bonilla-Moreno, Raúl; Martínez-Salazar, Martha; Martínez-Garcia, Martha; Berumen, Jaime; Villegas-Sepúlveda, Nicolás

2012-06-01

The HPV-16 E6/E7 genes, which contain intron 1, are processed by alternative splicing and its transcripts are detected with a heterogeneous profile in tumours cells. Frequently, the HPV-16 positive carcinoma cells bear viral variants that contain single nucleotide polymorphisms into its DNA sequence. We were interested in analysing the contribution of this polymorphism to the heterogeneity in the pattern of the E6/E7 spliced transcripts. Using the E6/E7 sequences from three closely related HPV-16 variants, we have shown that a few nucleotide changes are sufficient to produce heterogeneity in the splicing profile. Furthermore, using mutants that contained a single SNP, we also showed that one nucleotide change was sufficient to reproduce the heterogeneous splicing profile. Additionally, a difference of two or three SNPs among these viral sequences was sufficient to recruit differentially several splicing factors to the polymorphic E6/E7 transcripts. Moreover, only one SNP was sufficient to alter the binding site of at least one splicing factor, changing the ability of splicing factors to bind the transcript. Finally, the factors that were differentially bound to the short form of intron 1 of one of these E6/E7 variants were identified as TIA1 and/or TIAR and U1-70k, while U2AF65, U5-52k and PTB were preferentially bound to the transcript of the other variants. Copyright © 2012 Elsevier B.V. All rights reserved.

Preparation of Cu2O nanowire-blended polysulfone ultrafiltration membrane with improved stability and antimicrobial activity

NASA Astrophysics Data System (ADS)

Xu, Zehai; Ye, Shuaiju; Fan, Zheng; Ren, Fanghua; Gao, Congjie; Li, Qingbiao; Li, Guoqing; Zhang, Guoliang

2015-10-01

Polysulfone (PSF) membranes have been widely applied in water and wastewater treatment, food-processing and biomedical fields. In this study, we report the preparation of modified PSF membranes by blending PSF with Cu2O nanowires (NWs) to improve their stability and antifouling activity. Synthesis of novel Cu2O NWs/PSF-blended ultrafiltration membrane was achieved via phase inversion method by dispersing one-dimensional Cu2O nanowires in PSF casting solutions. Various techniques such as XRD, SEM, TEM, and EDS were applied to characterize and investigate the properties of nanowires and membranes. The introduced Cu2O nanowires can firmly be restricted into micropores of PSF membranes, and therefore, they can effectively prevent the serious leaking problem of inorganic substances in separation process. The blended PSF membranes also provided enhanced antimicrobial activity and superior permeation property compared to pure PSF membrane. The overall work can not only provide a new way for preparation of novel blended membranes with multidimensional nanomaterials, but can also be beneficial to solve the annoying problem of biofouling.

Mapping soil particle-size fractions: A comparison of compositional kriging and log-ratio kriging

NASA Astrophysics Data System (ADS)

Wang, Zong; Shi, Wenjiao

2017-03-01

Soil particle-size fractions (psf) as basic physical variables need to be accurately predicted for regional hydrological, ecological, geological, agricultural and environmental studies frequently. Some methods had been proposed to interpolate the spatial distributions of soil psf, but the performance of compositional kriging and different log-ratio kriging methods is still unclear. Four log-ratio transformations, including additive log-ratio (alr), centered log-ratio (clr), isometric log-ratio (ilr), and symmetry log-ratio (slr), combined with ordinary kriging (log-ratio kriging: alr_OK, clr_OK, ilr_OK and slr_OK) were selected to be compared with compositional kriging (CK) for the spatial prediction of soil psf in Tianlaochi of Heihe River Basin, China. Root mean squared error (RMSE), Aitchison's distance (AD), standardized residual sum of squares (STRESS) and right ratio of the predicted soil texture types (RR) were chosen to evaluate the accuracy for different interpolators. The results showed that CK had a better accuracy than the four log-ratio kriging methods. The RMSE (sand, 9.27%; silt, 7.67%; clay, 4.17%), AD (0.45), STRESS (0.60) of CK were the lowest and the RR (58.65%) was the highest in the five interpolators. The clr_OK achieved relatively better performance than the other log-ratio kriging methods. In addition, CK presented reasonable and smooth transition on mapping soil psf according to the environmental factors. The study gives insights for mapping soil psf accurately by comparing different methods for compositional data interpolation. Further researches of methods combined with ancillary variables are needed to be implemented to improve the interpolation performance.

PSF modeling by spikes simulations and wings measurements for the MOONS multi fiber spectrograph

NASA Astrophysics Data System (ADS)

Li Causi, G.; Lee, D.; Vitali, F.; Royer, F.; Oliva, E.

2016-08-01

The optical design of MOONS, the next generation thousand-fiber NIR spectrograph for the VLT, involves both on-axis reflective collimators and on-axis very fast reflective cameras, which yields both beam obstruction, due to fiber slit and detector support, and image spread, due to propagation within detector substrate. The need to model and control i) the effect of the diffraction spikes produced by these obstructions, ii) the detector-induced shape variation of the Point Spread Function (PSF), and iii) the intensity profile of the PSF wings, leads us to perform both simulations and lab measurements, in order to optimize the spider design and built a reliable PSF model, useful for simulate realistic raw images for testing the data reduction. Starting from the unobstructed PSF variation, as computed with the ZEMAX software, we numerically computed the diffraction spikes for different spider shapes, to which we added the PSF wing profile, as measured on a sample of the MOONS VPH diffraction grating. Finally, we implemented the PSF defocusing due to the thick detector (for the visible channel), we convolved the PSF with the fiber core image, and we added the optical ghosts, so finally obtaining a detailed and realistic PSF model, that we use for spectral extraction testing, cross talk estimation, and sensitivity predictions.

Transcriptome-wide analysis of alternative RNA splicing events in Epstein-Barr virus-associated gastric carcinomas

PubMed Central

Armero, Victoria E. S.; Tremblay, Marie-Pier; Allaire, Andréa; Boudreault, Simon; Martenon-Brodeur, Camille; Duval, Cyntia; Durand, Mathieu; Lapointe, Elvy; Thibault, Philippe; Tremblay-Létourneau, Maude; Perreault, Jean-Pierre; Scott, Michelle S.

2017-01-01

Multiple human diseases including cancer have been associated with a dysregulation in RNA splicing patterns. In the current study, modifications to the global RNA splicing landscape of cellular genes were investigated in the context of Epstein-Barr virus-associated gastric cancer. Global alterations to the RNA splicing landscape of cellular genes was examined in a large-scale screen from 295 primary gastric adenocarcinomas using high-throughput RNA sequencing data. RT-PCR analysis, mass spectrometry, and co-immunoprecipitation studies were also used to experimentally validate and investigate the differential alternative splicing (AS) events that were observed through RNA-seq studies. Our study identifies alterations in the AS patterns of approximately 900 genes such as tumor suppressor genes, transcription factors, splicing factors, and kinases. These findings allowed the identification of unique gene signatures for which AS is misregulated in both Epstein-Barr virus-associated gastric cancer and EBV-negative gastric cancer. Moreover, we show that the expression of Epstein–Barr nuclear antigen 1 (EBNA1) leads to modifications in the AS profile of cellular genes and that the EBNA1 protein interacts with cellular splicing factors. These findings provide insights into the molecular differences between various types of gastric cancer and suggest a role for the EBNA1 protein in the dysregulation of cellular AS. PMID:28493890

Transcriptome-wide analysis of alternative RNA splicing events in Epstein-Barr virus-associated gastric carcinomas.

PubMed

Armero, Victoria E S; Tremblay, Marie-Pier; Allaire, Andréa; Boudreault, Simon; Martenon-Brodeur, Camille; Duval, Cyntia; Durand, Mathieu; Lapointe, Elvy; Thibault, Philippe; Tremblay-Létourneau, Maude; Perreault, Jean-Pierre; Scott, Michelle S; Bisaillon, Martin

2017-01-01

Multiple human diseases including cancer have been associated with a dysregulation in RNA splicing patterns. In the current study, modifications to the global RNA splicing landscape of cellular genes were investigated in the context of Epstein-Barr virus-associated gastric cancer. Global alterations to the RNA splicing landscape of cellular genes was examined in a large-scale screen from 295 primary gastric adenocarcinomas using high-throughput RNA sequencing data. RT-PCR analysis, mass spectrometry, and co-immunoprecipitation studies were also used to experimentally validate and investigate the differential alternative splicing (AS) events that were observed through RNA-seq studies. Our study identifies alterations in the AS patterns of approximately 900 genes such as tumor suppressor genes, transcription factors, splicing factors, and kinases. These findings allowed the identification of unique gene signatures for which AS is misregulated in both Epstein-Barr virus-associated gastric cancer and EBV-negative gastric cancer. Moreover, we show that the expression of Epstein-Barr nuclear antigen 1 (EBNA1) leads to modifications in the AS profile of cellular genes and that the EBNA1 protein interacts with cellular splicing factors. These findings provide insights into the molecular differences between various types of gastric cancer and suggest a role for the EBNA1 protein in the dysregulation of cellular AS.

An EMT–Driven Alternative Splicing Program Occurs in Human Breast Cancer and Modulates Cellular Phenotype

PubMed Central

Flytzanis, Nicholas C.; Balsamo, Michele; Condeelis, John S.; Oktay, Maja H.; Burge, Christopher B.; Gertler, Frank B.

2011-01-01

Epithelial-mesenchymal transition (EMT), a mechanism important for embryonic development, plays a critical role during malignant transformation. While much is known about transcriptional regulation of EMT, alternative splicing of several genes has also been correlated with EMT progression, but the extent of splicing changes and their contributions to the morphological conversion accompanying EMT have not been investigated comprehensively. Using an established cell culture model and RNA–Seq analyses, we determined an alternative splicing signature for EMT. Genes encoding key drivers of EMT–dependent changes in cell phenotype, such as actin cytoskeleton remodeling, regulation of cell–cell junction formation, and regulation of cell migration, were enriched among EMT–associated alternatively splicing events. Our analysis suggested that most EMT–associated alternative splicing events are regulated by one or more members of the RBFOX, MBNL, CELF, hnRNP, or ESRP classes of splicing factors. The EMT alternative splicing signature was confirmed in human breast cancer cell lines, which could be classified into basal and luminal subtypes based exclusively on their EMT–associated splicing pattern. Expression of EMT–associated alternative mRNA transcripts was also observed in primary breast cancer samples, indicating that EMT–dependent splicing changes occur commonly in human tumors. The functional significance of EMT–associated alternative splicing was tested by expression of the epithelial-specific splicing factor ESRP1 or by depletion of RBFOX2 in mesenchymal cells, both of which elicited significant changes in cell morphology and motility towards an epithelial phenotype, suggesting that splicing regulation alone can drive critical aspects of EMT–associated phenotypic changes. The molecular description obtained here may aid in the development of new diagnostic and prognostic markers for analysis of breast cancer progression. PMID:21876675

Measurements of incoherent light and background structure at exo-Earth detection levels in the High Contrast Imaging Testbed

NASA Astrophysics Data System (ADS)

Cady, Eric; Shaklan, Stuart

2014-08-01

A major component of the estimation and correction of starlight at very high contrasts is the creation of a dark hole: a region in the vicinity of the core of the stellar point spread function (PSF) where speckles in the PSF wings have been greatly attenuated, up to a factor of 1010 for the imaging of terrestrial exoplanets. At these very high contrasts, removing these speckles requires distinguishing between light from the stellar PSF scattered by instrument imperfections, which may be partially corrected across a broad band using deformable mirrors in the system, from light from other sources which generally may not. These other sources may be external or internal to the instrument (e.g. planets, exozodiacal light), but in either case, their distinguishing characteristic is their inability to interfere coherently with the PSF. In the following we discuss the estimation, structure, and expected origin of this incoherent" signal, primarily in the context of a series of experiments made with a linear band-limited mask in Jan-Mar 2013. We find that the incoherent" signal at moderate contrasts is largely estimation error of the coherent signal, while at very high contrasts it represents a true floor which is stable over week-timescales.

Proceedings of Damping 󈨟, 13-15 February 1991, San Diego, California; Volume 1 (Pages AAC-1 through DCC-19)

DTIC Science & Technology

1991-08-01

typical flight conditions for which high load factor maneuver data was gathered. The range of dynamic pressure, 350 psf to 500 psf, for the 12...because PED systems do not outgas and are stable with respect to environmental temperature variations. In addition PED system performance is easily...0 (15) Equation (15) is rearranged to give: X2H2i + X3H21 + XAHAl + XsH5l + + + Xl+X2 + X3+X< + Xs X2 Y + X3(H2 + ^)+ XA(H2 + H3 + ^-) + XS (H2

LEDGF/p75 interacts with mRNA splicing factors and targets HIV-1 integration to highly spliced genes

PubMed Central

Singh, Parmit Kumar; Plumb, Matthew R.; Ferris, Andrea L.; Iben, James R.; Wu, Xiaolin; Fadel, Hind J.; Luke, Brian T.; Esnault, Caroline; Poeschla, Eric M.; Hughes, Stephen H.; Kvaratskhelia, Mamuka; Levin, Henry L.

2015-01-01

The host chromatin-binding factor LEDGF/p75 interacts with HIV-1 integrase and directs integration to active transcription units. To understand how LEDGF/p75 recognizes transcription units, we sequenced 1 million HIV-1 integration sites isolated from cultured HEK293T cells. Analysis of integration sites showed that cancer genes were preferentially targeted, raising concerns about using lentivirus vectors for gene therapy. Additional analysis led to the discovery that introns and alternative splicing contributed significantly to integration site selection. These correlations were independent of transcription levels, size of transcription units, and length of the introns. Multivariate analysis with five parameters previously found to predict integration sites showed that intron density is the strongest predictor of integration density in transcription units. Analysis of previously published HIV-1 integration site data showed that integration density in transcription units in mouse embryonic fibroblasts also correlated strongly with intron number, and this correlation was absent in cells lacking LEDGF. Affinity purification showed that LEDGF/p75 is associated with a number of splicing factors, and RNA sequencing (RNA-seq) analysis of HEK293T cells lacking LEDGF/p75 or the LEDGF/p75 integrase-binding domain (IBD) showed that LEDGF/p75 contributes to splicing patterns in half of the transcription units that have alternative isoforms. Thus, LEDGF/p75 interacts with splicing factors, contributes to exon choice, and directs HIV-1 integration to transcription units that are highly spliced. PMID:26545813

Rhythmic Behavior Is Controlled by the SRm160 Splicing Factor in Drosophila melanogaster.

PubMed

Beckwith, Esteban J; Hernando, Carlos E; Polcowñuk, Sofía; Bertolin, Agustina P; Mancini, Estefania; Ceriani, M Fernanda; Yanovsky, Marcelo J

2017-10-01

Circadian clocks organize the metabolism, physiology, and behavior of organisms throughout the day-night cycle by controlling daily rhythms in gene expression at the transcriptional and post-transcriptional levels. While many transcription factors underlying circadian oscillations are known, the splicing factors that modulate these rhythms remain largely unexplored. A genome-wide assessment of the alterations of gene expression in a null mutant of the alternative splicing regulator SR-related matrix protein of 160 kDa (SRm160) revealed the extent to which alternative splicing impacts on behavior-related genes. We show that SRm160 affects gene expression in pacemaker neurons of the Drosophila brain to ensure proper oscillations of the molecular clock. A reduced level of SRm160 in adult pacemaker neurons impairs circadian rhythms in locomotor behavior, and this phenotype is caused, at least in part, by a marked reduction in period ( per ) levels. Moreover, rhythmic accumulation of the neuropeptide PIGMENT DISPERSING FACTOR in the dorsal projections of these neurons is abolished after SRm160 depletion. The lack of rhythmicity in SRm160-downregulated flies is reversed by a fully spliced per construct, but not by an extra copy of the endogenous locus, showing that SRm160 positively regulates per levels in a splicing-dependent manner. Our findings highlight the significant effect of alternative splicing on the nervous system and particularly on brain function in an in vivo model. Copyright © 2017 by the Genetics Society of America.

Protein kinase WNK3 regulates the neuronal splicing factor Fox-1.

PubMed

Lee, A-Young; Chen, Wei; Stippec, Steve; Self, Jon; Yang, Fan; Ding, Xiaojun; Chen, She; Juang, Yu-Chi; Cobb, Melanie H

2012-10-16

We report an action of the protein kinase WNK3 on the neuronal mRNA splicing factor Fox-1. Fox-1 splices mRNAs encoding proteins important in synaptic transmission and membrane excitation. WNK3, implicated in the control of neuronal excitability through actions on ion transport, binds Fox-1 and inhibits its splicing activity in a kinase activity-dependent manner. Phosphorylation of Fox-1 by WNK3 does not change its RNA binding capacity; instead, WNK3 increases the cytoplasmic localization of Fox-1, thereby suppressing Fox-1-dependent splicing. These findings demonstrate a role of WNK3 in RNA processing. Considering the implication of WNK3 and Fox-1 in disorders of neuronal development such as autism, WNK3 may offer a target for treatment of Fox-1-induced disease.

Splicing predictions reliably classify different types of alternative splicing

PubMed Central

Busch, Anke; Hertel, Klemens J.

2015-01-01

Alternative splicing is a key player in the creation of complex mammalian transcriptomes and its misregulation is associated with many human diseases. Multiple mRNA isoforms are generated from most human genes, a process mediated by the interplay of various RNA signature elements and trans-acting factors that guide spliceosomal assembly and intron removal. Here, we introduce a splicing predictor that evaluates hundreds of RNA features simultaneously to successfully differentiate between exons that are constitutively spliced, exons that undergo alternative 5′ or 3′ splice-site selection, and alternative cassette-type exons. Surprisingly, the splicing predictor did not feature strong discriminatory contributions from binding sites for known splicing regulators. Rather, the ability of an exon to be involved in one or multiple types of alternative splicing is dictated by its immediate sequence context, mainly driven by the identity of the exon's splice sites, the conservation around them, and its exon/intron architecture. Thus, the splicing behavior of human exons can be reliably predicted based on basic RNA sequence elements. PMID:25805853

Boric acid reversibly inhibits the second step of pre-mRNA splicing.

PubMed

Shomron, Noam; Ast, Gil

2003-09-25

Several approaches have been used to identify the factors involved in mRNA splicing. None of them, however, comprises a straightforward reversible method for inhibiting the second step of splicing using an external reagent other than a chelator. This investigation demonstrates that the addition of boric acid to an in vitro pre-mRNA splicing reaction causes a dose-dependent reversible inhibition effect on the second step of splicing. The mechanism of action does not involve chelation of several metal ions; hindrance of 3' splice-site; or binding to hSlu7. This study presents a novel method for specific reversible inhibition of the second step of pre-mRNA splicing.

A mammalian germ cell-specific RNA-binding protein interacts with ubiquitously expressed proteins involved in splice site selection

NASA Astrophysics Data System (ADS)

Elliott, David J.; Bourgeois, Cyril F.; Klink, Albrecht; Stévenin, James; Cooke, Howard J.

2000-05-01

RNA-binding motif (RBM) genes are found on all mammalian Y chromosomes and are implicated in spermatogenesis. Within human germ cells, RBM protein shows a similar nuclear distribution to components of the pre-mRNA splicing machinery. To address the function of RBM, we have used protein-protein interaction assays to test for possible physical interactions between these proteins. We find that RBM protein directly interacts with members of the SR family of splicing factors and, in addition, strongly interacts with itself. We have mapped the protein domains responsible for mediating these interactions and expressed the mouse RBM interaction region as a bacterial fusion protein. This fusion protein can pull-down several functionally active SR protein species from cell extracts. Depletion and add-back experiments indicate that these SR proteins are the only splicing factors bound by RBM which are required for the splicing of a panel of pre-mRNAs. Our results suggest that RBM protein is an evolutionarily conserved mammalian splicing regulator which operates as a germ cell-specific cofactor for more ubiquitously expressed pre-mRNA splicing activators.

Radiographic and Clinical Outcomes of Robot-Assisted Posterior Pedicle Screw Fixation: Two-Year Results from a Randomized Controlled Trial.

PubMed

Park, Sang Min; Kim, Ho Joong; Lee, Se Yeon; Chang, Bong Soon; Lee, Choon Ki; Yeom, Jin S

2018-05-01

We prospectively assessed the early radiographic and clinical outcomes (minimum follow-up of 2 years) of robot-assisted pedicle screw fixation (Robot-PSF) and conventional freehand pedicle screw fixation (Conv-PSF). Patients were randomly assigned to Robot-PSF (37 patients) or Conv-PSF (41 patients) for posterior interbody fusion surgery. The Robot-PSF group underwent minimally invasive pedicle screw fixation using a pre-planned robot-guided screw trajectory. The Conv-PSF underwent screw fixation using the freehand technique. Radiographic adjacent segment degeneration (ASD) was measured on plain radiographs, and clinical outcomes were measured using visual analogue scale (VAS) and Oswestry disability index (ODI) scores regularly after surgery. The two groups had similar values for radiographic ASD, including University California at Los Angeles grade, vertebral translation, angular motion, and loss of disc height (p=0.320). At final follow-up, both groups had experienced significant improvements in back VAS, leg VAS, and ODI scores after surgery (p<0.001), although inter-group differences were not significant for back VAS (p=0.876), leg VAS (p=0.429), and ODI scores (p=0.952). In the Conv-PSF group, revision surgery was required for two of the 25 patients (8%), compared to no patients in the Robot-PSF group. There were no significant differences in radiographic ASD and clinical outcomes between Robot-PSF and Conv-PSF. Thus, the advantages of robot-assisted surgery (accurate pedicle screw insertion and minimal facet joint violation) do not appear to be clinically significant. © Copyright: Yonsei University College of Medicine 2018.

Induction of HoxB Transcription by Retinoic Acid Requires Actin Polymerization

PubMed Central

Ferrai, Carmelo; Naum-Onganía, Gabriela; Longobardi, Elena; Palazzolo, Martina; Disanza, Andrea; Diaz, Victor M.; Crippa, Massimo P.; Scita, Giorgio

2009-01-01

We have analyzed the role of actin polymerization in retinoic acid (RA)-induced HoxB transcription, which is mediated by the HoxB regulator Prep1. RA induction of the HoxB genes can be prevented by the inhibition of actin polymerization. Importantly, inhibition of actin polymerization specifically affects the transcription of inducible Hox genes, but not that of their transcriptional regulators, the RARs, nor of constitutively expressed, nor of actively transcribed Hox genes. RA treatment induces the recruitment to the HoxB2 gene enhancer of a complex composed of “elongating” RNAPII, Prep1, β-actin, and N-WASP as well as the accessory splicing components p54Nrb and PSF. We show that inhibition of actin polymerization prevents such recruitment. We conclude that inducible Hox genes are selectively sensitive to the inhibition of actin polymerization and that actin polymerization is required for the assembly of a transcription complex on the regulatory region of the Hox genes. PMID:19477923

Induction of HoxB transcription by retinoic acid requires actin polymerization.

PubMed

Ferrai, Carmelo; Naum-Onganía, Gabriela; Longobardi, Elena; Palazzolo, Martina; Disanza, Andrea; Diaz, Victor M; Crippa, Massimo P; Scita, Giorgio; Blasi, Francesco

2009-08-01

We have analyzed the role of actin polymerization in retinoic acid (RA)-induced HoxB transcription, which is mediated by the HoxB regulator Prep1. RA induction of the HoxB genes can be prevented by the inhibition of actin polymerization. Importantly, inhibition of actin polymerization specifically affects the transcription of inducible Hox genes, but not that of their transcriptional regulators, the RARs, nor of constitutively expressed, nor of actively transcribed Hox genes. RA treatment induces the recruitment to the HoxB2 gene enhancer of a complex composed of "elongating" RNAPII, Prep1, beta-actin, and N-WASP as well as the accessory splicing components p54Nrb and PSF. We show that inhibition of actin polymerization prevents such recruitment. We conclude that inducible Hox genes are selectively sensitive to the inhibition of actin polymerization and that actin polymerization is required for the assembly of a transcription complex on the regulatory region of the Hox genes.

Analytical model for effect of temperature variation on PSF consistency in wavefront coding infrared imaging system

NASA Astrophysics Data System (ADS)

Feng, Bin; Shi, Zelin; Zhang, Chengshuo; Xu, Baoshu; Zhang, Xiaodong

2016-05-01

The point spread function (PSF) inconsistency caused by temperature variation leads to artifacts in decoded images of a wavefront coding infrared imaging system. Therefore, this paper proposes an analytical model for the effect of temperature variation on the PSF consistency. In the proposed model, a formula for the thermal deformation of an optical phase mask is derived. This formula indicates that a cubic optical phase mask (CPM) is still cubic after thermal deformation. A proposed equivalent cubic phase mask (E-CPM) is a virtual and room-temperature lens which characterizes the optical effect of temperature variation on the CPM. Additionally, a calculating method for PSF consistency after temperature variation is presented. Numerical simulation illustrates the validity of the proposed model and some significant conclusions are drawn. Given the form parameter, the PSF consistency achieved by a Ge-material CPM is better than the PSF consistency by a ZnSe-material CPM. The effect of the optical phase mask on PSF inconsistency is much slighter than that of the auxiliary lens group. A large form parameter of the CPM will introduce large defocus-insensitive aberrations, which improves the PSF consistency but degrades the room-temperature MTF.

Persufflation (or gaseous oxygen perfusion) as a method of organ preservation.

PubMed

Suszynski, Thomas M; Rizzari, Michael D; Scott, William E; Tempelman, Linda A; Taylor, Michael J; Papas, Klearchos K

2012-06-01

Improved preservation techniques have the potential to improve transplant outcomes by better maintaining donor organ quality and by making more organs available for allotransplantation. Persufflation, (PSF, gaseous oxygen perfusion) is potentially one such technique that has been studied for over a century in a variety of tissues, but has yet to gain wide acceptance for a number of reasons. A principal barrier is the perception that ex vivo PSF will cause in vivo embolization post-transplant. This review summarizes the extensive published work on heart, liver, kidney, small intestine and pancreas PSF, discusses the differences between anterograde and retrograde PSF, and between PSF and other conventional methods of organ preservation (static cold storage, hypothermic machine perfusion). Prospective implications of PSF within the broader field of organ transplantation, and in the specific application with pancreatic islet isolation and transplant are also discussed. Finally, key issues that need to be addressed before PSF becomes a more widely utilized preservation strategy are summarized and discussed. Copyright © 2012 Elsevier Inc. All rights reserved.

Persufflation (or Gaseous Oxygen Perfusion) as a Method of Organ Preservation

PubMed Central

Suszynski, Thomas M.; Rizzari, Michael D.; Scott, William E.; Tempelman, Linda A.; Taylor, Michael J.; Papas, Klearchos K.

2012-01-01

Improved preservation techniques have the potential to improve transplant outcomes by better maintaining donor organ quality and by making more organs available for allotransplantation. Persufflation, (PSF, gaseous oxygen perfusion) is potentially one such technique that has been studied for over a century in a variety of tissues, but has yet to gain wide acceptance for a number of reasons. A principal barrier is the perception that ex vivo PSF will cause in vivo embolization post-transplant. This review summarizes the extensive published work on heart, liver, kidney, small intestine and pancreas PSF, discusses the differences between anterograde and retrograde PSF and between PSF and other conventional methods of organ preservation (static cold storage, hypothermic machine perfusion). Prospective implications of PSF within the broader field of organ transplantation, and in the specific application with pancreatic islet isolation and transplant are also discussed. Finally, key issues that need to be addressed before PSF becomes a more widely utilized preservation strategy are summarized and discussed. PMID:22301419

Global regulation of alternative RNA splicing by the SR-rich protein RBM39.

PubMed

Mai, Sanyue; Qu, Xiuhua; Li, Ping; Ma, Qingjun; Cao, Cheng; Liu, Xuan

2016-08-01

RBM39 is a serine/arginine-rich RNA-binding protein that is highly homologous to the splicing factor U2AF65. However, the role of RBM39 in alternative splicing is poorly understood. In this study, RBM39-mediated global alternative splicing was investigated using RNA-Seq and genome-wide RBM39-RNA interactions were mapped via cross-linking and immunoprecipitation coupled with deep sequencing (CLIP-Seq) in wild-type and RBM39-knockdown MCF-7 cells. RBM39 was involved in the up- or down-regulation of the transcript levels of various genes. Hundreds of alternative splicing events regulated by endogenous RBM39 were identified. The majority of these events were cassette exons. Genes containing RBM39-regulated alternative exons were found to be linked to G2/M transition, cellular response to DNA damage, adherens junctions and endocytosis. CLIP-Seq analysis showed that the binding site of RBM39 was mainly in proximity to 5' and 3' splicing sites. Considerable RBM39 binding to mRNAs encoding proteins involved in translation was observed. Of particular importance, ~20% of the alternative splicing events that were significantly regulated by RBM39 were similarly regulated by U2AF65. RBM39 is extensively involved in alternative splicing of RNA and helps regulate transcript levels. RBM39 may modulate alternative splicing similarly to U2AF65 by either directly binding to RNA or recruiting other splicing factors, such as U2AF65. The current study offers a genome-wide view of RBM39's regulatory function in alternative splicing. RBM39 may play important roles in multiple cellular processes by regulating both alternative splicing of RNA molecules and transcript levels. Copyright © 2016 Elsevier B.V. All rights reserved.

Interplay between estrogen receptor and AKT in Estradiol-induced alternative splicing

PubMed Central

2013-01-01

Background Alternative splicing is critical for generating complex proteomes in response to extracellular signals. Nuclear receptors including estrogen receptor alpha (ERα) and their ligands promote alternative splicing. The endogenous targets of ERα:estradiol (E2)-mediated alternative splicing and the influence of extracellular kinases that phosphorylate ERα on E2-induced splicing are unknown. Methods MCF-7 and its anti-estrogen derivatives were used for the majority of the assays. CD44 mini gene was used to measure the effect of E2 and AKT on alternative splicing. ExonHit array analysis was performed to identify E2 and AKT-regulated endogenous alternatively spliced apoptosis-related genes. Quantitative reverse transcription polymerase chain reaction was performed to verify alternative splicing. ERα binding to alternatively spliced genes was verified by chromatin immunoprecipitation assay. Bromodeoxyuridine incorporation-ELISA and Annexin V labeling assays were done to measure cell proliferation and apoptosis, respectively. Results We identified the targets of E2-induced alternative splicing and deconstructed some of the mechanisms surrounding E2-induced splicing by combining splice array with ERα cistrome and gene expression array. E2-induced alternatively spliced genes fall into at least two subgroups: coupled to E2-regulated transcription and ERα binding to the gene without an effect on rate of transcription. Further, AKT, which phosphorylates both ERα and splicing factors, influenced ERα:E2 dependent splicing in a gene-specific manner. Genes that are alternatively spliced include FAS/CD95, FGFR2, and AXIN-1. E2 increased the expression of FGFR2 C1 isoform but reduced C3 isoform at mRNA level. E2-induced alternative splicing of FAS and FGFR2 in MCF-7 cells correlated with resistance to FAS activation-induced apoptosis and response to keratinocyte growth factor (KGF), respectively. Resistance of MCF-7 breast cancer cells to the anti-estrogen tamoxifen was associated with ERα-dependent overexpression of FGFR2, whereas resistance to fulvestrant was associated with ERα-dependent isoform switching, which correlated with altered response to KGF. Conclusion E2 may partly alter cellular proteome through alternative splicing uncoupled to its effects on transcription initiation and aberration in E2-induced alternative splicing events may influence response to anti-estrogens. PMID:23758675

U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor of 65 kDa, U2AF65, can promote U1 snRNP recruitment to 5' splice sites.

PubMed Central

Förch, Patrik; Merendino, Livia; Martínez, Concepción; Valcárcel, Juan

2003-01-01

The splicing factor U2AF(65), U2 small nuclear ribonucleoprotein particle (snRNP) auxillary factor of 65 kDa, binds to pyrimidine-rich sequences at 3' splice sites to recruit U2 snRNP to pre-mRNAs. We report that U2AF(65) can also promote the recruitment of U1 snRNP to weak 5' splice sites that are followed by uridine-rich sequences. The arginine- and serine-rich domain of U2AF(65) is critical for U1 recruitment, and we discuss the role of its RNA-RNA annealing activity in this novel function of U2AF(65). PMID:12558503

Diversification of the muscle proteome through alternative splicing.

PubMed

Nakka, Kiran; Ghigna, Claudia; Gabellini, Davide; Dilworth, F Jeffrey

2018-03-06

Skeletal muscles express a highly specialized proteome that allows the metabolism of energy sources to mediate myofiber contraction. This muscle-specific proteome is partially derived through the muscle-specific transcription of a subset of genes. Surprisingly, RNA sequencing technologies have also revealed a significant role for muscle-specific alternative splicing in generating protein isoforms that give specialized function to the muscle proteome. In this review, we discuss the current knowledge with respect to the mechanisms that allow pre-mRNA transcripts to undergo muscle-specific alternative splicing while identifying some of the key trans-acting splicing factors essential to the process. The importance of specific splicing events to specialized muscle function is presented along with examples in which dysregulated splicing contributes to myopathies. Though there is now an appreciation that alternative splicing is a major contributor to proteome diversification, the emergence of improved "targeted" proteomic methodologies for detection of specific protein isoforms will soon allow us to better appreciate the extent to which alternative splicing modifies the activity of proteins (and their ability to interact with other proteins) in the skeletal muscle. In addition, we highlight a continued need to better explore the signaling pathways that contribute to the temporal control of trans-acting splicing factor activity to ensure specific protein isoforms are expressed in the proper cellular context. An understanding of the signal-dependent and signal-independent events driving muscle-specific alternative splicing has the potential to provide us with novel therapeutic strategies to treat different myopathies.

Attenuation of the suppressive activity of cellular splicing factor SRSF3 by Kaposi sarcoma–associated herpesvirus ORF57 protein is required for RNA splicing

PubMed Central

Majerciak, Vladimir; Lu, Mathew; Li, Xiaofan

2014-01-01

Kaposi sarcoma–associated herpesvirus (KSHV) ORF57 is a multifunctional post-transcriptional regulator essential for viral gene expression during KSHV lytic infection. ORF57 requires interactions with various cellular proteins for its function. Here, we identified serine/arginine-rich splicing factor 3 (SRSF3, formerly known as SRp20) as a cellular cofactor involved in ORF57-mediated splicing of KSHV K8β RNA. In the absence of ORF57, SRSF3 binds to a suboptimal K8β intron and inhibits K8β splicing. Knockdown of SRSF3 promotes K8β splicing, mimicking the effect of ORF57. The N-terminal half of ORF57 binds to the RNA recognition motif of SRSF3, which prevents SRSF3 from associating with the K8β intron RNA and therefore attenuates the suppressive effect of SRSF3 on K8β splicing. ORF57 also promotes splicing of heterologous non-KSHV transcripts that are negatively regulated by SRSF3, indicating that the effect of ORF57 on SRSF3 activity is independent of RNA target. SPEN proteins, previously identified as ORF57-interacting partners, suppress ORF57 splicing activity by displacing ORF57 from SRSF3–RNA complexes. In summary, we have identified modulation of SRSF3 activity as the molecular mechanism by which ORF57 promotes RNA splicing. PMID:25234929

Attenuation of the suppressive activity of cellular splicing factor SRSF3 by Kaposi sarcoma-associated herpesvirus ORF57 protein is required for RNA splicing.

PubMed

Majerciak, Vladimir; Lu, Mathew; Li, Xiaofan; Zheng, Zhi-Ming

2014-11-01

Kaposi sarcoma-associated herpesvirus (KSHV) ORF57 is a multifunctional post-transcriptional regulator essential for viral gene expression during KSHV lytic infection. ORF57 requires interactions with various cellular proteins for its function. Here, we identified serine/arginine-rich splicing factor 3 (SRSF3, formerly known as SRp20) as a cellular cofactor involved in ORF57-mediated splicing of KSHV K8β RNA. In the absence of ORF57, SRSF3 binds to a suboptimal K8β intron and inhibits K8β splicing. Knockdown of SRSF3 promotes K8β splicing, mimicking the effect of ORF57. The N-terminal half of ORF57 binds to the RNA recognition motif of SRSF3, which prevents SRSF3 from associating with the K8β intron RNA and therefore attenuates the suppressive effect of SRSF3 on K8β splicing. ORF57 also promotes splicing of heterologous non-KSHV transcripts that are negatively regulated by SRSF3, indicating that the effect of ORF57 on SRSF3 activity is independent of RNA target. SPEN proteins, previously identified as ORF57-interacting partners, suppress ORF57 splicing activity by displacing ORF57 from SRSF3-RNA complexes. In summary, we have identified modulation of SRSF3 activity as the molecular mechanism by which ORF57 promotes RNA splicing. Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Functional analysis of U1-70K interacting SR proteins in pre-mRNA splicing in Arabidopsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

A.S.N. Reddy

Proteins of a serine/arginine-rich (SR) family are part of the spliceosome and are implicated in both constitutive and alternative splicing of pre-mRNAs. With the funding from DOE we have been studying alternative of splicing of genes encoding serine/arginine-rich (SR) proteins and the roles of SR proteins that interact with U1-70K in regulating basic and alternative splicing. Alternative splicing of pre-mRNAs of Arabidopsis serine/arginine-rich proteins and its regulation by hormones and stresses: We analyzed the splicing of all 19 Arabidopsis genes in different tissues, during different seedling stages and in response to various hormonal and stress treatments. Remarkably, about 90 differentmore » transcripts are produced from 15 SR genes, thereby increasing the transcriptome complexity of SR genes by about five fold. Using the RNA isolated from polysomes we have shown that most of the splice variants are recruited for translation. Alternative splicing of some SR genes is controlled in a developmental and tissue-specific manner (Palusa et al., 2007). Interestingly, among the various hormones and abiotic stresses tested, temperature stress (cold and heat) and ultraviolet light dramatically altered alternative splicing of pre-mRNAs of several SR genes whereas hormones altered the splicing of only two SR genes (Palusa et al., 2007). Localization and dynamics of a novel serine/arginine-rich protein that interacts with U1-70K: We analyzed the intranuclear movement of SR45 fused to GFP by fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). We demonstrate that the movement of GFP-SR45 is ATP-dependent. Interestingly, inhibition of transcription or phosphorylation slowed the mobility of GFP-SR45 (Ali et al., 2006). Our studies have revealed that the nuclear localization signals are located in arg/ser-rich domains (RS) 1 and 2, whereas the speckle targeting signals are exclusively present in RS2 (Ali et al., 2006). The regulation of SR45 mobility by ATP and a transcriptional inhibitor is in contrast to the mobility of SR family splicing factors in animals and suggests fundamental differences in the movement of plant and animals splicing factors. In vivo interaction of U170K with SR45: To analyze the interaction of U170K with SR45, we expressed these proteins fused to RFP and GFP respectively, in protoplasts. Both the reporters co-localized to the same subnuclear domains. To determine direct interaction of these proteins, we fused full-length U170K to one part of split YFP and full-length or truncated version of SR45 to the second half of split YFP. Coexpession of these split YFP constructs resulted in reconstitution of YFP in speckles, suggesting direction interaction of these proteins in vivo (Ali et al., 2008). SR45 is a Novel Plant-Specific Splicing Factor and is Involved in Regulating Multiple Developmental Processes: Using an in vitro splicing complementation assay, we showed that SR45 is an essential splicing factor. The sr45-1 mutant exhibited a number of developmental abnormalities. Further analysis of flowering time has shown that the autonomous pathway of flowering is affected in the mutant. Expression analysis of several flowering genes has revealed that FLC, a key flowering repressor, is up-regulated in the SR45 mutant. Further, alternative splicing pattern of several other SR genes was altered in the sr45-1 mutant in a tissue-specific manner. Hence, the observed pleiotropic effects on various aspects of development are likely due to altered level of SR protein isoforms, which in turn regulate the splicing of other pre-mRNAs. Expression of wild-type SR45 in the mutant complemented the phenotypic defects and changes in alternative splicing of SR genes. SR45 thus is a novel plant-specific splicing factor and plays a crucial role in multiple developmental processes.« less

Removing the Impact of Correlated PSF Uncertainties in Weak Lensing

NASA Astrophysics Data System (ADS)

Lu, Tianhuan; Zhang, Jun; Dong, Fuyu; Li, Yingke; Liu, Dezi; Fu, Liping; Li, Guoliang; Fan, Zuhui

2018-05-01

Accurate reconstruction of the spatial distributions of the point-spread function (PSF) is crucial for high precision cosmic shear measurements. Nevertheless, current methods are not good at recovering the PSF fluctuations of high spatial frequencies. In general, the residual PSF fluctuations are spatially correlated, and therefore can significantly contaminate the correlation functions of the weak lensing signals. We propose a method to correct for this contamination statistically, without any assumptions on the PSF and galaxy morphologies or their spatial distribution. We demonstrate our idea with the data from the W2 field of CFHTLenS.

PKC-Theta is a Novel SC35 Splicing Factor Regulator in Response to T Cell Activation.

PubMed

McCuaig, Robert Duncan; Dunn, Jennifer; Li, Jasmine; Masch, Antonia; Knaute, Tobias; Schutkowski, Mike; Zerweck, Johannes; Rao, Sudha

2015-01-01

Alternative splicing of nuclear pre-mRNA is essential for generating protein diversity and regulating gene expression. While many immunologically relevant genes undergo alternative splicing, the role of regulated splicing in T cell immune responses is largely unexplored, and the signaling pathways and splicing factors that regulate alternative splicing in T cells are poorly defined. Here, we show using a combination of Jurkat T cells, human primary T cells, and ex vivo naïve and effector virus-specific T cells isolated after influenza A virus infection that SC35 phosphorylation is induced in response to stimulatory signals. We show that SC35 colocalizes with RNA polymerase II in activated T cells and spatially overlaps with H3K27ac and H3K4me3, which mark transcriptionally active genes. Interestingly, SC35 remains coupled to the active histone marks in the absence of continuing stimulatory signals. We show for the first time that nuclear PKC-θ co-exists with SC35 in the context of the chromatin template and is a key regulator of SC35 in T cells, directly phosphorylating SC35 peptide residues at RNA recognition motif and RS domains. Collectively, our findings suggest that nuclear PKC-θ is a novel regulator of the key splicing factor SC35 in T cells.

Preparation, characterisation and critical flux determination of graphene oxide blended polysulfone (PSf) membranes in an MBR system.

PubMed

Ravishankar, Harish; Roddick, Felicity; Navaratna, Dimuth; Jegatheesan, Veeriah

2018-05-01

Microfiltration membranes having different blends of graphene-oxide (GO) (0-1 wt%) and Polysulfone (PSf) (15-20 wt%) were prepared using the classical non-solvent induced phase inversion process. The prepared membranes were characterised for their structural morphology, surface properties, mechanical strength, porosity and pure water flux. Based on the initial characterisation results, four membranes (15 wt% PSf, 15 wt% PSf + 0.25 wt% GO, 15 wt% PSf + 1 wt% GO and 20 wt% PSf + 1 wt% GO) were chosen for critical flux study, that was conducted using flux-step method in a lab scale MBR system. In order to study the application potential of GO blended membranes, the critical flux of each membrane was evaluated in two operational modes i.e., continuous and intermittent modes with backwash. The membranes with maximal GO concentration (15 wt% PSf + 1 wt% GO and 20 wt% PSf + 1 wt% GO) showed higher critical flux (16.5, 12.8 L/m 2 h and 19, 15 L/m 2 h for continuous and intermittent mode, respectively). It was observed that the operational modes did not have a significant effect on the critical flux of the membranes with low GO concentration (15 wt% PSf and 15 wt% PSf + 0.25 wt% GO), indicating a minimal of 1 wt% GO was required for an observable effect that favoured intermittent mode of operation. Through these results, ideal operating condition was arrived (i.e., flux maintained at 6.4 L/m 2 h operated under intermittent mode) and the membranes 15 wt% PSf and 15 wt% PSf + 1 wt% GO were studied for their long-term operation. The positive effect of GO on filtration time, cleaning frequency and against fouling was demonstrated through long term TMP profile of the membranes, indicating the suitability of GO blended membrane for real time wastewater treatment. Copyright © 2018 Elsevier Ltd. All rights reserved.

Synthesis and luminescence properties of polymer-rare earth complexes containing salicylaldehyde-type bidentate Schiff base ligand.

PubMed

Zhang, Dandan; Gao, Baojiao; Li, Yanbin

2017-08-01

Using molecular design and polymer reactions, two types of bidentate Schiff base ligands, salicylaldehyde-aniline (SAN) and salicylaldehyde-cyclohexylamine (SCA), were synchronously synthesized and bonded onto the side chain of polysulfone (PSF), giving two bidentate Schiff base ligand-functionalized PSFs, PSF-SAN and PSF-SCA, referred to as macromolecular ligands. Following coordination reactions between the macromolecular ligands and Eu(III) and Tb(III) ions (the reaction occurred between the bonded ligands SAN or SCA and the lanthanide ion), two series of luminescent polymer-rare earth complexes, PSF-SAN-Eu(III) and PSF-SCA-Tb(III), were obtained. The two macromolecular ligands were fully characterized by Fourier transform infrared (FTIR), 1 H NMR and UV absorption spectroscopy, and the prepared complexes were also characterized by FTIR, UV absorption spectroscopy and thermo-gravity analysis. On this basis, the photoluminescence properties of these complexes and the relationships between their structure and luminescence were investigated in depth. The results show that the bonded bidentate Schiff base ligands, SAN and SCA, can effectively sensitize the fluorescence emission of Eu(III) and Tb(III) ions, respectively. PSF-SAN-Eu(III) series complexes, namely the binary complex PSF-(SAN) 3 -Eu(III) and the ternary complex PSF-(SAN) 3 -Eu(III)-(Phen) 1 (Phen is the small-molecule ligand 1,10-phenanthroline), produce strong red luminescence, suggesting that the triplet state energy level of SAN is lower and well matched with the resonant energy level of the Eu(III) ion. By contrast, PSF-SAN-Eu(III) series complexes, namely the binary complex PSF-(SCA) 3 -Tb(III) and the ternary complex PSF-(SCA) 3 -Tb(III)-(Phen) 1 , display strong green luminescence, suggesting that the triplet state energy level of SCA is higher and is well matched with the resonant energy level of Tb(III). Copyright © 2017 John Wiley & Sons, Ltd.

Exploiting differential RNA splicing patterns: a potential new group of therapeutic targets in cancer.

PubMed

Jyotsana, Nidhi; Heuser, Michael

2018-02-01

Mutations in genes associated with splicing have been found in hematologic malignancies, but also in solid cancers. Aberrant cancer specific RNA splicing either results from mutations or misexpression of the spliceosome genes directly, or from mutations in splice sites of oncogenes or tumor suppressors. Areas covered: In this review, we present molecular targets of aberrant splicing in various malignancies, information on existing and emerging therapeutics against such targets, and strategies for future drug development. Expert opinion: Alternative splicing is an important mechanism that controls gene expression, and hence pharmacologic and genetic control of aberrant alternative RNA splicing has been proposed as a potential therapy in cancer. To identify and validate aberrant RNA splicing patterns as therapeutic targets we need to (1) characterize the most common genetic aberrations of the spliceosome and of splice sites, (2) understand the dysregulated downstream pathways and (3) exploit in-vivo disease models of aberrant splicing. Antisense oligonucleotides show promising activity, but will benefit from improved delivery tools. Inhibitors of mutated splicing factors require improved specificity, as alternative and aberrant splicing are often intertwined like two sides of the same coin. In summary, targeting aberrant splicing is an early but emerging field in cancer treatment.

Pathological mechanisms of left main stent failure.

PubMed

Mori, Hiroyoshi; Torii, Sho; Harari, Emanuel; Jinnouchi, Hiroyuki; Brauman, Ryan; Smith, Samantha; Kutys, Robert; Fowler, David; Romero, Maria; Virmani, Renu; Finn, Aloke V

2018-07-15

Despite the increasing use of left main (LM) percutaneous coronary intervention (LM-PCI), there have been no pathological studies devoted to understanding the causes of LM stent failure. We aimed to systematically determine the pathological mechanisms of LM stent failure. From the CVPath Stent registry, a total of 46 lesions were identified to have LM-PCI. Pathologic stent failure (PSF) was defined as stent thrombosis, restenosis and in-stent chronic total occlusion (CTO). Failed and patent LM stented lesions were pathologically assessed to determine predictors of PSF. Malapposition and uncovered struts were numerically greater in the LM ostium, body, and bifurcation while neointimal thickness was relatively greater in bifurcation and proximal circumflex. In this study cohort, half of the lesions (n = 23) showed PSF. Stent thrombosis (ST, n = 18) was the major mode of PSF followed by in-stent CTO (n = 4) and restenosis (n = 1). Failed lesions showed significantly greater prevalence of malapposition >20% of struts/section (65% vs. 13%, P < 0.01), stent struts crossing an ostial side branch >30% of the circumference (48% vs. 13%, P < 0.01) and uncovered struts >30% (57% vs. 18%, P = 0.03). In multivariate analysis, the prevalence of malapposition >20% was the strongest risk factor for PSF (Odds ratio 8.0, 95% confidence interval 1.8-45.4, P < 0.01) followed by struts crossing an ostial side branch >30% (Odds ratio 4.2, 95% confidence interval 0.8-24.7, P = 0.09). Our data demonstrate the main pathological predictors for LM stent failure are malapposition and struts crossing an ostial side branch and suggest that imaging-guided PCI may be important. Copyright © 2018 Elsevier B.V. All rights reserved.

Two-dimensional PSF prediction of multiple-reflection optical systems with rough surfaces

NASA Astrophysics Data System (ADS)

Tayabaly, Kashmira; Spiga, Daniele; Sironi, Giorgia; Pareschi, Giovani; Lavagna, Michele

2016-09-01

The focusing accuracy in reflective optical systems, usually expressed in terms of the Point Spread Function (PSF) is chiefly determined by two factors: the deviation of the mirror shape from the nominal design and the surface finishing. While the effects of the former are usually well described by the geometrical optics, the latter is diffractive/interferential in nature and determined by a distribution of defects that cover several decades in the lateral scale (from a few millimeters to a few microns). Clearly, reducing the level of scattered light is crucial to improve the focusing of the collected radiation, particularly for astronomical telescopes that aim to detect faint light signals from our Universe. Telescopes are typically arranged in multiple reflections configuration and the behavior of the multiply-scattered radiation becomes difficult to predict and control. Also it is difficult to disentangle the effect of surface scattering from the PSF degradation caused by the shape deformation of the optical elements. This paper presents a simple and unifying method for evaluating the contribution of optical surfaces defects to the two-dimensional PSF of a multi-reflections system, regardless of the classification of a spectral range as "geometry" or "roughness". This method, entirely based on Huygens-Fresnel principle in the far-field approximation, was already applied in grazing-incidence X-ray mirrors and experimentally validated for a single reflection system, accounting for the real surface topography of the optics. In this work we show the extension of this formalism to a double reflection system and introducing real microroughness data. The formalism is applied to a MAGIC-I panel mirror that was fully characterized, allowing us to predict the PSF and the validation with real measurements of the double reflection ASTRI telescope, a prototype of CTA-SST telescope.

Monte Carlo investigation of backscatter point spread function for x-ray imaging examinations

NASA Astrophysics Data System (ADS)

Xiong, Zhenyu; Vijayan, Sarath; Rudin, Stephen; Bednarek, Daniel R.

2017-03-01

X-ray imaging examinations, especially complex interventions, may result in relatively high doses to the patient's skin inducing skin injuries. A method was developed to determine the skin-dose distribution for non-uniform x-ray beams by convolving the backscatter point-spread-function (PSF) with the primary-dose distribution to generate the backscatter distribution that, when added to the primary dose, gives the total-dose distribution. This technique was incorporated in the dose-tracking system (DTS), which provides a real-time color-coded 3D-mapping of skin dose during fluoroscopic procedures. The aim of this work is to investigate the variation of the backscatter PSF with different parameters. A backscatter PSF of a 1-mm x-ray beam was generated by EGSnrc Monte-Carlo code for different x-ray beam energies, different soft-tissue thickness above bone, different bone thickness and different entrance-beam angles, as well as for different locations on the SK-150 anthropomorphic head phantom. The results show a reduction of the peak scatter to primary dose ratio of 48% when X-ray beam voltage is increased from 40 keV to 120 keV. The backscatter dose was reduced when bone was beneath the soft tissue layer and this reduction increased with thinner soft tissue and thicker bone layers. The backscatter factor increased about 21% as the angle of incidence of the beam with the entrance surface decreased from 90° (perpendicular) to 30°. The backscatter PSF differed for different locations on the SK-150 phantom by up to 15%. The results of this study can be used to improve the accuracy of dose calculation when using PSF convolution in the DTS.

Structure and Function of the Two Tandem WW Domains of the Pre-mRNA Splicing Factor FBP21 (Formin-binding Protein 21)*

PubMed Central

Huang, Xiaojuan; Beullens, Monique; Zhang, Jiahai; Zhou, Yi; Nicolaescu, Emilia; Lesage, Bart; Hu, Qi; Wu, Jihui; Bollen, Mathieu; Shi, Yunyu

2009-01-01

Human FBP21 (formin-binding protein 21) contains a matrin-type zinc finger and two tandem WW domains. It is a component of the spliceosomes and interacts with several established splicing factors. Here we demonstrate for the first time that FBP21 is an activator of pre-mRNA splicing in vivo and that its splicing activation function and interaction with the splicing factor SIPP1 (splicing factor that interacts with PQBP1 and PP1) are both mediated by the two tandem WW domains of group III. We determined the solution structure of the tandem WW domains of FBP21 and found that the WW domains recognize peptide ligands containing either group II (PPLP) or group III (PPR) motifs. The binding interfaces involve both the XP and XP2 grooves of the two WW domains. Significantly, the tandem WW domains of FBP21 are connected by a highly flexible region, enabling their simultaneous interaction with two proline-rich motifs of SIPP1. The strong interaction between SIPP1 and FBP21 can be explained by the conjugation of two low affinity interactions with the tandem WW domains. Our study provides a structural basis for understanding the molecular mechanism underlying the functional implication of FBP21 and the biological specificity of tandem WW domains. PMID:19592703

Dynamic integration of splicing within gene regulatory pathways

PubMed Central

Braunschweig, Ulrich; Gueroussov, Serge; Plocik, Alex; Graveley, Brenton R.; Blencowe, Benjamin J.

2013-01-01

Precursor mRNA splicing is one of the most highly regulated processes in metazoan species. In addition to generating vast repertoires of RNAs and proteins, splicing has a profound impact on other gene regulatory layers, including mRNA transcription, turnover, transport and translation. Conversely, factors regulating chromatin and transcription complexes impact the splicing process. This extensive cross-talk between gene regulatory layers takes advantage of dynamic spatial, physical and temporal organizational properties of the cell nucleus, and further emphasizes the importance of developing a multidimensional understanding of splicing control. PMID:23498935

Polyoma virus small tumor antigen pre-mRNA splicing requires cooperation between two 3' splice sites.

PubMed Central

Ge, H; Noble, J; Colgan, J; Manley, J L

1990-01-01

We have studied splicing of the polyoma virus early region pre-mRNA in vitro. This RNA is alternatively spliced in vivo to produce mRNA encoding the large, middle-sized (MTAg), and small (StAg) tumor antigens. Our primary interest was to learn how the 48-nucleotide StAg intron is excised, because the length of this intron is significantly less than the apparent minimum established for mammalian introns. Although the products of all three splices are detected in vitro, characterization of the pathway and sequence requirements of StAg splicing suggests that splicing factors interact with the precursor RNA in an unexpected way to catalyze removal of this intron. Specifically, StAg splicing uses either of two lariat branch points, one of which is located only 4 nucleotides from the 3' splice site. Furthermore, the StAg splice absolutely requires that the alternative MTAg 3' splice site, located 14 nucleotides downstream of the StAg 3' splice site, be intact. Insertion mutations that increase or decrease the quality of the MTAg pyrimidine stretch enhance or repress StAg as well as MTAg splicing, and a single-base change in the MTAg AG splice acceptor totally blocks both splices. These results demonstrate the ability of two 3' splice sites to cooperate with each other to bring about removal of a single intron. Images PMID:2159146

Implications of a wavelength-dependent PSF for weak lensing measurements

NASA Astrophysics Data System (ADS)

Eriksen, Martin; Hoekstra, Henk

2018-07-01

The convolution of galaxy images by the point spread function (PSF) is the dominant source of bias for weak gravitational lensing studies, and an accurate estimate of the PSF is required to obtain unbiased shape measurements. The PSF estimate for a galaxy depends on its spectral energy distribution (SED), because the instrumental PSF is generally a function of the wavelength. In this paper we explore various approaches to determine the resulting `effective' PSF using broad-band data. Considering the Euclid mission as a reference, we find that standard SED template fitting methods result in biases that depend on source redshift, although this may be remedied if the algorithms can be optimized for this purpose. Using a machine learning algorithm we show that, at least in principle, the required accuracy can be achieved with the current survey parameters. It is also possible to account for the correlations between photometric redshift and PSF estimates that arise from the use of the same photometry. We explore the impact of errors in photometric calibration, errors in the assumed wavelength dependence of the PSF model, and limitations of the adopted template libraries. Our results indicate that the required accuracy for Euclid can be achieved using the data that are planned to determine photometric redshifts.

Implications of a wavelength dependent PSF for weak lensing measurements.

NASA Astrophysics Data System (ADS)

Eriksen, Martin; Hoekstra, Henk

2018-05-01

The convolution of galaxy images by the point-spread function (PSF) is the dominant source of bias for weak gravitational lensing studies, and an accurate estimate of the PSF is required to obtain unbiased shape measurements. The PSF estimate for a galaxy depends on its spectral energy distribution (SED), because the instrumental PSF is generally a function of the wavelength. In this paper we explore various approaches to determine the resulting `effective' PSF using broad-band data. Considering the Euclid mission as a reference, we find that standard SED template fitting methods result in biases that depend on source redshift, although this may be remedied if the algorithms can be optimised for this purpose. Using a machine-learning algorithm we show that, at least in principle, the required accuracy can be achieved with the current survey parameters. It is also possible to account for the correlations between photometric redshift and PSF estimates that arise from the use of the same photometry. We explore the impact of errors in photometric calibration, errors in the assumed wavelength dependence of the PSF model and limitations of the adopted template libraries. Our results indicate that the required accuracy for Euclid can be achieved using the data that are planned to determine photometric redshifts.

RNA splicing process analysis for identifying antisense oligonucleotide inhibitors with padlock probe-based isothermal amplification.

PubMed

Ren, Xiaojun; Deng, Ruijie; Wang, Lida; Zhang, Kaixiang; Li, Jinghong

2017-08-01

RNA splicing, which mainly involves two transesterification steps, is a fundamental process of gene expression and its abnormal regulation contributes to serious genetic diseases. Antisense oligonucleotides (ASOs) are genetic control tools that can be used to specifically control genes through alteration of the RNA splicing pathway. Despite intensive research, how ASOs or various other factors influence the multiple processes of RNA splicing still remains obscure. This is largely due to an inability to analyze the splicing efficiency of each step in the RNA splicing process with high sensitivity. We addressed this limitation by introducing a padlock probe-based isothermal amplification assay to achieve quantification of the specific products in different splicing steps. With this amplified assay, the roles that ASOs play in RNA splicing inhibition in the first and second steps could be distinguished. We identified that 5'-ASO could block RNA splicing by inhibiting the first step, while 3'-ASO could block RNA splicing by inhibiting the second step. This method provides a versatile tool for assisting efficient ASO design and discovering new splicing modulators and therapeutic drugs.

Small-Grid Dithers for the JWST Coronagraphs

NASA Technical Reports Server (NTRS)

Lajoie, Charles-Philippe; Soummer, Remi; Pueyo, Laurent; Hines, Dean C.; Nelan, Edmund P.; Perrin, Marshall; Clampin, Mark; Isaacs, John C.

2016-01-01

We discuss new results of coronagraphic simulations demonstrating a novel mode for JWST that utilizes sub-pixel dithered reference images, called Small-Grid Dithers, to optimize coronagraphic PSF subtraction. These sub-pixel dithers are executed with the Fine Steering Mirror under fine guidance, are accurate to approx.2-3 milliarcseconds (1-s/axis), and provide ample speckle diversity to reconstruct an optimized synthetic reference PSF using LOCI or KLIP. We also discuss the performance gains of Small-Grid Dithers compared to the standard undithered scenario, and show potential contrast gain factors for the NIRCam and MIRI coronagraphs ranging from 2 to more than 10, respectively.

Mechanisms and Regulation of Alternative Pre-mRNA Splicing

PubMed Central

Lee, Yeon

2015-01-01

Precursor messenger RNA (pre-mRNA) splicing is a critical step in the posttranscriptional regulation of gene expression, providing significant expansion of the functional proteome of eukaryotic organisms with limited gene numbers. Split eukaryotic genes contain intervening sequences or introns disrupting protein-coding exons, and intron removal occurs by repeated assembly of a large and highly dynamic ribonucleoprotein complex termed the spliceosome, which is composed of five small nuclear ribonucleoprotein particles, U1, U2, U4/U6, and U5. Biochemical studies over the past 10 years have allowed the isolation as well as compositional, functional, and structural analysis of splicing complexes at distinct stages along the spliceosome cycle. The average human gene contains eight exons and seven introns, producing an average of three or more alternatively spliced mRNA isoforms. Recent high-throughput sequencing studies indicate that 100% of human genes produce at least two alternative mRNA isoforms. Mechanisms of alternative splicing include RNA–protein interactions of splicing factors with regulatory sites termed silencers or enhancers, RNA–RNA base-pairing interactions, or chromatin-based effects that can change or determine splicing patterns. Disease-causing mutations can often occur in splice sites near intron borders or in exonic or intronic RNA regulatory silencer or enhancer elements, as well as in genes that encode splicing factors. Together, these studies provide mechanistic insights into how spliceosome assembly, dynamics, and catalysis occur; how alternative splicing is regulated and evolves; and how splicing can be disrupted by cis- and trans-acting mutations leading to disease states. These findings make the spliceosome an attractive new target for small-molecule, antisense, and genome-editing therapeutic interventions. PMID:25784052

The effect of hydrophilicity of graphene oxide as additive towards performance of polysulfone membrane

NASA Astrophysics Data System (ADS)

Jamalludin, Mohd Riduan; Harun, Zawati; Zakaria, M. S.; Rahim, Wan Mohd Faizal Wan Abd; Khor, C. Y.; Rosli, M. U.; Ishak, Muhammad Ikman; Nawi, M. A. M.; Shahrin, Suhaimi

2017-09-01

This study investigates the effect of hydrophilicity of graphene oxide as additive towards properties and performance on polysulfone (PSf) membrane. A polysulfone (PSf) membrane containing graphene oxide (GO) with different content was prepared via phase inversion technique. Characterization on the fabricated membrane were water contact angle measurement, porosity and mean pore size. The effects of PSf/GO were further investigated in terms of permeation test, rejection test and antifouling test. Based on the result obtained, PSf membrane incorporating with GO additive shows better capability to permeate more water compared to unfilled PSf membrane. The PSf/GO membrane were able to increase the permeate rate rapidly with the highest value is given by sample 4 (consist of 0.7 g of GO) at 571.78 J/m2h. Furthermore, water contact angle confirmed the hydrophilicity of this membrane at 62°. The transmembrane fouling activity showed that GO embedded in PSf membrane has improved tremendously. In addition, this study revealed that 0.7g of GO had the highest permeation test, rejection test and antifouling test.

Deconvolution enhanced direction of arrival estimation using one- and three-component seismic arrays applied to ocean induced microseisms

NASA Astrophysics Data System (ADS)

Gal, M.; Reading, A. M.; Ellingsen, S. P.; Koper, K. D.; Burlacu, R.; Gibbons, S. J.

2016-07-01

Microseisms in the period of 2-10 s are generated in deep oceans and near coastal regions. It is common for microseisms from multiple sources to arrive at the same time at a given seismometer. It is therefore desirable to be able to measure multiple slowness vectors accurately. Popular ways to estimate the direction of arrival of ocean induced microseisms are the conventional (fk) or adaptive (Capon) beamformer. These techniques give robust estimates, but are limited in their resolution capabilities and hence do not always detect all arrivals. One of the limiting factors in determining direction of arrival with seismic arrays is the array response, which can strongly influence the estimation of weaker sources. In this work, we aim to improve the resolution for weaker sources and evaluate the performance of two deconvolution algorithms, Richardson-Lucy deconvolution and a new implementation of CLEAN-PSF. The algorithms are tested with three arrays of different aperture (ASAR, WRA and NORSAR) using 1 month of real data each and compared with the conventional approaches. We find an improvement over conventional methods from both algorithms and the best performance with CLEAN-PSF. We then extend the CLEAN-PSF framework to three components (3C) and evaluate 1 yr of data from the Pilbara Seismic Array in northwest Australia. The 3C CLEAN-PSF analysis is capable in resolving a previously undetected Sn phase.

Development of Potent, Selective SRPK1 Inhibitors as Potential Topical Therapeutics for Neovascular Eye Disease.

PubMed

Batson, Jennifer; Toop, Hamish D; Redondo, Clara; Babaei-Jadidi, Roya; Chaikuad, Apirat; Wearmouth, Stephen F; Gibbons, Brian; Allen, Claire; Tallant, Cynthia; Zhang, Jingxue; Du, Chunyun; Hancox, Jules C; Hawtrey, Tom; Da Rocha, Joana; Griffith, Renate; Knapp, Stefan; Bates, David O; Morris, Jonathan C

2017-03-17

Serine/arginine-protein kinase 1 (SRPK1) regulates alternative splicing of VEGF-A to pro-angiogenic isoforms and SRPK1 inhibition can restore the balance of pro/antiangiogenic isoforms to normal physiological levels. The lack of potency and selectivity of available compounds has limited development of SRPK1 inhibitors, with the control of alternative splicing by splicing factor-specific kinases yet to be translated. We present here compounds that occupy a binding pocket created by the unique helical insert of SRPK1, and trigger a backbone flip in the hinge region, that results in potent (<10 nM) and selective inhibition of SRPK1 kinase activity. Treatment with these inhibitors inhibited SRPK1 activity and phosphorylation of serine/arginine splicing factor 1 (SRSF1), resulting in alternative splicing of VEGF-A from pro-angiogenic to antiangiogenic isoforms. This property resulted in potent inhibition of blood vessel growth in models of choroidal angiogenesis in vivo. This work identifies tool compounds for splice isoform selective targeting of pro-angiogenic VEGF, which may lead to new therapeutic strategies for a diversity of diseases where dysfunctional splicing drives disease development.

Quaking and PTB control overlapping splicing regulatory networks during muscle cell differentiation

PubMed Central

Hall, Megan P.; Nagel, Roland J.; Fagg, W. Samuel; Shiue, Lily; Cline, Melissa S.; Perriman, Rhonda J.; Donohue, John Paul; Ares, Manuel

2013-01-01

Alternative splicing contributes to muscle development, but a complete set of muscle-splicing factors and their combinatorial interactions are unknown. Previous work identified ACUAA (“STAR” motif) as an enriched intron sequence near muscle-specific alternative exons such as Capzb exon 9. Mass spectrometry of myoblast proteins selected by the Capzb exon 9 intron via RNA affinity chromatography identifies Quaking (QK), a protein known to regulate mRNA function through ACUAA motifs in 3′ UTRs. We find that QK promotes inclusion of Capzb exon 9 in opposition to repression by polypyrimidine tract-binding protein (PTB). QK depletion alters inclusion of 406 cassette exons whose adjacent intron sequences are also enriched in ACUAA motifs. During differentiation of myoblasts to myotubes, QK levels increase two- to threefold, suggesting a mechanism for QK-responsive exon regulation. Combined analysis of the PTB- and QK-splicing regulatory networks during myogenesis suggests that 39% of regulated exons are under the control of one or both of these splicing factors. This work provides the first evidence that QK is a global regulator of splicing during muscle development in vertebrates and shows how overlapping splicing regulatory networks contribute to gene expression programs during differentiation. PMID:23525800

29 CFR 1918.62 - Miscellaneous auxiliary gear.

Code of Federal Regulations, 2010 CFR

2010-07-01

... appendix II to this part. A design safety factor of at least five shall be maintained for the common sizes... cranes, designed to be used with lesser wire rope safety factors; (ii) According to design factors in... pre-slung drafts. (3) Eye splices shall consist of at least three full tucks. Short splices shall...

29 CFR 1918.62 - Miscellaneous auxiliary gear.

Code of Federal Regulations, 2011 CFR

2011-07-01

... appendix II to this part. A design safety factor of at least five shall be maintained for the common sizes... cranes, designed to be used with lesser wire rope safety factors; (ii) According to design factors in... pre-slung drafts. (3) Eye splices shall consist of at least three full tucks. Short splices shall...

Methods to calibrate and scale axial distances in confocal microscopy as a function of refractive index.

PubMed

Besseling, T H; Jose, J; Van Blaaderen, A

2015-02-01

Accurate distance measurement in 3D confocal microscopy is important for quantitative analysis, volume visualization and image restoration. However, axial distances can be distorted by both the point spread function (PSF) and by a refractive-index mismatch between the sample and immersion liquid, which are difficult to separate. Additionally, accurate calibration of the axial distances in confocal microscopy remains cumbersome, although several high-end methods exist. In this paper we present two methods to calibrate axial distances in 3D confocal microscopy that are both accurate and easily implemented. With these methods, we measured axial scaling factors as a function of refractive-index mismatch for high-aperture confocal microscopy imaging. We found that our scaling factors are almost completely linearly dependent on refractive index and that they were in good agreement with theoretical predictions that take the full vectorial properties of light into account. There was however a strong deviation with the theoretical predictions using (high-angle) geometrical optics, which predict much lower scaling factors. As an illustration, we measured the PSF of a correctly calibrated point-scanning confocal microscope and showed that a nearly index-matched, micron-sized spherical object is still significantly elongated due to this PSF, which signifies that care has to be taken when determining axial calibration or axial scaling using such particles. © 2014 The Authors Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.

Fox-2 Splicing Factor Binds to a Conserved Intron Motif to PromoteInclusion of Protein 4.1R Alternative Exon 16

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ponthier, Julie L.; Schluepen, Christina; Chen, Weiguo

Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of hnRNP A/B proteins to silencer elements in the exon and that downregulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This paper demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding tomore » the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox-2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2, but not Fox-1. These findings suggest that Fox-2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gehrmann, Thies; Pelkmans, Jordi F.; Lugones, Luis G.

Recent genome-wide studies have demonstrated that fungi possess the machinery to alternatively splice pre-mRNA. However, there has not been a systematic categorization of the functional impact of alternative splicing in a fungus. We investigate alternative splicing and its functional consequences in the model mushroom forming fungus Schizophyllum commune. Alternative splicing was demonstrated for 2,285 out of 12,988 expressed genes, resulting in 20% additional transcripts. Intron retentions were the most common alternative splicing events, accounting for 33% of all splicing events, and 43% of the events in coding regions. On the other hand, exon skipping events were rare in coding regionsmore » (1%) but enriched in UTRs where they accounted for 57% of the events. Specific functional groups, including transcription factors, contained alternatively spliced genes. Alternatively spliced transcripts were regulated differently throughout development in 19% of the 2,285 alternatively spliced genes. Notably, 69% of alternatively spliced genes have predicted alternative functionality by loss or gain of functional domains, or by acquiring alternative subcellular locations. S. commune exhibits more alternative splicing than any other studied fungus. Finally, taken together, alternative splicing increases the complexity of the S. commune proteome considerably and provides it with a rich repertoire of alternative functionality that is exploited dynamically.« less

Ultraconserved elements are associated with homeostatic control of splicing regulators by alternative splicing and nonsense-mediated decay

PubMed Central

Ni, Julie Z.; Grate, Leslie; Donohue, John Paul; Preston, Christine; Nobida, Naomi; O’Brien, Georgeann; Shiue, Lily; Clark, Tyson A.; Blume, John E.; Ares, Manuel

2007-01-01

Many alternative splicing events create RNAs with premature stop codons, suggesting that alternative splicing coupled with nonsense-mediated decay (AS-NMD) may regulate gene expression post-transcriptionally. We tested this idea in mice by blocking NMD and measuring changes in isoform representation using splicing-sensitive microarrays. We found a striking class of highly conserved stop codon-containing exons whose inclusion renders the transcript sensitive to NMD. A genomic search for additional examples identified >50 such exons in genes with a variety of functions. These exons are unusually frequent in genes that encode splicing activators and are unexpectedly enriched in the so-called “ultraconserved” elements in the mammalian lineage. Further analysis show that NMD of mRNAs for splicing activators such as SR proteins is triggered by splicing activation events, whereas NMD of the mRNAs for negatively acting hnRNP proteins is triggered by splicing repression, a polarity consistent with widespread homeostatic control of splicing regulator gene expression. We suggest that the extreme genomic conservation surrounding these regulatory splicing events within splicing factor genes demonstrates the evolutionary importance of maintaining tightly tuned homeostasis of RNA-binding protein levels in the vertebrate cell. PMID:17369403

Persufflation (gaseous oxygen perfusion) as a method of heart preservation.

PubMed

Suszynski, Thomas M; Rizzari, Michael D; Scott, William E; Eckman, Peter M; Fonger, James D; John, Ranjit; Chronos, Nicolas; Tempelman, Linda A; Sutherland, David E R; Papas, Klearchos K

2013-04-22

Persufflation (PSF; gaseous oxygen perfusion) is an organ preservation technique with a potential for use in donor heart preservation. Improved heart preservation with PSF may improve outcomes by maintaining cardiac tissue quality in the setting of longer cold ischemia times and possibly increasing the number of donor hearts available for allotransplant. Published data suggests that PSF is able to extend the cold storage times for porcine hearts up to 14 hours without compromising viability and function, and has been shown to resuscitate porcine hearts following donation after cardiac death. This review summarizes key published work on heart PSF, including prospective implications and future directions for PSF in heart transplantation. We emphasize the potential impact of extending preservation times and expanding donor selection criteria in heart allotransplant. Additionally, the key issues that need to be addressed before PSF were to become a widely utilized preservation strategy prior to clinical heart transplantation are summarized and discussed.

The emerging role of alternative splicing in senescence and aging.

PubMed

Deschênes, Mathieu; Chabot, Benoit

2017-10-01

Deregulation of precursor mRNA splicing is associated with many illnesses and has been linked to age-related chronic diseases. Here we review recent progress documenting how defects in the machinery that performs intron removal and controls splice site selection contribute to cellular senescence and organismal aging. We discuss the functional association linking p53, IGF-1, SIRT1, and ING-1 splice variants with senescence and aging, and review a selection of splicing defects occurring in accelerated aging (progeria), vascular aging, and Alzheimer's disease. Overall, it is becoming increasingly clear that changes in the activity of splicing factors and in the production of key splice variants can impact cellular senescence and the aging phenotype. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

The group II intron maturase: a reverse transcriptase and splicing factor go hand in hand.

PubMed

Zhao, Chen; Pyle, Anna Marie

2017-12-01

The splicing of group II introns in vivo requires the assistance of a multifunctional intron encoded protein (IEP, or maturase). Each IEP is also a reverse-transcriptase enzyme that enables group II introns to behave as mobile genetic elements. During splicing or retro-transposition, each group II intron forms a tight, specific complex with its own encoded IEP, resulting in a highly reactive holoenzyme. This review focuses on the structural basis for IEP function, as revealed by recent crystal structures of an IEP reverse transcriptase domain and cryo-EM structures of an IEP-intron complex. These structures explain how the same IEP scaffold is utilized for intron recognition, splicing and reverse transcription, while providing a physical basis for understanding the evolutionary transformation of the IEP into the eukaryotic splicing factor Prp8. Copyright © 2017 Elsevier Ltd. All rights reserved.

A Conserved Nuclear Cyclophilin Is Required for Both RNA Polymerase II Elongation and Co-transcriptional Splicing in Caenorhabditis elegans

PubMed Central

Ahn, Jeong H.; Rechsteiner, Andreas; Strome, Susan; Kelly, William G.

2016-01-01

The elongation phase of transcription by RNA Polymerase II (Pol II) involves numerous events that are tightly coordinated, including RNA processing, histone modification, and chromatin remodeling. RNA splicing factors are associated with elongating Pol II, and the interdependent coupling of splicing and elongation has been documented in several systems. Here we identify a conserved, multi-domain cyclophilin family member, SIG-7, as an essential factor for both normal transcription elongation and co-transcriptional splicing. In embryos depleted for SIG-7, RNA levels for over a thousand zygotically expressed genes are substantially reduced, Pol II becomes significantly reduced at the 3’ end of genes, marks of transcription elongation are reduced, and unspliced mRNAs accumulate. Our findings suggest that SIG-7 plays a central role in both Pol II elongation and co-transcriptional splicing and may provide an important link for their coordination and regulation. PMID:27541139

Alternative splicing at the intersection of biological timing, development, and stress responses.

PubMed

Staiger, Dorothee; Brown, John W S

2013-10-01

High-throughput sequencing for transcript profiling in plants has revealed that alternative splicing (AS) affects a much higher proportion of the transcriptome than was previously assumed. AS is involved in most plant processes and is particularly prevalent in plants exposed to environmental stress. The identification of mutations in predicted splicing factors and spliceosomal proteins that affect cell fate, the circadian clock, plant defense, and tolerance/sensitivity to abiotic stress all point to a fundamental role of splicing/AS in plant growth, development, and responses to external cues. Splicing factors affect the AS of multiple downstream target genes, thereby transferring signals to alter gene expression via splicing factor/AS networks. The last two to three years have seen an ever-increasing number of examples of functional AS. At a time when the identification of AS in individual genes and at a global level is exploding, this review aims to bring together such examples to illustrate the extent and importance of AS, which are not always obvious from individual publications. It also aims to ensure that plant scientists are aware that AS is likely to occur in the genes that they study and that dynamic changes in AS and its consequences need to be considered routinely.

Alternative Splicing at the Intersection of Biological Timing, Development, and Stress Responses[OPEN

PubMed Central

Staiger, Dorothee; Brown, John W.S.

2013-01-01

High-throughput sequencing for transcript profiling in plants has revealed that alternative splicing (AS) affects a much higher proportion of the transcriptome than was previously assumed. AS is involved in most plant processes and is particularly prevalent in plants exposed to environmental stress. The identification of mutations in predicted splicing factors and spliceosomal proteins that affect cell fate, the circadian clock, plant defense, and tolerance/sensitivity to abiotic stress all point to a fundamental role of splicing/AS in plant growth, development, and responses to external cues. Splicing factors affect the AS of multiple downstream target genes, thereby transferring signals to alter gene expression via splicing factor/AS networks. The last two to three years have seen an ever-increasing number of examples of functional AS. At a time when the identification of AS in individual genes and at a global level is exploding, this review aims to bring together such examples to illustrate the extent and importance of AS, which are not always obvious from individual publications. It also aims to ensure that plant scientists are aware that AS is likely to occur in the genes that they study and that dynamic changes in AS and its consequences need to be considered routinely. PMID:24179132

Hypoxia regulates alternative splicing of HIF and non-HIF target genes.

PubMed

Sena, Johnny A; Wang, Liyi; Heasley, Lynn E; Hu, Cheng-Jun

2014-09-01

Hypoxia is a common characteristic of many solid tumors. The hypoxic microenvironment stabilizes hypoxia-inducible transcription factor 1α (HIF1α) and 2α (HIF2α/EPAS1) to activate gene transcription, which promotes tumor cell survival. The majority of human genes are alternatively spliced, producing RNA isoforms that code for functionally distinct proteins. Thus, an effective hypoxia response requires increased HIF target gene expression as well as proper RNA splicing of these HIF-dependent transcripts. However, it is unclear if and how hypoxia regulates RNA splicing of HIF targets. This study determined the effects of hypoxia on alternative splicing (AS) of HIF and non-HIF target genes in hepatocellular carcinoma cells and characterized the role of HIF in regulating AS of HIF-induced genes. The results indicate that hypoxia generally promotes exon inclusion for hypoxia-induced, but reduces exon inclusion for hypoxia-reduced genes. Mechanistically, HIF activity, but not hypoxia per se is found to be necessary and sufficient to increase exon inclusion of several HIF targets, including pyruvate dehydrogenase kinase 1 (PDK1). PDK1 splicing reporters confirm that transcriptional activation by HIF is sufficient to increase exon inclusion of PDK1 splicing reporter. In contrast, transcriptional activation of a PDK1 minigene by other transcription factors in the absence of endogenous HIF target gene activation fails to alter PDK1 RNA splicing. This study demonstrates a novel function of HIF in regulating RNA splicing of HIF target genes. ©2014 American Association for Cancer Research.

18F-FDG PET/CT heterogeneity quantification through textural features in the era of harmonisation programs: a focus on lung cancer.

PubMed

Lasnon, Charline; Majdoub, Mohamed; Lavigne, Brice; Do, Pascal; Madelaine, Jeannick; Visvikis, Dimitris; Hatt, Mathieu; Aide, Nicolas

2016-12-01

Quantification of tumour heterogeneity in PET images has recently gained interest, but has been shown to be dependent on image reconstruction. This study aimed to evaluate the impact of the EANM/EARL accreditation program on selected 18 F-FDG heterogeneity metrics. To carry out our study, we prospectively analysed 71 tumours in 60 biopsy-proven lung cancer patient acquisitions reconstructed with unfiltered point spread function (PSF) positron emission tomography (PET) images (optimised for diagnostic purposes), PSF-reconstructed images with a 7-mm Gaussian filter (PSF 7 ) chosen to meet European Association of Nuclear Medicine (EANM) 1.0 harmonising standards, and EANM Research Ltd. (EARL)-compliant ordered subset expectation maximisation (OSEM) images. Delineation was performed with fuzzy locally adaptive Bayesian (FLAB) algorithm on PSF images and reported on PSF 7 and OSEM ones, and with a 50 % standardised uptake values (SUV) max threshold (SUV max50% ) applied independently to each image. Robust and repeatable heterogeneity metrics including 1st-order [area under the curve of the cumulative histogram (CH AUC )], 2nd-order (entropy, correlation, and dissimilarity), and 3rd-order [high-intensity larger area emphasis (HILAE) and zone percentage (ZP)] textural features (TF) were statistically compared. Volumes obtained with SUV max50% were significantly smaller than FLAB-derived ones, and were significantly smaller in PSF images compared to OSEM and PSF 7 images. PSF-reconstructed images showed significantly higher SUVmax and SUVmean values, as well as heterogeneity for CH AUC , dissimilarity, correlation, and HILAE, and a wider range of heterogeneity values than OSEM images for most of the metrics considered, especially when analysing larger tumours. Histological subtypes had no impact on TF distribution. No significant difference was observed between any of the considered metrics (SUV or heterogeneity features) that we extracted from OSEM and PSF 7 reconstructions. Furthermore, the distributions of TF for OSEM and PSF 7 reconstructions according to tumour volumes were similar for all ranges of volumes. PSF reconstruction with Gaussian filtering chosen to meet harmonising standards resulted in similar SUV values and heterogeneity information as compared to OSEM images, which validates its use within the harmonisation strategy context. However, unfiltered PSF-reconstructed images also showed higher heterogeneity according to some metrics, as well as a wider range of heterogeneity values than OSEM images for most of the metrics considered, especially when analysing larger tumours. This suggests that, whenever available, unfiltered PSF images should also be exploited to obtain the most discriminative quantitative heterogeneity features.

Analysis of point source size on measurement accuracy of lateral point-spread function of confocal Raman microscopy

NASA Astrophysics Data System (ADS)

Fu, Shihang; Zhang, Li; Hu, Yao; Ding, Xiang

2018-01-01

Confocal Raman Microscopy (CRM) has matured to become one of the most powerful instruments in analytical science because of its molecular sensitivity and high spatial resolution. Compared with conventional Raman Microscopy, CRM can perform three dimensions mapping of tiny samples and has the advantage of high spatial resolution thanking to the unique pinhole. With the wide application of the instrument, there is a growing requirement for the evaluation of the imaging performance of the system. Point-spread function (PSF) is an important approach to the evaluation of imaging capability of an optical instrument. Among a variety of measurement methods of PSF, the point source method has been widely used because it is easy to operate and the measurement results are approximate to the true PSF. In the point source method, the point source size has a significant impact on the final measurement accuracy. In this paper, the influence of the point source sizes on the measurement accuracy of PSF is analyzed and verified experimentally. A theoretical model of the lateral PSF for CRM is established and the effect of point source size on full-width at half maximum of lateral PSF is simulated. For long-term preservation and measurement convenience, PSF measurement phantom using polydimethylsiloxane resin, doped with different sizes of polystyrene microspheres is designed. The PSF of CRM with different sizes of microspheres are measured and the results are compared with the simulation results. The results provide a guide for measuring the PSF of the CRM.

A novel TFF2 splice variant (ΔEX2TFF2) correlates with longer overall survival time in cholangiocarcinoma

PubMed Central

KAMLUA, SURASEE; PATRAKITKOMJORN, SIRIPORN; JEARANAIKOON, PATCHAREE; MENHENIOTT, TREVELYAN R.; GIRAUD, ANDREW S.; LIMPAIBOON, TEMDUANG

2012-01-01

Trefoil factor 2 (TFF2) is a member of trefoil factor family found to be overexpressed in many cancers including cholangiocarcinoma (CCA). The majority of studies have focused on wild-type TFF2 (wtTFF2) expression, but information regarding alternative splicing variants of TFF2 mRNA has not been reported. In this study, we aimed to identify and quantify a novel TFF2 splice variant in cholangiocarcinoma (CCA). Seventy-eight tumors and 15 normal adjacent tissues were quantified for the expression of the TFF2 splice variant relative to wild-type (wt) TFF2 mRNA using quantitative reverse transcriptase polymerase chain reaction (QRT-PCR). The ratio of TFF2 splice variant against wtTFF2 was analyzed for associations with clinical parameters. We found a novel TFF2 splice variant, exon 2 skipping (ΔEX2TFF2), resulting in a stop codon (TAG) at exon 1. The ΔEX2TFF2/wtTFF2 ratio in tumors was significantly higher than in normal tissue (P<0.01). Interestingly, high ΔEX2TFF2/wtTFF2 ratio was significantly associated with good prognosis compared with low ratio (P=0.017). In contrast, the presence of wtTFF2 protein was associated with poor survival of CCA patients (P=0.034). This is the first report of a trefoil factor splice variant and its potential application as a prognostic biomarker in CCA. PMID:22159958

Schizophyllum commune has an extensive and functional alternative splicing repertoire

PubMed Central

Gehrmann, Thies; Pelkmans, Jordi F.; Lugones, Luis G.; Wösten, Han A. B.; Abeel, Thomas; Reinders, Marcel J. T.

2016-01-01

Recent genome-wide studies have demonstrated that fungi possess the machinery to alternatively splice pre-mRNA. However, there has not been a systematic categorization of the functional impact of alternative splicing in a fungus. We investigate alternative splicing and its functional consequences in the model mushroom forming fungus Schizophyllum commune. Alternative splicing was demonstrated for 2,285 out of 12,988 expressed genes, resulting in 20% additional transcripts. Intron retentions were the most common alternative splicing events, accounting for 33% of all splicing events, and 43% of the events in coding regions. On the other hand, exon skipping events were rare in coding regions (1%) but enriched in UTRs where they accounted for 57% of the events. Specific functional groups, including transcription factors, contained alternatively spliced genes. Alternatively spliced transcripts were regulated differently throughout development in 19% of the 2,285 alternatively spliced genes. Notably, 69% of alternatively spliced genes have predicted alternative functionality by loss or gain of functional domains, or by acquiring alternative subcellular locations. S. commune exhibits more alternative splicing than any other studied fungus. Taken together, alternative splicing increases the complexity of the S. commune proteome considerably and provides it with a rich repertoire of alternative functionality that is exploited dynamically. PMID:27659065

Widespread Use of Non-productive Alternative Splice Sites in Saccharomyces cerevisiae

PubMed Central

Kawashima, Tadashi; Douglass, Stephen; Gabunilas, Jason; Pellegrini, Matteo; Chanfreau, Guillaume F.

2014-01-01

Saccharomyces cerevisiae has been used as a model system to investigate the mechanisms of pre-mRNA splicing but only a few examples of alternative splice site usage have been described in this organism. Using RNA-Seq analysis of nonsense-mediated mRNA decay (NMD) mutant strains, we show that many S. cerevisiae intron-containing genes exhibit usage of alternative splice sites, but many transcripts generated by splicing at these sites are non-functional because they introduce premature termination codons, leading to degradation by NMD. Analysis of splicing mutants combined with NMD inactivation revealed the role of specific splicing factors in governing the use of these alternative splice sites and identified novel functions for Prp17p in enhancing the use of branchpoint-proximal upstream 3′ splice sites and for Prp18p in suppressing the usage of a non-canonical AUG 3′-splice site in GCR1. The use of non-productive alternative splice sites can be increased in stress conditions in a promoter-dependent manner, contributing to the down-regulation of genes during stress. These results show that alternative splicing is frequent in S. cerevisiae but masked by RNA degradation and that the use of alternative splice sites in this organism is mostly aimed at controlling transcript levels rather than increasing proteome diversity. PMID:24722551

Schizophyllum commune has an extensive and functional alternative splicing repertoire

DOE PAGES

Gehrmann, Thies; Pelkmans, Jordi F.; Lugones, Luis G.; ...

2016-09-23

Recent genome-wide studies have demonstrated that fungi possess the machinery to alternatively splice pre-mRNA. However, there has not been a systematic categorization of the functional impact of alternative splicing in a fungus. We investigate alternative splicing and its functional consequences in the model mushroom forming fungus Schizophyllum commune. Alternative splicing was demonstrated for 2,285 out of 12,988 expressed genes, resulting in 20% additional transcripts. Intron retentions were the most common alternative splicing events, accounting for 33% of all splicing events, and 43% of the events in coding regions. On the other hand, exon skipping events were rare in coding regionsmore » (1%) but enriched in UTRs where they accounted for 57% of the events. Specific functional groups, including transcription factors, contained alternatively spliced genes. Alternatively spliced transcripts were regulated differently throughout development in 19% of the 2,285 alternatively spliced genes. Notably, 69% of alternatively spliced genes have predicted alternative functionality by loss or gain of functional domains, or by acquiring alternative subcellular locations. S. commune exhibits more alternative splicing than any other studied fungus. Finally, taken together, alternative splicing increases the complexity of the S. commune proteome considerably and provides it with a rich repertoire of alternative functionality that is exploited dynamically.« less

Quantitative optical imaging and sensing by joint design of point spread functions and estimation algorithms

NASA Astrophysics Data System (ADS)

Quirin, Sean Albert

The joint application of tailored optical Point Spread Functions (PSF) and estimation methods is an important tool for designing quantitative imaging and sensing solutions. By enhancing the information transfer encoded by the optical waves into an image, matched post-processing algorithms are able to complete tasks with improved performance relative to conventional designs. In this thesis, new engineered PSF solutions with image processing algorithms are introduced and demonstrated for quantitative imaging using information-efficient signal processing tools and/or optical-efficient experimental implementations. The use of a 3D engineered PSF, the Double-Helix (DH-PSF), is applied as one solution for three-dimensional, super-resolution fluorescence microscopy. The DH-PSF is a tailored PSF which was engineered to have enhanced information transfer for the task of localizing point sources in three dimensions. Both an information- and optical-efficient implementation of the DH-PSF microscope are demonstrated here for the first time. This microscope is applied to image single-molecules and micro-tubules located within a biological sample. A joint imaging/axial-ranging modality is demonstrated for application to quantifying sources of extended transverse and axial extent. The proposed implementation has improved optical-efficiency relative to prior designs due to the use of serialized cycling through select engineered PSFs. This system is demonstrated for passive-ranging, extended Depth-of-Field imaging and digital refocusing of random objects under broadband illumination. Although the serialized engineered PSF solution is an improvement over prior designs for the joint imaging/passive-ranging modality, it requires the use of multiple PSFs---a potentially significant constraint. Therefore an alternative design is proposed, the Single-Helix PSF, where only one engineered PSF is necessary and the chromatic behavior of objects under broadband illumination provides the necessary information transfer. The matched estimation algorithms are introduced along with an optically-efficient experimental system to image and passively estimate the distance to a test object. An engineered PSF solution is proposed for improving the sensitivity of optical wave-front sensing using a Shack-Hartmann Wave-front Sensor (SHWFS). The performance limits of the classical SHWFS design are evaluated and the engineered PSF system design is demonstrated to enhance performance. This system is fabricated and the mechanism for additional information transfer is identified.

Corkscrew point spread function for far-field three-dimensional nanoscale localization of pointlike objects

PubMed Central

Lew, Matthew D.; Lee, Steven F.; Badieirostami, Majid; Moerner, W. E.

2011-01-01

We describe the corkscrew point spread function (PSF), which can localize objects in three dimensions throughout a 3.2 µm depth of field with nanometer precision. The corkscrew PSF rotates as a function of the axial (z) position of an emitter. Fisher information calculations show that the corkscrew PSF can achieve nanometer localization precision with limited numbers of photons. We demonstrate three-dimensional super-resolution microscopy with the corkscrew PSF by imaging beads on the surface of a triangular polydimethylsiloxane (PDMS) grating. With 99,000 photons detected, the corkscrew PSF achieves a localization precision of 2.7 nm in x, 2.1 nm in y, and 5.7 nm in z. PMID:21263500

Corkscrew point spread function for far-field three-dimensional nanoscale localization of pointlike objects.

PubMed

Lew, Matthew D; Lee, Steven F; Badieirostami, Majid; Moerner, W E

2011-01-15

We describe the corkscrew point spread function (PSF), which can localize objects in three dimensions throughout a 3.2 μm depth of field with nanometer precision. The corkscrew PSF rotates as a function of the axial (z) position of an emitter. Fisher information calculations show that the corkscrew PSF can achieve nanometer localization precision with limited numbers of photons. We demonstrate three-dimensional super-resolution microscopy with the corkscrew PSF by imaging beads on the surface of a triangular polydimethylsiloxane (PDMS) grating. With 99,000 photons detected, the corkscrew PSF achieves a localization precision of 2.7 nm in x, 2.1 nm in y, and 5.7 nm in z.

Projections onto Convex Sets Super-Resolution Reconstruction Based on Point Spread Function Estimation of Low-Resolution Remote Sensing Images

PubMed Central

Fan, Chong; Wu, Chaoyun; Li, Grand; Ma, Jun

2017-01-01

To solve the problem on inaccuracy when estimating the point spread function (PSF) of the ideal original image in traditional projection onto convex set (POCS) super-resolution (SR) reconstruction, this paper presents an improved POCS SR algorithm based on PSF estimation of low-resolution (LR) remote sensing images. The proposed algorithm can improve the spatial resolution of the image and benefit agricultural crop visual interpolation. The PSF of the high-resolution (HR) image is unknown in reality. Therefore, analysis of the relationship between the PSF of the HR image and the PSF of the LR image is important to estimate the PSF of the HR image by using multiple LR images. In this study, the linear relationship between the PSFs of the HR and LR images can be proven. In addition, the novel slant knife-edge method is employed, which can improve the accuracy of the PSF estimation of LR images. Finally, the proposed method is applied to reconstruct airborne digital sensor 40 (ADS40) three-line array images and the overlapped areas of two adjacent GF-2 images by embedding the estimated PSF of the HR image to the original POCS SR algorithm. Experimental results show that the proposed method yields higher quality of reconstructed images than that produced by the blind SR method and the bicubic interpolation method. PMID:28208837

Organellar maturases: A window into the evolution of the spliceosome.

PubMed

Schmitz-Linneweber, Christian; Lampe, Marie-Kristin; Sultan, Laure D; Ostersetzer-Biran, Oren

2015-09-01

During the evolution of eukaryotic genomes, many genes have been interrupted by intervening sequences (introns) that must be removed post-transcriptionally from RNA precursors to form mRNAs ready for translation. The origin of nuclear introns is still under debate, but one hypothesis is that the spliceosome and the intron-exon structure of genes have evolved from bacterial-type group II introns that invaded the eukaryotic genomes. The group II introns were most likely introduced into the eukaryotic genome from an α-proteobacterial predecessor of mitochondria early during the endosymbiosis event. These self-splicing and mobile introns spread through the eukaryotic genome and later degenerated. Pieces of introns became part of the general splicing machinery we know today as the spliceosome. In addition, group II introns likely brought intron maturases with them to the nucleus. Maturases are found in most bacterial introns, where they act as highly specific splicing factors for group II introns. In the spliceosome, the core protein Prp8 shows homology to group II intron-encoded maturases. While maturases are entirely intron specific, their descendant of the spliceosomal machinery, the Prp8 protein, is an extremely versatile splicing factor with multiple interacting proteins and RNAs. How could such a general player in spliceosomal splicing evolve from the monospecific bacterial maturases? Analysis of the organellar splicing machinery in plants may give clues on the evolution of nuclear splicing. Plants encode various proteins which are closely related to bacterial maturases. The organellar genomes contain one maturase each, named MatK in chloroplasts and MatR in mitochondria. In addition, several maturase genes have been found in the nucleus as well, which are acting on mitochondrial pre-RNAs. All plant maturases show sequence deviation from their progenitor bacterial maturases, and interestingly are all acting on multiple organellar group II intron targets. Moreover, they seem to function in the splicing of group II introns together with a number of additional nuclear-encoded splicing factors, possibly acting as an organellar proto-spliceosome. Together, this makes them interesting models for the early evolution of nuclear spliceosomal splicing. In this review, we summarize recent advances in our understanding of the role of plant maturases and their accessory factors in plants. This article is part of a Special Issue entitled: Chloroplast Biogenesis. Copyright © 2015 Elsevier B.V. All rights reserved.

RBFOX2 Is an Important Regulator of Mesenchymal Tissue-Specific Splicing in both Normal and Cancer Tissues

PubMed Central

Venables, Julian P.; Brosseau, Jean-Philippe; Gadea, Gilles; Klinck, Roscoe; Prinos, Panagiotis; Beaulieu, Jean-François; Lapointe, Elvy; Durand, Mathieu; Thibault, Philippe; Tremblay, Karine; Rousset, François; Tazi, Jamal; Abou Elela, Sherif

2013-01-01

Alternative splicing provides a critical and flexible layer of regulation intervening in many biological processes to regulate the diversity of proteins and impact cell phenotype. To identify alternative splicing differences that distinguish epithelial from mesenchymal tissues, we have investigated hundreds of cassette exons using a high-throughput reverse transcription-PCR (RT-PCR) platform. Extensive changes in splicing were noted between epithelial and mesenchymal tissues in both human colon and ovarian tissues, with many changes from mostly one splice variant to predominantly the other. Remarkably, many of the splicing differences that distinguish normal mesenchymal from normal epithelial tissues matched those that differentiate normal ovarian tissues from ovarian cancer. Furthermore, because splicing profiling could classify cancer cell lines according to their epithelial/mesenchymal characteristics, we used these cancer cell lines to identify regulators for these specific splicing signatures. By knocking down 78 potential splicing factors in five cell lines, we provide an extensive view of the complex regulatory landscape associated with the epithelial and mesenchymal states, thus revealing that RBFOX2 is an important driver of mesenchymal tissue-specific splicing. PMID:23149937

Alternative Splicing as a Target for Cancer Treatment.

PubMed

Martinez-Montiel, Nancy; Rosas-Murrieta, Nora Hilda; Anaya Ruiz, Maricruz; Monjaraz-Guzman, Eduardo; Martinez-Contreras, Rebeca

2018-02-11

Alternative splicing is a key mechanism determinant for gene expression in metazoan. During alternative splicing, non-coding sequences are removed to generate different mature messenger RNAs due to a combination of sequence elements and cellular factors that contribute to splicing regulation. A different combination of splicing sites, exonic or intronic sequences, mutually exclusive exons or retained introns could be selected during alternative splicing to generate different mature mRNAs that could in turn produce distinct protein products. Alternative splicing is the main source of protein diversity responsible for 90% of human gene expression, and it has recently become a hallmark for cancer with a full potential as a prognostic and therapeutic tool. Currently, more than 15,000 alternative splicing events have been associated to different aspects of cancer biology, including cell proliferation and invasion, apoptosis resistance and susceptibility to different chemotherapeutic drugs. Here, we present well established and newly discovered splicing events that occur in different cancer-related genes, their modification by several approaches and the current status of key tools developed to target alternative splicing with diagnostic and therapeutic purposes.

Alternative Splicing in Neurogenesis and Brain Development.

PubMed

Su, Chun-Hao; D, Dhananjaya; Tarn, Woan-Yuh

2018-01-01

Alternative splicing of precursor mRNA is an important mechanism that increases transcriptomic and proteomic diversity and also post-transcriptionally regulates mRNA levels. Alternative splicing occurs at high frequency in brain tissues and contributes to every step of nervous system development, including cell-fate decisions, neuronal migration, axon guidance, and synaptogenesis. Genetic manipulation and RNA sequencing have provided insights into the molecular mechanisms underlying the effects of alternative splicing in stem cell self-renewal and neuronal fate specification. Timely expression and perhaps post-translational modification of neuron-specific splicing regulators play important roles in neuronal development. Alternative splicing of many key transcription regulators or epigenetic factors reprograms the transcriptome and hence contributes to stem cell fate determination. During neuronal differentiation, alternative splicing also modulates signaling activity, centriolar dynamics, and metabolic pathways. Moreover, alternative splicing impacts cortical lamination and neuronal development and function. In this review, we focus on recent progress toward understanding the contributions of alternative splicing to neurogenesis and brain development, which has shed light on how splicing defects may cause brain disorders and diseases.

Molecular dissection of step 2 catalysis of yeast pre-mRNA splicing investigated in a purified system

PubMed Central

Ohrt, Thomas; Odenwälder, Peter; Dannenberg, Julia; Prior, Mira; Warkocki, Zbigniew; Schmitzová, Jana; Karaduman, Ramazan; Gregor, Ingo; Enderlein, Jörg; Fabrizio, Patrizia; Lührmann, Reinhard

2013-01-01

Step 2 catalysis of pre-mRNA splicing entails the excision of the intron and ligation of the 5′ and 3′ exons. The tasks of the splicing factors Prp16, Slu7, Prp18, and Prp22 in the formation of the step 2 active site of the spliceosome and in exon ligation, and the timing of their recruitment, remain poorly understood. Using a purified yeast in vitro splicing system, we show that only the DEAH-box ATPase Prp16 is required for formation of a functional step 2 active site and for exon ligation. Efficient docking of the 3′ splice site (3′SS) to the active site requires only Slu7/Prp18 but not Prp22. Spliceosome remodeling by Prp16 appears to be subtle as only the step 1 factor Cwc25 is dissociated prior to step 2 catalysis, with its release dependent on docking of the 3′SS to the active site and Prp16 action. We show by fluorescence cross-correlation spectroscopy that Slu7/Prp18 and Prp16 bind early to distinct, low-affinity binding sites on the step-1-activated B* spliceosome, which are subsequently converted into high-affinity sites. Our results shed new light on the factor requirements for step 2 catalysis and the dynamics of step 1 and 2 factors during the catalytic steps of splicing. PMID:23685439

CYCLIN-DEPENDENT KINASE G1 Is Associated with the Spliceosome to Regulate CALLOSE SYNTHASE5 Splicing and Pollen Wall Formation in Arabidopsis[C][W][OA

PubMed Central

Huang, Xue-Yong; Niu, Jin; Sun, Ming-Xi; Zhu, Jun; Gao, Ju-Fang; Yang, Jun; Zhou, Que; Yang, Zhong-Nan

2013-01-01

Arabidopsis thaliana CYCLIN-DEPEDENT KINASE G1 (CDKG1) belongs to the family of cyclin-dependent protein kinases that were originally characterized as cell cycle regulators in eukaryotes. Here, we report that CDKG1 regulates pre-mRNA splicing of CALLOSE SYNTHASE5 (CalS5) and, therefore, pollen wall formation. The knockout mutant cdkg1 exhibits reduced male fertility with impaired callose synthesis and abnormal pollen wall formation. The sixth intron in CalS5 pre-mRNA, a rare type of intron with a GC 5′ splice site, is abnormally spliced in cdkg1. RNA immunoprecipitation analysis suggests that CDKG1 is associated with this intron. CDKG1 contains N-terminal Ser/Arg (RS) motifs and interacts with splicing factor Arginine/Serine-Rich Zinc Knuckle-Containing Protein33 (RSZ33) through its RS region to regulate proper splicing. CDKG1 and RS-containing Zinc Finger Protein22 (SRZ22), a splicing factor interacting with RSZ33 and U1 small nuclear ribonucleoprotein particle (snRNP) component U1-70k, colocalize in nuclear speckles and reside in the same complex. We propose that CDKG1 is recruited to U1 snRNP through RSZ33 to facilitate the splicing of the sixth intron of CalS5. PMID:23404887

Arrest is a regulator of fiber-specific alternative splicing in the indirect flight muscles of Drosophila

PubMed Central

Oas, Sandy T.

2014-01-01

Drosophila melanogaster flight muscles are distinct from other skeletal muscles, such as jump muscles, and express several uniquely spliced muscle-associated transcripts. We sought to identify factors mediating splicing differences between the flight and jump muscle fiber types. We found that the ribonucleic acid–binding protein Arrest (Aret) is expressed in flight muscles: in founder cells, Aret accumulates in a novel intranuclear compartment that we termed the Bruno body, and after the onset of muscle differentiation, Aret disperses in the nucleus. Down-regulation of the aret gene led to ultrastructural changes and functional impairment of flight muscles, and transcripts of structural genes expressed in the flight muscles became spliced in a manner characteristic of jump muscles. Aret also potently promoted flight muscle splicing patterns when ectopically expressed in jump muscles or tissue culture cells. Genetically, aret is located downstream of exd (extradenticle), hth (homothorax), and salm (spalt major), transcription factors that control fiber identity. Our observations provide insight into a transcriptional and splicing regulatory network for muscle fiber specification. PMID:25246617

Implementing a rational and consistent nomenclature for serine/arginine-rich protein splicing factors (SR proteins) in plants.

PubMed

Barta, Andrea; Kalyna, Maria; Reddy, Anireddy S N

2010-09-01

Growing interest in alternative splicing in plants and the extensive sequencing of new plant genomes necessitate more precise definition and classification of genes coding for splicing factors. SR proteins are a family of RNA binding proteins, which function as essential factors for constitutive and alternative splicing. We propose a unified nomenclature for plant SR proteins, taking into account the newly revised nomenclature of the mammalian SR proteins and a number of plant-specific properties of the plant proteins. We identify six subfamilies of SR proteins in Arabidopsis thaliana and rice (Oryza sativa), three of which are plant specific. The proposed subdivision of plant SR proteins into different subfamilies will allow grouping of paralogous proteins and simple assignment of newly discovered SR orthologs from other plant species and will promote functional comparisons in diverse plant species.

Prognostic alternative mRNA splicing signature in non-small cell lung cancer.

PubMed

Li, Yuan; Sun, Nan; Lu, Zhiliang; Sun, Shouguo; Huang, Jianbing; Chen, Zhaoli; He, Jie

2017-05-01

Alternative splicing provides a major mechanism to generate protein diversity. Increasing evidence suggests a link of dysregulation of splicing associated with cancer. Genome-wide alternative splicing profiling in lung cancer remains largely unstudied. We generated alternative splicing profiles in 491 lung adenocarcinoma (LUAD) and 471 lung squamous cell carcinoma (LUSC) patients in TCGA using RNA-seq data, prognostic models and splicing networks were built by integrated bioinformatics analysis. A total of 3691 and 2403 alternative splicing events were significantly associated with patient survival in LUAD and LUSC, respectively, including EGFR, CD44, PIK3C3, RRAS2, MAPKAP1 and FGFR2. The area under the curve of the receiver-operator characteristic curve for prognostic predictor in NSCLC was 0.817 at 2000 days of overall survival which were also over 0.8 in LUAD and LUSC, separately. Interestingly, splicing correlation networks uncovered opposite roles of splicing factors in LUAD and LUSC. We created prognostic predictors based on alternative splicing events with high performances for risk stratification in NSCLC patients and uncovered interesting splicing networks in LUAD and LUSC which could be underlying mechanisms. Copyright © 2017 Elsevier B.V. All rights reserved.

A user-friendly modified pore-solid fractal model

PubMed Central

Ding, Dian-yuan; Zhao, Ying; Feng, Hao; Si, Bing-cheng; Hill, Robert Lee

2016-01-01

The primary objective of this study was to evaluate a range of calculation points on water retention curves (WRC) instead of the singularity point at air-entry suction in the pore-solid fractal (PSF) model, which additionally considered the hysteresis effect based on the PSF theory. The modified pore-solid fractal (M-PSF) model was tested using 26 soil samples from Yangling on the Loess Plateau in China and 54 soil samples from the Unsaturated Soil Hydraulic Database. The derivation results showed that the M-PSF model is user-friendly and flexible for a wide range of calculation point options. This model theoretically describes the primary differences between the soil moisture desorption and the adsorption processes by the fractal dimensions. The M-PSF model demonstrated good performance particularly at the calculation points corresponding to the suctions from 100 cm to 1000 cm. Furthermore, the M-PSF model, used the fractal dimension of the particle size distribution, exhibited an accepted performance of WRC predictions for different textured soils when the suction values were ≥100 cm. To fully understand the function of hysteresis in the PSF theory, the role of allowable and accessible pores must be examined. PMID:27996013

IRAS: High-Throughput Identification of Novel Alternative Splicing Regulators.

PubMed

Zheng, S

2016-01-01

Alternative splicing is a fundamental regulatory process of gene expression. Defects in alternative splicing can lead to various diseases, and modification of disease-causing splicing events presents great therapeutic promise. Splicing outcome is commonly affected by extracellular stimuli and signaling cascades that converge on RNA-binding splicing regulators. These trans-acting factors recognize cis-elements in pre-mRNA transcripts to affect spliceosome assembly and splice site choices. Identification of these splicing regulators and/or upstream modulators has been difficult and traditionally done by piecemeal. High-throughput screening strategies to find multiple regulators of exon splicing have great potential to accelerate the discovery process, but typically confront low sensitivity and low specificity of screening assays. Here we describe a unique screening strategy, IRAS (identifying regulators of alternative splicing), using a pair of dual-output minigene reporters to allow for sensitive detection of exon splicing changes. Each dual-output reporter produces green fluorescent protein (GFP) and red fluorescent protein (RFP) fluorescent signals to assay the two spliced isoforms exclusively. The two complementary minigene reporters alter GFP/RFP output ratios in the opposite direction in response to splicing change. Applying IRAS in cell-based high-throughput screens allows sensitive and specific identification of splicing regulators and modulators for any alternative exons of interest. In comparison to previous high-throughput screening methods, IRAS substantially enhances the specificity of the screening assay. This strategy significantly eliminates false positives without sacrificing sensitive identification of true regulators of splicing. © 2016 Elsevier Inc. All rights reserved.

Methodology of splicing large air filling factor suspended core photonic crystal fibres

NASA Astrophysics Data System (ADS)

Jaroszewicz, L. R.; Murawski, M.; Nasilowski, T.; Stasiewicz, K.; Marć, P.; Szymański, M.; Mergo, P.

2011-06-01

We report the methodology of effective low-loss fusion splicing a photonic crystal fibre (PCF) to itself as well as to a standard single mode fibre (SMF). Distinctly from other papers in this area, we report on the results for splicing suspended core (SC) PCF having tiny core and non-Gaussian shape of guided beam. We show that studied splices exhibit transmission losses strongly dispersive and non-reciprocal in view of light propagation direction. Achieved splicing losses, defined as larger decrease in transmitted optical power comparing both propagation directions, are equal to 2.71 ±0.25 dB, 1.55 ±0.25 dB at 1550 nm for fibre SC PCF spliced to itself and to SMF, respectively.

Detecting unresolved binary stars in Euclid VIS images

NASA Astrophysics Data System (ADS)

Kuntzer, T.; Courbin, F.

2017-10-01

Measuring a weak gravitational lensing signal to the level required by the next generation of space-based surveys demands exquisite reconstruction of the point-spread function (PSF). However, unresolved binary stars can significantly distort the PSF shape. In an effort to mitigate this bias, we aim at detecting unresolved binaries in realistic Euclid stellar populations. We tested methods in numerical experiments where (I) the PSF shape is known to Euclid requirements across the field of view; and (II) the PSF shape is unknown. We drew simulated catalogues of PSF shapes for this proof-of-concept paper. Following the Euclid survey plan, the objects were observed four times. We propose three methods to detect unresolved binary stars. The detection is based on the systematic and correlated biases between exposures of the same object. One method is a simple correlation analysis, while the two others use supervised machine-learning algorithms (random forest and artificial neural network). In both experiments, we demonstrate the ability of our methods to detect unresolved binary stars in simulated catalogues. The performance depends on the level of prior knowledge of the PSF shape and the shape measurement errors. Good detection performances are observed in both experiments. Full complexity, in terms of the images and the survey design, is not included, but key aspects of a more mature pipeline are discussed. Finding unresolved binaries in objects used for PSF reconstruction increases the quality of the PSF determination at arbitrary positions. We show, using different approaches, that we are able to detect at least binary stars that are most damaging for the PSF reconstruction process. The code corresponding to the algorithms used in this work and all scripts to reproduce the results are publicly available from a GitHub repository accessible via http://lastro.epfl.ch/software

Cellular organization of pre-mRNA splicing factors in several tissues. Changes in the uterus by hormone action.

PubMed

George-Téllez, R; Segura-Valdez, M L; González-Santos, L; Jiménez-García, L F

2002-05-01

In the mammalian cell nucleus, splicing factors are distributed in nuclear domains known as speckles or splicing factor compartments (SFCs). In cultured cells, these domains are dynamic and reflect transcriptional and splicing activities. We used immunofluorescence and confocal microscopy to monitor whether splicing factors in differentiated cells display similar features. Speckled patterns are observed in rat hepatocytes, beta-cells, bronchial and intestine epithelia and also in three cell types of the uterus. Moreover, the number, distribution and sizes of the speckles vary among them. In addition, we studied variations in the circular form (shape) of speckles in uterine cells that are transcriptionally modified by a hormone action. During proestrus of the estral cycle, speckles are irregular in shape while in diestrus I they are circular. Experimentally, in castrated rats luminal epithelial cells show a pattern where speckles are dramatically rounded, but they recover their irregular shape rapidly after an injection of estradiol. The same results were observed in muscle and gland epithelial cells of the uterus. We concluded that different speckled patterns are present in various cells types in differentiated tissues and that these patterns change in the uterus depending upon the presence or absence of hormones such as estradiol.

Characterization of a spliced variant of human IRF-3 promoter and its regulation by the transcription factor Sp1.

PubMed

Ren, Wei; Zhu, Liang-Hua; Xu, Hua-Guo; Jin, Rui; Zhou, Guo-Ping

2012-06-01

Interferon regulatory factor 3 (IRF-3), an essential transcriptional regulator of the interferon genes, plays an important role in host defense against viral and microbial infection as well as in cell growth regulation. Promoter plays a crucial role in gene transcription. We have reported the characterization of the wide type of human IRF-3 promoter, but the characterization of the spliced variant of human IRF-3 Int2V1 promoter has not been systematically analyzed. To observe the spliced variant of human IRF-3 promoter, we have cloned the human IRF-3 gene promoter region containing 300 nucleotides upstream the transcription start site (TSS). Transient transfection of 5' deleted promoter-reporter constructs and luciferase assay illustrated the region -159/-100 relative to the TSS is sufficient for full promoter activity. This region contains GATA1 and specific protein-1 (Sp1) transcription factor binding sites. Interestingly, mutation of this Sp1 site reduced the promoter activity by 50%. However, overexpression of Sp1 increased the transcription activity by 2.4-fold. These results indicated that the spliced variant of human IRF-3 gene core promoter was located within the region -159/-100 relative to the TSS. Sp1 transcription factor upregulates the spliced variant of human IRF-3 gene promoter.

Model-based restoration using light vein for range-gated imaging systems.

PubMed

Wang, Canjin; Sun, Tao; Wang, Tingfeng; Wang, Rui; Guo, Jin; Tian, Yuzhen

2016-09-10

The images captured by an airborne range-gated imaging system are degraded by many factors, such as light scattering, noise, defocus of the optical system, atmospheric disturbances, platform vibrations, and so on. The characteristics of low illumination, few details, and high noise make the state-of-the-art restoration method fail. In this paper, we present a restoration method especially for range-gated imaging systems. The degradation process is divided into two parts: the static part and the dynamic part. For the static part, we establish the physical model of the imaging system according to the laser transmission theory, and estimate the static point spread function (PSF). For the dynamic part, a so-called light vein feature extraction method is presented to estimate the fuzzy parameter of the atmospheric disturbance and platform movement, which make contributions to the dynamic PSF. Finally, combined with the static and dynamic PSF, an iterative updating framework is used to restore the image. Compared with the state-of-the-art methods, the proposed method can effectively suppress ringing artifacts and achieve better performance in a range-gated imaging system.

Tratamiento formal de imágenes astronómicas con PSF espacialmente variable

NASA Astrophysics Data System (ADS)

Sánchez, B. O.; Domínguez, M. J.; Lares, M.

2017-10-01

We present a python implementation of a method for PSF determination in the context of optimal subtraction of astronomical images. We introduce an expansion of the spatially variant point spread function (PSF) in terms of the Karhunen Loève basis. The advantage of this approach is that the basis is able to naturally adapt to the data, instead of imposing a fixed ad-hoc analytic form. Simulated image reconstruction was analyzed, by using the measured PSF, with good agreement in terms of sky background level between the reconstructed and original images. The technique is simple enough to be implemented on more sophisticated image subtraction methods, since it improves its results without extra computational cost in a spatially variant PSF environment.

The DNA replication licensing factor miniature chromosome maintenance 7 is essential for RNA splicing of epidermal growth factor receptor, c-Met, and platelet-derived growth factor receptor.

PubMed

Chen, Zhang-Hui; Yu, Yan P; Michalopoulos, George; Nelson, Joel; Luo, Jian-Hua

2015-01-16

Miniature chromosome maintenance 7 (MCM7) is an essential component of DNA replication licensing complex. Recent studies indicate that MCM7 is amplified and overexpressed in a variety of human malignancies. In this report, we show that MCM7 binds SF3B3. The binding motif is located in the N terminus (amino acids 221-248) of MCM7. Knockdown of MCM7 or SF3B3 significantly increased unspliced RNA of epidermal growth factor receptor, platelet-derived growth factor receptor, and c-Met. A dramatic drop of reporter gene expression of the oxytocin exon 1-intron-exon 2-EGFP construct was also identified in SF3B3 and MCM7 knockdown PC3 and DU145 cells. The MCM7 or SF3B3 depleted cell extract failed to splice reporter RNA in in vitro RNA splicing analyses. Knockdown of SF3B3 and MCM7 leads to an increase of cell death of both PC3 and DU145 cells. Such cell death induction is partially rescued by expressing spliced c-Met. To our knowledge, this is the first report suggesting that MCM7 is a critical RNA splicing factor, thus giving significant new insight into the oncogenic activity of this protein. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Polysulfone ultrafiltration membrane incorporated with Ag-SiO2 nanohybrid for effective fouling control.

PubMed

Wu, Huiqing; Huang, Jing; Liu, Yuejun

2017-06-01

An anti-fouling hybrid membrane was prepared by incorporating Ag-SiO 2 nanohybrid into a polysulfone (PSf) matrix. The addition of Ag-SiO 2 can significantly improve the hydrophilicity, separation property, anti-fouling ability, and especially anti-bacterial activity of hybrid membranes. The optimum performance of the Ag-SiO 2 /PSf hybrid membrane is achieved when the concentration of Ag-SiO 2 is as low as 0.45 wt%. Compared with PSf membrane and SiO 2 /PSf hybrid membrane, the Ag-SiO 2 /PSf hybrid membrane displays the best overall properties. The excellent performance of the Ag-SiO 2 /PSf hybrid membrane can be attributed to the well-tailored structure and unique property of Ag-SiO 2 nanohybrid, where nanosized Ag (∼5 nm) can densely and uniformly disperse on the surface of silica spheres. The obtained membrane could be a promising material for water treatment.

Region-specific expression of brain-derived neurotrophic factor splice variants in morphine conditioned place preference in mice.

PubMed

Meng, Min; Zhao, Xinhan; Dang, Yonghui; Ma, Jingyuan; Li, Lixu; Gu, Shanzhi

2013-06-26

It is well established that brain-derived neurotrophic factor (BDNF) plays a pivotal role in brain plasticity-related processes, such as learning, memory and drug addiction. However, changes in expression of BDNF splice variants after acquisition, extinction and reinstatement of cue-elicited morphine seeking behavior have not yet been investigated. Real-time PCR was used to assess BDNF splice variants (I, II, IV and VI) in various brain regions during acquisition, extinction and reinstatement of morphine-conditioned place preference (CPP) in mice. Repeated morphine injections (10mg/kg, i.p.) increased expression of BDNF splice variants II, IV and VI in the hippocampus, caudate putamen (CPu) and nucleus accumbens (NAcc). Levels of BDNF splice variants decreased after extinction training and continued to decrease during reinstatement induced by a morphine priming injection (10mg/kg, i.p.). However, after reinstatement induced by exposure to 6 min of forced swimming (FS), expression of BDNF splice variants II, IV and VI was increased in the hippocampus, CPu, NAcc and prefrontal cortex (PFC). After reinstatement induced by 40 min of restraint, expression of BDNF splice variants was increased in PFC. These results show that exposure to either morphine or acute stress can induce reinstatement of drug-seeking, but expression of BDNF splice variants is differentially affected by chronic morphine and acute stress. Furthermore, BDNF splice variants II, IV and VI may play a role in learning and memory for morphine addiction in the hippocampus, CPu and NAcc. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

Distortion correction in EPI at ultra-high-field MRI using PSF mapping with optimal combination of shift detection dimension.

PubMed

Oh, Se-Hong; Chung, Jun-Young; In, Myung-Ho; Zaitsev, Maxim; Kim, Young-Bo; Speck, Oliver; Cho, Zang-Hee

2012-10-01

Despite its wide use, echo-planar imaging (EPI) suffers from geometric distortions due to off-resonance effects, i.e., strong magnetic field inhomogeneity and susceptibility. This article reports a novel method for correcting the distortions observed in EPI acquired at ultra-high-field such as 7 T. Point spread function (PSF) mapping methods have been proposed for correcting the distortions in EPI. The PSF shift map can be derived either along the nondistorted or the distorted coordinates. Along the nondistorted coordinates more information about compressed areas is present but it is prone to PSF-ghosting artifacts induced by large k-space shift in PSF encoding direction. In contrast, shift maps along the distorted coordinates contain more information in stretched areas and are more robust against PSF-ghosting. In ultra-high-field MRI, an EPI contains both compressed and stretched regions depending on the B0 field inhomogeneity and local susceptibility. In this study, we present a new geometric distortion correction scheme, which selectively applies the shift map with more information content. We propose a PSF-ghost elimination method to generate an artifact-free pixel shift map along nondistorted coordinates. The proposed method can correct the effects of the local magnetic field inhomogeneity induced by the susceptibility effects along with the PSF-ghost artifact cancellation. We have experimentally demonstrated the advantages of the proposed method in EPI data acquisitions in phantom and human brain using 7-T MRI. Copyright © 2011 Wiley Periodicals, Inc.

On-orbit point spread function estimation for THEOS imaging system

NASA Astrophysics Data System (ADS)

Khetkeeree, Suphongsa; Liangrocapart, Sompong

2018-03-01

In this paper, we present two approaches for net Point Spread Function (net-PSF) estimation of Thailand Earth Observation System (THEOS) imaging system. In the first approach, we estimate the net- PSF by employing the specification information of the satellite. The analytic model of the net- PSF based on the simple model of push-broom imaging system. This model consists of a scanner, optical system, detector and electronics system. The mathematical PSF model of each component is demonstrated in spatial domain. In the second approach, the specific target images from THEOS imaging system are analyzed to determine the net-PSF. For panchromatic imaging system, the images of the checkerboard target at Salon de Provence airport are used to analysis the net-PSF by slant-edge method. For multispectral imaging system, the new man-made targets are proposed. It is a pier bridge in Lamchabang, Chonburi, Thailand. This place has had a lot of bridges which have several width sizes and orientation. The pulse method is used to analysis the images of this bridge for estimating the net-PSF. Finally, the Full Width at Half Maximums (FWHMs) of the net-PSF of both approaches is compared. The results show that both approaches coincide and all Modulation Transfer Functions (MTFs) at Nyquist of both approaches are better than the requirement. However, the FWHM of multispectral system more deviate than panchromatic system, because the targets are not specially constructed for estimating the characteristics of the satellite imaging system.

Polysaccharides from Ganoderma formosanum function as a Th1 adjuvant and stimulate cytotoxic T cell response in vivo.

PubMed

Pi, Chia-Chen; Chu, Ching-Liang; Lu, Chu-Ying; Zhuang, Yu-Jing; Wang, Cheng-Li; Yu, Yao-Hsuan; Wang, Hui-Yi; Lin, Chih-Chung; Chen, Chun-Jen

2014-01-09

The fungus of Ganoderma is a basidiomycete that possesses a variety of pharmacological effects and has been used in traditional Asian medicine for centuries. Ganoderma formosanum is a native Ganoderma species isolated in Taiwan, and we have previously demonstrated that PS-F2, a polysaccharide fraction purified from the submerged culture broth of G. formosanum, exhibits immunostimulatory properties in macrophages. In this study, we further characterized the adjuvant functions of PS-F2. In vitro, PS-F2 stimulated dendritic cells (DCs) to produce proinflammatory cytokines, including TNF-α, interleukin (IL)-6, and IL-12/IL-23 p40. PS-F2 also stimulated DCs to express the maturation markers CD40, CD80, CD86, and MHC class II. In a murine splenocyte culture, PS-F2 treatment resulted in elevated expression of T-bet and interferon (IFN)-γ in T lymphocytes. When used as an adjuvant in vivo with the ovalbumin (OVA) antigen, PS-F2 stimulated OVA-specific antibody production and primed IFN-γ production in OVA-specific T lymphocytes. PS-F2-adjuvated immunization also induced OVA-specific CTLs, which protected mice from a challenge with tumor cells expressing OVA. Collectively, our data show that PS-F2 functions as an adjuvant capable of inducing a Th1-polarized adaptive immune response, which would be useful in vaccines against viruses and tumors. Copyright © 2013 Elsevier Ltd. All rights reserved.

Splicing factor SFRS1 recognizes a functionally diverse landscape of RNA transcripts.

PubMed

Sanford, Jeremy R; Wang, Xin; Mort, Matthew; Vanduyn, Natalia; Cooper, David N; Mooney, Sean D; Edenberg, Howard J; Liu, Yunlong

2009-03-01

Metazoan genes are encrypted with at least two superimposed codes: the genetic code to specify the primary structure of proteins and the splicing code to expand their proteomic output via alternative splicing. Here, we define the specificity of a central regulator of pre-mRNA splicing, the conserved, essential splicing factor SFRS1. Cross-linking immunoprecipitation and high-throughput sequencing (CLIP-seq) identified 23,632 binding sites for SFRS1 in the transcriptome of cultured human embryonic kidney cells. SFRS1 was found to engage many different classes of functionally distinct transcripts including mRNA, miRNA, snoRNAs, ncRNAs, and conserved intergenic transcripts of unknown function. The majority of these diverse transcripts share a purine-rich consensus motif corresponding to the canonical SFRS1 binding site. The consensus site was not only enriched in exons cross-linked to SFRS1 in vivo, but was also enriched in close proximity to splice sites. mRNAs encoding RNA processing factors were significantly overrepresented, suggesting that SFRS1 may broadly influence the post-transcriptional control of gene expression in vivo. Finally, a search for the SFRS1 consensus motif within the Human Gene Mutation Database identified 181 mutations in 82 different genes that disrupt predicted SFRS1 binding sites. This comprehensive analysis substantially expands the known roles of human SR proteins in the regulation of a diverse array of RNA transcripts.

Can the HIV-1 splicing machinery be targeted for drug discovery?

PubMed Central

Dlamini, Zodwa; Hull, Rodney

2017-01-01

HIV-1 is able to express multiple protein types and isoforms from a single 9 kb mRNA transcript. These proteins are also expressed at particular stages of viral development, and this is achieved through the control of alternative splicing and the export of these transcripts from the nucleus. The nuclear export is controlled by the HIV protein Rev being required to transport incompletely spliced and partially spliced mRNA from the nucleus where they are normally retained. This implies a close relationship between the control of alternate splicing and the nuclear export of mRNA in the control of HIV-1 viral proliferation. This review discusses both the processes. The specificity and regulation of splicing in HIV-1 is controlled by the use of specific splice sites as well as exonic splicing enhancer and exonic splicing silencer sequences. The use of these silencer and enhancer sequences is dependent on the serine arginine family of proteins as well as the heterogeneous nuclear ribonucleoprotein family of proteins that bind to these sequences and increase or decrease splicing. Since alternative splicing is such a critical factor in viral development, it presents itself as a promising drug target. This review aims to discuss the inhibition of splicing, which would stall viral development, as an anti-HIV therapeutic strategy. In this review, the most recent knowledge of splicing in human immunodeficiency viral development and the latest therapeutic strategies targeting human immunodeficiency viral splicing are discussed. PMID:28331370

An alternative splicing program promotes adipose tissue thermogenesis

PubMed Central

Vernia, Santiago; Edwards, Yvonne JK; Han, Myoung Sook; Cavanagh-Kyros, Julie; Barrett, Tamera; Kim, Jason K; Davis, Roger J

2016-01-01

Alternative pre-mRNA splicing expands the complexity of the transcriptome and controls isoform-specific gene expression. Whether alternative splicing contributes to metabolic regulation is largely unknown. Here we investigated the contribution of alternative splicing to the development of diet-induced obesity. We found that obesity-induced changes in adipocyte gene expression include alternative pre-mRNA splicing. Bioinformatics analysis associated part of this alternative splicing program with sequence specific NOVA splicing factors. This conclusion was confirmed by studies of mice with NOVA deficiency in adipocytes. Phenotypic analysis of the NOVA-deficient mice demonstrated increased adipose tissue thermogenesis and improved glycemia. We show that NOVA proteins mediate a splicing program that suppresses adipose tissue thermogenesis. Together, these data provide quantitative analysis of gene expression at exon-level resolution in obesity and identify a novel mechanism that contributes to the regulation of adipose tissue function and the maintenance of normal glycemia. DOI: http://dx.doi.org/10.7554/eLife.17672.001 PMID:27635635

Aberrant and alternative splicing in skeletal system disease.

PubMed

Fan, Xin; Tang, Liling

2013-10-01

The main function of skeletal system is to support the body and help movement. A variety of factors can lead to skeletal system disease, including age, exercise, and of course genetic makeup and expression. Pre-mRNA splicing plays a crucial role in gene expression, by creating multiple protein variants with different biological functions. The recent studies show that several skeletal system diseases are related to pre-mRNA splicing. This review focuses on the relationship between pre-mRNA splicing and skeletal system disease. On the one hand, splice site mutation that leads to aberrant splicing often causes genetic skeletal system disease, like COL1A1, SEDL and LRP5. On the other hand, alternative splicing without genomic mutation may generate some marker protein isoforms, for example, FN, VEGF and CD44. Therefore, understanding the relationship between pre-mRNA splicing and skeletal system disease will aid in uncovering the mechanism of disease and contribute to the future development of gene therapy. © 2013 Elsevier B.V. All rights reserved.

Small-Grid Dithering Strategy for Improved Coronagraphic Performance with JWST

NASA Astrophysics Data System (ADS)

Lajoie, Charles-Philippe; Soummer, Remi; Pueyo, Laurent; Hines, Dean C.; Nelan, Edmund P.; JWST Coronagraphs Working Group

2015-01-01

Contrast performances for most coronagraph designs typically depend rather strongly on the accuracy of target acquisition. For JWST, target acquisition away from the center of the coronagraphs will allow for centroid measurement, which will in turn be used to command a small-angle maneuver (SAM) to accurately place the star behind the coronagraphic mask. With this approach, the SAM accuracy inherently limits the contrast performance of the coronagraphs, especially given that a reference star (or self-reference after telescope roll) might also be required. For such differential measurements, the reproducibility of the TA is therefore a very important factor. Here, we propose a novel coronagraphic observation concept whereby the reference PSF is first acquired using a standard TA, followed by coronagraphic observations on a small grid of dithered positions. Sub-pixel dithers (5-10mas each) provide a small reference PSF library that sample the possible variations in the PSF shape due to imperfect TAs. This small library can then be used for example with principal component analysis for PSF subtraction (e.g; LOCI or KLIP algorithms). Such very small dithers can be achieved with the JWST attitude control system without overhead and with higher accuracy than a SAM since they take advantage of the fine steering mirror under closed-loop fine guidance. We discuss and evaluate the performance gains from this observation scenario compared to the standard TA for MIRI Four-Quadrant Phase Mask coronagraphs and provide numerical simulations for a some astrophysical targets of interest.

Fluorescence microscopy point spread function model accounting for aberrations due to refractive index variability within a specimen.

PubMed

Ghosh, Sreya; Preza, Chrysanthe

2015-07-01

A three-dimensional (3-D) point spread function (PSF) model for wide-field fluorescence microscopy, suitable for imaging samples with variable refractive index (RI) in multilayered media, is presented. This PSF model is a key component for accurate 3-D image restoration of thick biological samples, such as lung tissue. Microscope- and specimen-derived parameters are combined with a rigorous vectorial formulation to obtain a new PSF model that accounts for additional aberrations due to specimen RI variability. Experimental evaluation and verification of the PSF model was accomplished using images from 175-nm fluorescent beads in a controlled test sample. Fundamental experimental validation of the advantage of using improved PSFs in depth-variant restoration was accomplished by restoring experimental data from beads (6  μm in diameter) mounted in a sample with RI variation. In the investigated study, improvement in restoration accuracy in the range of 18 to 35% was observed when PSFs from the proposed model were used over restoration using PSFs from an existing model. The new PSF model was further validated by showing that its prediction compares to an experimental PSF (determined from 175-nm beads located below a thick rat lung slice) with a 42% improved accuracy over the current PSF model prediction.

Relative importance of competition and plant-soil feedback, their synergy, context dependency and implications for coexistence.

PubMed

Lekberg, Ylva; Bever, James D; Bunn, Rebecca A; Callaway, Ragan M; Hart, Miranda M; Kivlin, Stephanie N; Klironomos, John; Larkin, Beau G; Maron, John L; Reinhart, Kurt O; Remke, Michael; van der Putten, Wim H

2018-06-12

Plants interact simultaneously with each other and with soil biota, yet the relative importance of competition vs. plant-soil feedback (PSF) on plant performance is poorly understood. Using a meta-analysis of 38 published studies and 150 plant species, we show that effects of interspecific competition (either growing plants with a competitor or singly, or comparing inter- vs. intraspecific competition) and PSF (comparing home vs. away soil, live vs. sterile soil, or control vs. fungicide-treated soil) depended on treatments but were predominantly negative, broadly comparable in magnitude, and additive or synergistic. Stronger competitors experienced more negative PSF than weaker competitors when controlling for density (inter- to intraspecific competition), suggesting that PSF could prevent competitive dominance and promote coexistence. When competition was measured against plants growing singly, the strength of competition overwhelmed PSF, indicating that the relative importance of PSF may depend not only on neighbour identity but also density. We evaluate how competition and PSFs might interact across resource gradients; PSF will likely strengthen competitive interactions in high resource environments and enhance facilitative interactions in low-resource environments. Finally, we provide a framework for filling key knowledge gaps and advancing our understanding of how these biotic interactions influence community structure. © 2018 John Wiley & Sons Ltd/CNRS.

Effect of stride frequency on metabolic costs and rating of perceived exertion during walking in water.

PubMed

Masumoto, Kenji; Nishizaki, Yoshiko; Hamada, Ayako

2013-06-01

We investigated the effect of stride frequency (SF) on metabolic costs and rating of perceived exertion (RPE) during walking in water and on dry land. Eleven male subjects walked on a treadmill on dry land and on an underwater treadmill at their preferred SF (PSF) and walked at an SF which was lower and higher than the PSF (i.e., PSF ± 5, 10, and 15 strides min(-1)). Walking speed was kept constant at each subject's preferred walking speed in water and on dry land. Oxygen uptake, heart rate, RPE, PSF and preferred walking speeds were measured. Metabolic costs and RPE were significantly higher when walking at low and high SF conditions than when walking at the PSF condition both in water and on dry land (P<0.05). Additionally, the high SF condition produced significantly higher metabolic costs and RPE than the equivalent low SF condition during walking in water (P<0.01). Furthermore, metabolic costs, RPE, PSF, and the preferred walking speed were significantly lower in water than on dry land when walking at the PSF (P<0.05). These observations indicate that a change in SF influences metabolic costs and RPE during walking in water. Copyright © 2012 Elsevier B.V. All rights reserved.

The role of Amicar in same-day anterior and posterior spinal fusion for idiopathic scoliosis.

PubMed

Thompson, George H; Florentino-Pineda, Ivan; Poe-Kochert, Connie; Armstrong, Douglas G; Son-Hing, Jochen P

2008-09-15

A retrospective study of the effectiveness of Amicar (epsilon aminocaproic acid). Evaluate the effectiveness of Amicar in decreasing perioperative blood loss and transfusion requirements in same-day anterior (ASF) and posterior spinal fusion (PSF) with segmental spinal instrumentation (SSI) for idiopathic scoliosis. Preliminary prospective, prospective randomized double-blind, and fibrinogen studies have demonstrated Amicar to be effective in decreasing perioperative blood loss in patients with idiopathic scoliosis undergoing PSF with SSI. Increased fibrinogen secretion is a possible explanation. There were 73 consecutive patients divided into 3 study groups based on the administration of Amicar: Group 1 (n = 16), no Amicar; Group 2 (n = 18), Amicar for the PSF with SSI only; and Group 3 (n = 39), Amicar for both ASF and PSF with SSI. All patients were managed using the same general anesthesia technique, intraoperative procedure, postoperative care path, and indications for transfusion (hemoglobin <7 g/dL). Total perioperative blood loss (estimated intraoperative blood loss for both procedures and measured postoperative chest tube and PSF wound suction drainage) and total transfusion requirements between groups were compared using one-way ANOVA. There were statistically significant decreases in mean estimated intraoperative PSF with SSI, total perioperative blood loss, and transfusion requirements in the 2 Amicar groups. However, Amicar had no significant effect on estimated intraoperative ASF blood loss, chest tube drainage, or PSF wound suction drainage. Total perioperative blood loss and transfusion requirements (cell saver, autologous, directed, and allogeneic blood) were: 3442.8 +/- 1344.0 mL and 1537.1 +/- 905.1 mL in Group 1; 2089.8 +/- 684.0 mL and 485.2 +/- 349.8 mL in Group 2; and 2184.1 +/- 1163.7 mL and 531.5 +/- 510.5 mL in Group 3. There were no Amicar related complications. Amicar was highly effective in decreasing total perioperative blood loss and transfusion requirements in same-day ASF and PSF with SSI for idiopathic scoliosis. It results in less preoperative autologous blood donation, perioperative blood transfusion, costs, and potential transfusion-related complications. It was most effective in decreasing intraoperative estimated PSF with SSI blood loss. It had no significant effect during the ASF, postoperative chest tube, or PSF wound suction drainage. We now recommend that it be used for the PSF with SSI procedure only.

RNA splicing process analysis for identifying antisense oligonucleotide inhibitors with padlock probe-based isothermal amplification† †Electronic supplementary information (ESI) available: Additional experimental materials, methods, DNA sequences and supplementary figures and tables. See DOI: 10.1039/c7sc01336a Click here for additional data file.

PubMed Central

Ren, Xiaojun; Deng, Ruijie; Wang, Lida; Zhang, Kaixiang

2017-01-01

RNA splicing, which mainly involves two transesterification steps, is a fundamental process of gene expression and its abnormal regulation contributes to serious genetic diseases. Antisense oligonucleotides (ASOs) are genetic control tools that can be used to specifically control genes through alteration of the RNA splicing pathway. Despite intensive research, how ASOs or various other factors influence the multiple processes of RNA splicing still remains obscure. This is largely due to an inability to analyze the splicing efficiency of each step in the RNA splicing process with high sensitivity. We addressed this limitation by introducing a padlock probe-based isothermal amplification assay to achieve quantification of the specific products in different splicing steps. With this amplified assay, the roles that ASOs play in RNA splicing inhibition in the first and second steps could be distinguished. We identified that 5′-ASO could block RNA splicing by inhibiting the first step, while 3′-ASO could block RNA splicing by inhibiting the second step. This method provides a versatile tool for assisting efficient ASO design and discovering new splicing modulators and therapeutic drugs. PMID:28989608

Evolution of Nova-Dependent Splicing Regulation in the Brain

PubMed Central

Živin, Marko; Darnell, Robert B

2007-01-01

A large number of alternative exons are spliced with tissue-specific patterns, but little is known about how such patterns have evolved. Here, we study the conservation of the neuron-specific splicing factors Nova1 and Nova2 and of the alternatively spliced exons they regulate in mouse brain. Whereas Nova RNA binding domains are 94% identical across vertebrate species, Nova-dependent splicing silencer and enhancer elements (YCAY clusters) show much greater divergence, as less than 50% of mouse YCAY clusters are conserved at orthologous positions in the zebrafish genome. To study the relation between the evolution of tissue-specific splicing and YCAY clusters, we compared the brain-specific splicing of Nova-regulated exons in zebrafish, chicken, and mouse. The presence of YCAY clusters in lower vertebrates invariably predicted conservation of brain-specific splicing across species, whereas their absence in lower vertebrates correlated with a loss of alternative splicing. We hypothesize that evolution of Nova-regulated splicing in higher vertebrates proceeds mainly through changes in cis-acting elements, that tissue-specific splicing might in some cases evolve in a single step corresponding to evolution of a YCAY cluster, and that the conservation level of YCAY clusters relates to the functions encoded by the regulated RNAs. PMID:17937501

Effects of secondary structure on pre-mRNA splicing: hairpins sequestering the 5' but not the 3' splice site inhibit intron processing in Nicotiana plumbaginifolia.

PubMed

Liu, H X; Goodall, G J; Kole, R; Filipowicz, W

1995-01-16

We have performed a systematic study of the effect of artificial hairpins on pre-mRNA splicing in protoplasts of a dicot plant, Nicotiana plumbaginifolia. Hairpins with a potential to form 18 or 24 bp stems strongly inhibit splicing when they sequester the 5' splice site or are placed in the middle of short introns. However, similar 24 bp hairpins sequestering the 3' splice site do not prevent this site from being used as an acceptor. Utilization of the stem-located 3' site requires that the base of the stem is separated from the upstream 5' splice site by a minimum of approximately 45 nucleotides and that another 'helper' 3' splice site is present downstream of the stem. The results indicate that the spliceosome or factors associated with it may have a potential to unfold secondary structure present in the downstream portion of the intron, prior to or at the step of the 3' splice site selection. The finding that the helper 3' site is required for utilization of the stem-located acceptor confirms and extends previous observations, obtained with HeLa cell in vitro splicing systems, indicating that the 3' splice site may be recognized at least twice during spliceosome assembly.

Alternative Splicing Control of Abiotic Stress Responses.

PubMed

Laloum, Tom; Martín, Guiomar; Duque, Paula

2018-02-01

Alternative splicing, which generates multiple transcripts from the same gene, is an important modulator of gene expression that can increase proteome diversity and regulate mRNA levels. In plants, this post-transcriptional mechanism is markedly induced in response to environmental stress, and recent studies have identified alternative splicing events that allow rapid adjustment of the abundance and function of key stress-response components. In agreement, plant mutants defective in splicing factors are severely impaired in their response to abiotic stress. Notably, mounting evidence indicates that alternative splicing regulates stress responses largely by targeting the abscisic acid (ABA) pathway. We review here current understanding of post-transcriptional control of plant stress tolerance via alternative splicing and discuss research challenges for the near future. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Clk/Sty protein kinase phosphorylates SR splicing factors and regulates their intranuclear distribution.

PubMed Central

Colwill, K; Pawson, T; Andrews, B; Prasad, J; Manley, J L; Bell, J C; Duncan, P I

1996-01-01

Mammalian Clk/Sty is the prototype for a family of dual specificity kinases (termed LAMMER kinases) that have been conserved in evolution, but whose physiological substrates are unknown. In a yeast two-hybrid screen, the Clk/Sty kinase specifically interacted with RNA binding proteins, particularly members of the serine/arginine-rich (SR) family of splicing factors. Clk/Sty itself has an serine/arginine-rich non-catalytic N-terminal region which is important for its association with SR splicing factors. In vitro, Clk/Sty efficiently phosphorylated the SR family member ASF/SF2 on serine residues located within its serine/arginine-rich region (the RS domain). Tryptic phosphopeptide mapping demonstrated that the sites on ASF/SF2 phosphorylated in vitro overlap with those phosphorylated in vivo. Immunofluorescence studies showed that a catalytically inactive form of Clk/Sty co-localized with SR proteins in nuclear speckles. Overexpression of the active Clk/Sty kinase caused a redistribution of SR proteins within the nucleus. These results suggest that Clk/Sty kinase directly regulates the activity and compartmentalization of SR splicing factors. Images PMID:8617202

Clinical evaluation of whole-body oncologic PET with time-of-flight and point-spread function for the hybrid PET/MR system.

PubMed

Shang, Kun; Cui, Bixiao; Ma, Jie; Shuai, Dongmei; Liang, Zhigang; Jansen, Floris; Zhou, Yun; Lu, Jie; Zhao, Guoguang

2017-08-01

Hybrid positron emission tomography/magnetic resonance (PET/MR) imaging is a new multimodality imaging technology that can provide structural and functional information simultaneously. The aim of this study was to investigate the effects of the time-of-flight (TOF) and point-spread function (PSF) on small lesions observed in PET/MR images from clinical patient image sets. This study evaluated 54 small lesions in 14 patients who had undergone 18 F-fluorodeoxyglucose (FDG) PET/MR. Lesions up to 30mm in diameter were included. The PET data were reconstructed with a baseline ordered-subsets expectation-maximization (OSEM) algorithm, OSEM+PSF, OSEM+TOF and OSEM+TOF+PSF. PET image quality and small lesions were visually evaluated and scored by a 3-point scale. A quantitative analysis was then performed using the mean and maximum standardized uptake value (SUV) of the small lesions (SUV mean and SUV max ). The lesions were divided into two groups according to the long-axis diameter and the location respectively and evaluated with each reconstruction algorithm. We also evaluated the background signal by analyzing the SUV liver . OSEM+TOF+PSF provided the highest value and OSEM+TOF or PSF showed a higher value than OSEM for the visual assessment and quantitative analysis. The combination of TOF and PSF increased the SUV mean by 26.6% and the SUV max by 30.0%. The SUV liver was not influenced by PSF or TOF. For the OSEM+TOF+PSF model, the change in SUV mean and SUV max for lesions <10mm in diameter was 31.9% and 35.8%, and 24.5% and 27.6% for lesions 10-30mm in diameter, respectively. The abdominal lesions obtained the higher SUV than those of chest on the images with TOF and/or PSF. Application of TOF and PSF significantly increased the SUV of small lesions in hybrid PET/MR images, potentially improving small lesion detectability. Copyright © 2017 Elsevier B.V. All rights reserved.

An in vivo genetic screen for genes involved in spliced leader trans-splicing indicates a crucial role for continuous de novo spliced leader RNP assembly.

PubMed

Philippe, Lucas; Pandarakalam, George C; Fasimoye, Rotimi; Harrison, Neale; Connolly, Bernadette; Pettitt, Jonathan; Müller, Berndt

2017-08-21

Spliced leader (SL) trans-splicing is a critical element of gene expression in a number of eukaryotic groups. This process is arguably best understood in nematodes, where biochemical and molecular studies in Caenorhabditis elegans and Ascaris suum have identified key steps and factors involved. Despite this, the precise details of SL trans-splicing have yet to be elucidated. In part, this is because the systematic identification of the molecules involved has not previously been possible due to the lack of a specific phenotype associated with defects in this process. We present here a novel GFP-based reporter assay that can monitor SL1 trans-splicing in living C. elegans. Using this assay, we have identified mutants in sna-1 that are defective in SL trans-splicing, and demonstrate that reducing function of SNA-1, SNA-2 and SUT-1, proteins that associate with SL1 RNA and related SmY RNAs, impairs SL trans-splicing. We further demonstrate that the Sm proteins and pICln, SMN and Gemin5, which are involved in small nuclear ribonucleoprotein assembly, have an important role in SL trans-splicing. Taken together these results provide the first in vivo evidence for proteins involved in SL trans-splicing, and indicate that continuous replacement of SL ribonucleoproteins consumed during trans-splicing reactions is essential for effective trans-splicing. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Study of GLAO-corrected PSF evolution for the MUSE Wide Field Mode. Expected performance and requirements for PSF reconstruction

NASA Astrophysics Data System (ADS)

Fusco, T.; Villecroze, R.; Jarno, A.; Bacon, R.

2011-09-01

The second generation instrument MUSE for the VLT has been designed to profit of the ESO Adaptive Optics Facility (AOF). The two Adaptive Optics (AO) modes (GLAO in Wide Field Mode [WFM] and LTAO in Narrow Field Mode [NFM]) will be used. To achieve its key science goals, MUSE will require information on the full system (Atmosphere, AO, telescope and instrument) image quality and its variation with Field position and wavelength. For example, optimal summation of a large number of deep field exposures in WFM will require a good knowledge of the PSF. In this paper, we will present an exhaustive analysis of the MUSE Wide Field Mode PSF evolution both spatially and spectrally. For that purpose we have coupled a complete AO simulation tool developed at ONERA with the MUSE instrumental PSF simulation. Relative impact of atmospheric and system parameters (seeing, Cn^2, LGS and NGS positions etc ...) with respect to differential MUSE aberrations per channel (i.e. slicer and IFU) is analysed. The results allow us (in close collaboration with astronomers) to define pertinent parameters (fit parameters using a Moffat function) for a PSF reconstruction process (estimation of this parameters using GLAO telemetry) and to propose an efficient and robust algorithm to be implemented in the MUSE pipeline. The extension of the spatial and spectral PSF analysis to the NFM case is discussed and preliminary results are given. Some specific requirements for the generalisation of the GLAO PSF reconstruction process to the LTAO case are derived from these early results.

Selective aggregation of the splicing factor Hsh155 suppresses splicing upon genotoxic stress.

PubMed

Mathew, Veena; Tam, Annie S; Milbury, Karissa L; Hofmann, Analise K; Hughes, Christopher S; Morin, Gregg B; Loewen, Christopher J R; Stirling, Peter C

2017-12-04

Upon genotoxic stress, dynamic relocalization events control DNA repair as well as alterations of the transcriptome and proteome, enabling stress recovery. How these events may influence one another is only partly known. Beginning with a cytological screen of genome stability proteins, we find that the splicing factor Hsh155 disassembles from its partners and localizes to both intranuclear and cytoplasmic protein quality control (PQC) aggregates under alkylation stress. Aggregate sequestration of Hsh155 occurs at nuclear and then cytoplasmic sites in a manner that is regulated by molecular chaperones and requires TORC1 activity signaling through the Sfp1 transcription factor. This dynamic behavior is associated with intron retention in ribosomal protein gene transcripts, a decrease in splicing efficiency, and more rapid recovery from stress. Collectively, our analyses suggest a model in which some proteins evicted from chromatin and undergoing transcriptional remodeling during stress are targeted to PQC sites to influence gene expression changes and facilitate stress recovery. © 2017 Mathew et al.

Regulation of human adenovirus alternative RNA splicing by the adenoviral L4-33K and L4-22K proteins.

PubMed

Biasiotto, Roberta; Akusjärvi, Göran

2015-01-28

Adenovirus makes extensive use of alternative RNA splicing to produce a complex set of spliced viral mRNAs. Studies aimed at characterizing the interactions between the virus and the host cell RNA splicing machinery have identified three viral proteins of special significance for the control of late viral gene expression: L4-33K, L4-22K, and E4-ORF4. L4-33K is a viral alternative RNA splicing factor that controls L1 alternative splicing via an interaction with the cellular protein kinases Protein Kinase A (PKA) and DNA-dependent protein kinase (DNA-PK). L4-22K is a viral transcription factor that also has been implicated in the splicing of a subset of late viral mRNAs. E4-ORF4 is a viral protein that binds the cellular protein phosphatase IIA (PP2A) and controls Serine/Arginine (SR)-rich protein activity by inducing SR protein dephosphorylation. The L4-33K, and most likely also the L4-22K protein, are highly phosphorylated in vivo. Here we will review the function of these viral proteins in the post-transcriptional control of adenoviral gene expression and further discuss the significance of potential protein kinases phosphorylating the L4-33K and/or L4-22K proteins.

Drug discovery with an RBM20 dependent titin splice reporter identifies cardenolides as lead structures to improve cardiac filling.

PubMed

Liss, Martin; Radke, Michael H; Eckhard, Jamina; Neuenschwander, Martin; Dauksaite, Vita; von Kries, Jens-Peter; Gotthardt, Michael

2018-01-01

Diastolic dysfunction is increasingly prevalent in our ageing society and an important contributor to heart failure. The giant protein titin could serve as a therapeutic target, as its elastic properties are a main determinant of cardiac filling in diastole. This study aimed to develop a high throughput pharmacological screen to identify small molecules that affect titin isoform expression through differential inclusion of exons encoding the elastic PEVK domains. We used a dual luciferase splice reporter assay that builds on the titin splice factor RBM20 to screen ~34,000 small molecules and identified several compounds that inhibit the exclusion of PEVK exons. These compounds belong to the class of cardenolides and affect RBM20 dependent titin exon exclusion but did not affect RBFOX1 mediated splicing of FMNL3. We provide evidence that cardenolides do not bind to the RNA interacting domain of RBM20, but reduce RBM20 protein levels and alter transcription of select splicing factors that interact with RBM20. Cardenolides affect titin isoform expression. Understanding their mode of action and harnessing the splice effects through chemical modifications that suppress the effects on ion homeostasis and more selectively affect cardiac splicing has the potential to improve cardiac filling and thus help patients with diastolic heart failure, for which currently no targeted therapy exists.

CDKL5 influences RNA splicing activity by its association to the nuclear speckle molecular machinery.

PubMed

Ricciardi, Sara; Kilstrup-Nielsen, Charlotte; Bienvenu, Thierry; Jacquette, Aurélia; Landsberger, Nicoletta; Broccoli, Vania

2009-12-01

Mutations in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been shown to cause severe neurodevelopmental disorders including infantile spasms, encephalopathy, West-syndrome and an early-onset variant of Rett syndrome. CDKL5 is a serine/threonine kinase whose involvement in Rett syndrome can be inferred by its ability to directly bind and mediate phosphorylation of MeCP2. However, it remains to be elucidated how CDKL5 exerts its function. Here, we report that CDKL5 localizes to specific nuclear foci referred to as nuclear speckles in both cell lines and tissues. These sub-nuclear structures are traditionally considered as storage/modification sites of pre-mRNA splicing factors. Interestingly, we provide evidence that CDKL5 regulates the dynamic behaviour of nuclear speckles. Indeed, CDKL5 overexpression leads to nuclear speckle disassembly, and this event is strictly dependent on its kinase activity. Conversely, its down-regulation affects nuclear speckle morphology leading to abnormally large and uneven speckles. Similar results were obtained for primary adult fibroblasts isolated from CDKL5-mutated patients. Altogether, these findings indicate that CDKL5 controls nuclear speckle morphology probably by regulating the phosphorylation state of splicing regulatory proteins. Nuclear speckles are dynamic sites that can continuously supply splicing factors to active transcription sites, where splicing occurs. Notably, we proved that CDKL5 influences alternative splicing, at least as proved in heterologous minigene assays. In conclusion, we provide evidence that CDKL5 is involved indirectly in pre-mRNA processing, by controlling splicing factor dynamics. These findings identify a biological process whose disregulation might affect neuronal maturation and activity in CDKL5-related disorders.

Vimentin and PSF act in concert to regulate IbeA+ E. coli K1 induced activation and nuclear translocation of NF-κB in human brain endothelial cells.

PubMed

Chi, Feng; Bo, Tao; Wu, Chun-Hua; Jong, Ambrose; Huang, Sheng-He

2012-01-01

IbeA-induced NF-κB signaling through its primary receptor vimentin as well as its co-receptor PSF is required for meningitic E. coli K1 penetration and leukocyte transmigration across the blood-brain barrier (BBB), which are the hallmarks of bacterial meningitis. However, it is unknown how vimentin and PSF cooperatively contribute to IbeA-induced cytoplasmic activation and nuclear translocation of NF-κB, which are required for bacteria-mediated pathogenicities. IbeA-induced E. coli K1 invasion, polymorphonuclear leukocyte (PMN) transmigration and IKK/NF-κB activation are blocked by Caffeic acid phenethyl ester (CAPE), an inhibitor of NF-κB. IKKα/β phosphorylation is blocked by ERK inhibitors. Co-immunoprecipitation analysis shows that vimentin forms a complex with IκB, NF-κB and tubulins in the resting cells. A dissociation of this complex and a simultaneous association of PSF with NF-κB could be induced by IbeA in a time-dependent manner. The head domain of vimentin is required for the complex formation. Two cytoskeletal components, vimentin filaments and microtubules, contribute to the regulation of NF-κB. SiRNA-mediated knockdown studies demonstrate that IKKα/β phosphorylation is completely abolished in HBMECs lacking vimentin and PSF. Phosphorylation of ERK and nuclear translocation of NF-κB are entirely dependent on PSF. These findings suggest that vimentin and PSF cooperatively contribute to IbeA-induced cytoplasmic activation and nuclear translocation of NF-κB activation. PSF is essential for translocation of NF-κB and ERK to the nucleus. These findings reveal previously unappreciated facets of the IbeA-binding proteins. Cooperative contributions of vimentin and PSF to IbeA-induced cytoplasmic activation and nuclear translocation of NF-κB may represent a new paradigm in pathogen-induced signal transduction and lead to the development of novel strategies for the prevention and treatment of bacterial meningitis.

Prolyl hydroxylation regulates protein degradation, synthesis, and splicing in human induced pluripotent stem cell-derived cardiomyocytes

PubMed Central

Stoehr, Andrea; Yang, Yanqin; Patel, Sajni; Evangelista, Alicia M.; Aponte, Angel; Wang, Guanghui; Liu, Poching; Boylston, Jennifer; Kloner, Philip H.; Lin, Yongshun; Gucek, Marjan; Zhu, Jun; Murphy, Elizabeth

2016-01-01

Aims Protein hydroxylases are oxygen- and α-ketoglutarate-dependent enzymes that catalyse hydroxylation of amino acids such as proline, thus linking oxygen and metabolism to enzymatic activity. Prolyl hydroxylation is a dynamic post-translational modification that regulates protein stability and protein–protein interactions; however, the extent of this modification is largely uncharacterized. The goals of this study are to investigate the biological consequences of prolyl hydroxylation and to identify new targets that undergo prolyl hydroxylation in human cardiomyocytes. Methods and results We used human induced pluripotent stem cell-derived cardiomyocytes in combination with pulse-chase amino acid labelling and proteomics to analyse the effects of prolyl hydroxylation on protein degradation and synthesis. We identified 167 proteins that exhibit differences in degradation with inhibition of prolyl hydroxylation by dimethyloxalylglycine (DMOG); 164 were stabilized. Proteins involved in RNA splicing such as serine/arginine-rich splicing factor 2 (SRSF2) and splicing factor and proline- and glutamine-rich (SFPQ) were stabilized with DMOG. DMOG also decreased protein translation of cytoskeletal and sarcomeric proteins such as α-cardiac actin. We searched the mass spectrometry data for proline hydroxylation and identified 134 high confidence peptides mapping to 78 unique proteins. We identified SRSF2, SFPQ, α-cardiac actin, and cardiac titin as prolyl hydroxylated. We identified 29 prolyl hydroxylated proteins that showed a significant difference in either protein degradation or synthesis. Additionally, we performed next-generation RNA sequencing and showed that the observed decrease in protein synthesis was not due to changes in mRNA levels. Because RNA splicing factors were prolyl hydroxylated, we investigated splicing ± inhibition of prolyl hydroxylation and detected 369 alternative splicing events, with a preponderance of exon skipping. Conclusions This study provides the first extensive characterization of the cardiac prolyl hydroxylome and demonstrates that inhibition of α-ketoglutarate hydroxylases alters protein stability, translation, and splicing. PMID:27095734

High resolution multiple excitation spot optical microscopy

NASA Astrophysics Data System (ADS)

Dilipkumar, Shilpa; Mondal, Partha Pratim

2011-06-01

We propose fundamental improvements in three-dimensional (3D) resolution of multiple excitation spot optical microscopy. The excitation point spread function (PSF) is generated by two interfering counter-propagating depth-of-focus beams along the optical axis. Detection PSF is obtained by coherently interfering the emitted fluorescent light (collected by both the objectives) at the detector. System PSF shows upto 14-fold reduction in focal volume as compared to confocal, and almost 2-fold improvement in lateral resolution. Proposed PSF has the ability to simultaneously excite multiple 3D-spots of sub-femtoliter volume. Potential applications are in fluorescence microscopy and nanobioimaging.

C3G dynamically associates with nuclear speckles and regulates mRNA splicing.

PubMed

Shakyawar, Dhruv Kumar; Muralikrishna, Bhattiprolu; Radha, Vegesna

2018-05-01

C3G (Crk SH3 domain binding guanine nucleotide releasing factor) (Rap guanine nucleotide exchange factor 1), essential for mammalian embryonic development, is ubiquitously expressed and undergoes regulated nucleocytoplasmic exchange. Here we show that C3G localizes to SC35-positive nuclear speckles and regulates splicing activity. Reversible association of C3G with speckles was seen on inhibition of transcription and splicing. C3G shows partial colocalization with SC35 and is recruited to a chromatin and RNase-sensitive fraction of speckles. Its presence in speckles is dependent on intact cellular actin cytoskeleton and is lost on expression of the kinase Clk1. Rap1, a substrate of C3G, is also present in nuclear speckles, and inactivation of Rap signaling by expression of GFP-Rap1GAP alters speckle morphology and number. Enhanced association of C3G with speckles is seen on glycogen synthase kinase 3 beta inhibition or differentiation of C2C12 cells to myotubes. CRISPR/Cas9-mediated knockdown of C3G resulted in altered splicing activity of an artificial gene as well as endogenous CD44. C3G knockout clones of C2C12 as well as MDA-MB-231 cells showed reduced protein levels of several splicing factors compared with control cells. Our results identify C3G and Rap1 as novel components of nuclear speckles and a role for C3G in regulating cellular RNA splicing activity.

Validation and Interrogation of Differentially Expressed and Alternately Spliced Genes in African American Prostate Cancer

DTIC Science & Technology

2017-10-01

aggressive disease. 15. SUBJECT TERMS Prostate cancer, health disparities among racial groups, molecular mechanisms, differential gene expression...identify molecular mechanisms of tumor aggressiveness. The studies proposed here address the urgent need to elucidate the molecular mechanisms underlying... genetic /epigenetic/post-transcriptional factors in AA prostate cancer and Gleason grade and 2) manipulate splicing using novel splice-switching

Functional impact of splice isoform diversity in individual cells

PubMed Central

Yap, Karen; Makeyev, Eugene V.

2016-01-01

Alternative pre-mRNA splicing provides an effective means for expanding coding capacity of eukaryotic genomes. Recent studies suggest that co-expression of different splice isoforms may increase diversity of RNAs and proteins at a single-cell level. A pertinent question in the field is whether such co-expression is biologically meaningful or, rather, represents insufficiently stringent splicing regulation. Here we argue that isoform co-expression may produce functional outcomes that are difficult and sometimes impossible to achieve using other regulation strategies. Far from being a ‘splicing noise’, co-expression is often established through co-ordinated activity of specific cis-elements and trans-acting factors. Further work in this area may uncover new biological functions of alternative splicing (AS) and generate important insights into mechanisms allowing different cell types to attain their unique molecular identities. PMID:27528755

Functional impact of splice isoform diversity in individual cells.

PubMed

Yap, Karen; Makeyev, Eugene V

2016-08-15

Alternative pre-mRNA splicing provides an effective means for expanding coding capacity of eukaryotic genomes. Recent studies suggest that co-expression of different splice isoforms may increase diversity of RNAs and proteins at a single-cell level. A pertinent question in the field is whether such co-expression is biologically meaningful or, rather, represents insufficiently stringent splicing regulation. Here we argue that isoform co-expression may produce functional outcomes that are difficult and sometimes impossible to achieve using other regulation strategies. Far from being a 'splicing noise', co-expression is often established through co-ordinated activity of specific cis-elements and trans-acting factors. Further work in this area may uncover new biological functions of alternative splicing (AS) and generate important insights into mechanisms allowing different cell types to attain their unique molecular identities. © 2016 The Author(s).

Point spread functions for earthquake source imaging: An interpretation based on seismic interferometry

USGS Publications Warehouse

Nakahara, Hisashi; Haney, Matt

2015-01-01

Recently, various methods have been proposed and applied for earthquake source imaging, and theoretical relationships among the methods have been studied. In this study, we make a follow-up theoretical study to better understand the meanings of earthquake source imaging. For imaging problems, the point spread function (PSF) is used to describe the degree of blurring and degradation in an obtained image of a target object as a response of an imaging system. In this study, we formulate PSFs for earthquake source imaging. By calculating the PSFs, we find that waveform source inversion methods remove the effect of the PSF and are free from artifacts. However, the other source imaging methods are affected by the PSF and suffer from the effect of blurring and degradation due to the restricted distribution of receivers. Consequently, careful treatment of the effect is necessary when using the source imaging methods other than waveform inversions. Moreover, the PSF for source imaging is found to have a link with seismic interferometry with the help of the source-receiver reciprocity of Green’s functions. In particular, the PSF can be related to Green’s function for cases in which receivers are distributed so as to completely surround the sources. Furthermore, the PSF acts as a low-pass filter. Given these considerations, the PSF is quite useful for understanding the physical meaning of earthquake source imaging.

Computational-optical microscopy for 3D biological imaging beyond the diffraction limit

NASA Astrophysics Data System (ADS)

Grover, Ginni

In recent years, super-resolution imaging has become an important fluorescent microscopy tool. It has enabled imaging of structures smaller than the optical diffraction limit with resolution less than 50 nm. Extension to high-resolution volume imaging has been achieved by integration with various optical techniques. In this thesis, development of a fluorescent microscope to enable high resolution, extended depth, three dimensional (3D) imaging is discussed; which is achieved by integration of computational methods with optical systems. In the first part of the thesis, point spread function (PSF) engineering for volume imaging is discussed. A class of PSFs, referred to as double-helix (DH) PSFs, is generated. The PSFs exhibit two focused spots in the image plane which rotate about the optical axis, encoding depth in rotation of the image. These PSFs extend the depth-of-field up to a factor of ˜5. Precision performance of the DH-PSFs, based on an information theoretical analysis, is compared with other 3D methods with conclusion that the DH-PSFs provide the best precision and the longest depth-of-field. Out of various possible DH-PSFs, a suitable PSF is obtained for super-resolution microscopy. The DH-PSFs are implemented in imaging systems, such as a microscope, with a special phase modulation at the pupil plane. Surface-relief elements which are polarization-insensitive and ˜90% light efficient are developed for phase modulation. The photon-efficient DH-PSF microscopes thus developed are used, along with optimal position estimation algorithms, for tracking and super-resolution imaging in 3D. Imaging at depths-of-field of up to 2.5 microm is achieved without focus scanning. Microtubules were imaged with 3D resolution of (6, 9, 39) nm, which is in close agreement with the theoretical limit. A quantitative study of co-localization of two proteins in volume was conducted in live bacteria. In the last part of the thesis practical aspects of the DH-PSF microscope are discussed. A method to stabilize it, for extended periods of time, with 3-4 nm precision in 3D is developed. 3D Super-resolution is demonstrated without drift. A PSF correction algorithm is demonstrated to improve characteristics of the DH-PSF in an experiment, where it is implemented with a polarization-insensitive liquid crystal spatial light modulator.

U2AF1 mutations alter splice site recognition in hematological malignancies.

PubMed

Ilagan, Janine O; Ramakrishnan, Aravind; Hayes, Brian; Murphy, Michele E; Zebari, Ahmad S; Bradley, Philip; Bradley, Robert K

2015-01-01

Whole-exome sequencing studies have identified common mutations affecting genes encoding components of the RNA splicing machinery in hematological malignancies. Here, we sought to determine how mutations affecting the 3' splice site recognition factor U2AF1 alter its normal role in RNA splicing. We find that U2AF1 mutations influence the similarity of splicing programs in leukemias, but do not give rise to widespread splicing failure. U2AF1 mutations cause differential splicing of hundreds of genes, affecting biological pathways such as DNA methylation (DNMT3B), X chromosome inactivation (H2AFY), the DNA damage response (ATR, FANCA), and apoptosis (CASP8). We show that U2AF1 mutations alter the preferred 3' splice site motif in patients, in cell culture, and in vitro. Mutations affecting the first and second zinc fingers give rise to different alterations in splice site preference and largely distinct downstream splicing programs. These allele-specific effects are consistent with a computationally predicted model of U2AF1 in complex with RNA. Our findings suggest that U2AF1 mutations contribute to pathogenesis by causing quantitative changes in splicing that affect diverse cellular pathways, and give insight into the normal function of U2AF1's zinc finger domains. © 2015 Ilagan et al.; Published by Cold Spring Harbor Laboratory Press.

A Systems-Level Analysis Reveals Circadian Regulation of Splicing in Colorectal Cancer.

PubMed

El-Athman, Rukeia; Fuhr, Luise; Relógio, Angela

2018-06-20

Accumulating evidence points to a significant role of the circadian clock in the regulation of splicing in various organisms, including mammals. Both dysregulated circadian rhythms and aberrant pre-mRNA splicing are frequently implicated in human disease, in particular in cancer. To investigate the role of the circadian clock in the regulation of splicing in a cancer progression context at the systems-level, we conducted a genome-wide analysis and compared the rhythmic transcriptional profiles of colon carcinoma cell lines SW480 and SW620, derived from primary and metastatic sites of the same patient, respectively. We identified spliceosome components and splicing factors with cell-specific circadian expression patterns including SRSF1, HNRNPLL, ESRP1, and RBM 8A, as well as altered alternative splicing events and circadian alternative splicing patterns of output genes (e.g., VEGFA, NCAM1, FGFR2, CD44) in our cellular model. Our data reveals a remarkable interplay between the circadian clock and pre-mRNA splicing with putative consequences in tumor progression and metastasis. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Splicing regulatory factors, ageing and age-related disease.

PubMed

Latorre, Eva; Harries, Lorna W

2017-07-01

Alternative splicing is a co-transcriptional process, which allows for the production of multiple transcripts from a single gene and is emerging as an important control point for gene expression. Alternatively expressed isoforms often have antagonistic function and differential temporal or spatial expression patterns, yielding enormous plasticity and adaptability to cells and increasing their ability to respond to environmental challenge. The regulation of alternative splicing is critical for numerous cellular functions in both pathological and physiological conditions, and deregulated alternative splicing is a key feature of common chronic diseases. Isoform choice is controlled by a battery of splicing regulatory proteins, which include the serine arginine rich (SRSF) proteins and the heterogeneous ribonucleoprotein (hnRNP) classes of genes. These important splicing regulators have been implicated in age-related disease, and in the ageing process itself. This review will outline the important contribution of splicing regulator proteins to ageing and age-related disease. Copyright © 2017 Elsevier B.V. All rights reserved.

Genome-wide analysis of alternative splicing during dendritic cell response to a bacterial challenge.

PubMed

Rodrigues, Raquel; Grosso, Ana Rita; Moita, Luís

2013-01-01

The immune system relies on the plasticity of its components to produce appropriate responses to frequent environmental challenges. Dendritic cells (DCs) are critical initiators of innate immunity and orchestrate the later and more specific adaptive immunity. The generation of diversity in transcriptional programs is central for effective immune responses. Alternative splicing is widely considered a key generator of transcriptional and proteomic complexity, but its role has been rarely addressed systematically in immune cells. Here we used splicing-sensitive arrays to assess genome-wide gene- and exon-level expression profiles in human DCs in response to a bacterial challenge. We find widespread alternative splicing events and splicing factor transcriptional signatures induced by an E. coli challenge to human DCs. Alternative splicing acts in concert with transcriptional modulation, but these two mechanisms of gene regulation affect primarily distinct functional gene groups. Alternative splicing is likely to have an important role in DC immunobiology because it affects genes known to be involved in DC development, endocytosis, antigen presentation and cell cycle arrest.

WebbPSF: Updated PSF Models Based on JWST Ground Testing Results

NASA Astrophysics Data System (ADS)

Osborne, Shannon; Perrin, Marshall D.; Melendez Hernandez, Marcio

2018-06-01

WebbPSF is a widely-used package that allows astronomers to create simulated point spread functions (PSFs) for the James Webb Space Telescope (JWST). WebbPSF provides the user with the flexibility to produce PSFs for direct imaging and coronographic modes, for a range of filters and masks, and across all the JWST instruments. These PSFs can then be analyzed with built-in evaluation tools or can be output to be used with users’ own tools. In the most recent round of updates, the accuracy of the PSFs have been improved with updated analyses of the instrument test data from NASA Goddard and with the new data from the testing of the combined Optical Telescope Element and Integrated Science Instrument Module (OTIS) at NASA Johnson. A post-processing function applying detector effects and pupil distortions to input PSFs has also been added to the WebbPSF package.

Obtaining the phase in the star test using genetic algorithms

NASA Astrophysics Data System (ADS)

Salazar Romero, Marcos A.; Vazquez-Montiel, Sergio; Cornejo-Rodriguez, Alejandro

2004-10-01

The star test is conceptually perhaps the most basic and simplest of all methods of testing image-forming optical systems, the irradiance distribution at the image of a point source (such as a star) is give for the Point Spread Function, PSF. The PSF is very sensitive to aberrations. One way to quantify the PSF is measuring the irradiance distribution on the image of the source point. On the other hand, if we know the aberrations introduced by the optical systems and utilizing the diffraction theory then we can calculate the PSF. In this work we propose a method in order to find the wavefront aberrations starting from the PSF, transforming the problem of fitting a polynomial of aberrations in a problem of optimization using Genetic Algorithm. Also, we show that this method is immune to the noise introduced in the register or recording of the image. Results of these methods are shown.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fu, Houqiang; Lu, Zhijian; Zhao, Yuji

We study the low efficiency droop characteristics of semipolar InGaN light-emitting diodes (LEDs) using modified rate equation incoporating the phase-space filling (PSF) effect where the results on c-plane LEDs are also obtained and compared. Internal quantum efficiency (IQE) of LEDs was simulated using a modified ABC model with different PSF filling (n{sub 0}), Shockley-Read-Hall (A), radiative (B), Auger (C) coefficients and different active layer thickness (d), where the PSF effect showed a strong impact on the simulated LED efficiency results. A weaker PSF effect was found for low-droop semipolar LEDs possibly due to small quantum confined Stark effect, short carriermore » lifetime, and small average carrier density. A very good agreement between experimental data and the theoretical modeling was obtained for low-droop semipolar LEDs with weak PSF effect. These results suggest the low droop performance may be explained by different mechanisms for semipolar LEDs.« less

Three dimensional single molecule localization using a phase retrieved pupilfunction

PubMed Central

Liu, Sheng; Kromann, Emil B.; Krueger, Wesley D.; Bewersdorf, Joerg; Lidke, Keith A.

2013-01-01

Localization-based superresolution imaging is dependent on finding the positions of individualfluorophores in a sample by fitting the observed single-molecule intensity pattern to the microscopepoint spread function (PSF). For three-dimensional imaging, system-specific aberrations of theoptical system can lead to inaccurate localizations when the PSF model does not account for theseaberrations. Here we describe the use of phase-retrieved pupil functions to generate a more accuratePSF and therefore more accurate 3D localizations. The complex-valued pupil function containsinformation about the system-specific aberrations and can thus be used to generate the PSF forarbitrary defocus. Further, it can be modified to include depth dependent aberrations. We describethe phase retrieval process, the method for including depth dependent aberrations, and a fastfitting algorithm using graphics processing units. The superior localization accuracy of the pupilfunction generated PSF is demonstrated with dual focal plane 3D superresolution imaging ofbiological structures. PMID:24514501

A genome-wide aberrant RNA splicing in patients with acute myeloid leukemia identifies novel potential disease markers and therapeutic targets.

PubMed

Adamia, Sophia; Haibe-Kains, Benjamin; Pilarski, Patrick M; Bar-Natan, Michal; Pevzner, Samuel; Avet-Loiseau, Herve; Lode, Laurence; Verselis, Sigitas; Fox, Edward A; Burke, John; Galinsky, Ilene; Dagogo-Jack, Ibiayi; Wadleigh, Martha; Steensma, David P; Motyckova, Gabriela; Deangelo, Daniel J; Quackenbush, John; Stone, Richard; Griffin, James D

2014-03-01

Despite new treatments, acute myeloid leukemia (AML) remains an incurable disease. More effective drug design requires an expanded view of the molecular complexity that underlies AML. Alternative splicing of RNA is used by normal cells to generate protein diversity. Growing evidence indicates that aberrant splicing of genes plays a key role in cancer. We investigated genome-wide splicing abnormalities in AML and based on these abnormalities, we aimed to identify novel potential biomarkers and therapeutic targets. We used genome-wide alternative splicing screening to investigate alternative splicing abnormalities in two independent AML patient cohorts [Dana-Farber Cancer Institute (DFCI) (Boston, MA) and University Hospital de Nantes (UHN) (Nantes, France)] and normal donors. Selected splicing events were confirmed through cloning and sequencing analysis, and than validated in 193 patients with AML. Our results show that approximately 29% of expressed genes genome-wide were differentially and recurrently spliced in patients with AML compared with normal donors bone marrow CD34(+) cells. Results were reproducible in two independent AML cohorts. In both cohorts, annotation analyses indicated similar proportions of differentially spliced genes encoding several oncogenes, tumor suppressor proteins, splicing factors, and heterogeneous-nuclear-ribonucleoproteins, proteins involved in apoptosis, cell proliferation, and spliceosome assembly. Our findings are consistent with reports for other malignances and indicate that AML-specific aberrations in splicing mechanisms are a hallmark of AML pathogenesis. Overall, our results suggest that aberrant splicing is a common characteristic for AML. Our findings also suggest that splice variant transcripts that are the result of splicing aberrations create novel disease markers and provide potential targets for small molecules or antibody therapeutics for this disease. ©2013 AACR

Sequential recognition of the pre-mRNA branch point by U2AF65 and a novel spliceosome-associated 28-kDa protein.

PubMed Central

Gaur, R K; Valcárcel, J; Green, M R

1995-01-01

Splicing of pre-mRNAs occurs via a lariat intermediate in which an intronic adenosine, embedded within a branch point sequence, forms a 2',5'-phosphodiester bond (RNA branch) with the 5' end of the intron. How the branch point is recognized and activated remains largely unknown. Using site-specific photochemical cross-linking, we have identified two proteins that specifically interact with the branch point during the splicing reaction. U2AF65, an essential splicing factor that binds to the adjacent polypyrimidine tract, crosslinks to the branch point at the earliest stage of spliceosome formation in an ATP-independent manner. A novel 28-kDa protein, which is a constituent of the mature spliceosome, contacts the branch point after the first catalytic step. Our results indicate that the branch point is sequentially recognized by distinct splicing factors in the course of the splicing reaction. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 FIGURE 7 FIGURE 8 FIGURE 9 PMID:7493318

Do Androgen Receptor Splice Variants Facilitate Growth of Bone Metastases

DTIC Science & Technology

2016-11-01

therapy is expression of constitutively active AR splice variants, which lack the carboxyl terminal hormone binding domain. The best characterized...resistance is expression of constitutively active AR splice variants, which lack the carboxyl terminal hormone binding domain. Of these, the most...removes hormone , but also many other factors. We plan to retest the effects of AR-V7 in the complete medium to determine whether effects would be

Identification of an Intronic Splicing Enhancer Essential for the Inclusion of FGFR2 Exon IIIc*S⃞

PubMed Central

Seth, Puneet; Miller, Heather B.; Lasda, Erika L.; Pearson, James L.; Garcia-Blanco, Mariano A.

2008-01-01

The ligand specificity of fibroblast growth factor receptor 2 (FGFR2) is determined by the alternative splicing of exons 8 (IIIb) or 9 (IIIc). Exon IIIb is included in epithelial cells, whereas exon IIIc is included in mesenchymal cells. Although a number of cis elements and trans factors have been identified that play a role in exon IIIb inclusion in epithelium, little is known about the activation of exon IIIc in mesenchyme. We report here the identification of a splicing enhancer required for IIIc inclusion. This 24-nucleotide (nt) downstream intronic splicing enhancer (DISE) is located within intron 9 immediately downstream of exon IIIc. DISE was able to activate the inclusion of heterologous exons rat FGFR2 IIIb and human β-globin exon 2 in cell lines from different tissues and species and also in HeLa cell nuclear extracts in vitro. DISE was capable of replacing the intronic activator sequence 1 (IAS1), a known IIIb splicing enhancer and vice versa. This fact, together with the requirement for DISE to be close to the 5′-splice site and the ability of DISE to promote binding of U1 snRNP, suggested that IAS1 and DISE belong to the same class of cis-acting elements. PMID:18256031

Intron-mediated alternative splicing of WOOD-ASSOCIATED NAC TRANSCRIPTION FACTOR1B regulates cell wall thickening during fiber development in Populus species.

PubMed

Zhao, Yunjun; Sun, Jiayan; Xu, Peng; Zhang, Rui; Li, Laigeng

2014-02-01

Alternative splicing is an important mechanism involved in regulating the development of multicellular organisms. Although many genes in plants undergo alternative splicing, little is understood of its significance in regulating plant growth and development. In this study, alternative splicing of black cottonwood (Populus trichocarpa) wood-associated NAC domain transcription factor (PtrWNDs), PtrWND1B, is shown to occur exclusively in secondary xylem fiber cells. PtrWND1B is expressed with a normal short-transcript PtrWND1B-s as well as its alternative long-transcript PtrWND1B-l. The intron 2 structure of the PtrWND1B gene was identified as a critical sequence that causes PtrWND1B alternative splicing. Suppression of PtrWND1B expression specifically inhibited fiber cell wall thickening. The two PtrWND1B isoforms play antagonistic roles in regulating cell wall thickening during fiber cell differentiation in Populus spp. PtrWND1B-s overexpression enhanced fiber cell wall thickening, while overexpression of PtrWND1B-l repressed fiber cell wall thickening. Alternative splicing may enable more specific regulation of processes such as fiber cell wall thickening during wood formation.

Intron-Mediated Alternative Splicing of WOOD-ASSOCIATED NAC TRANSCRIPTION FACTOR1B Regulates Cell Wall Thickening during Fiber Development in Populus Species1[W

PubMed Central

Zhao, Yunjun; Sun, Jiayan; Xu, Peng; Zhang, Rui; Li, Laigeng

2014-01-01

Alternative splicing is an important mechanism involved in regulating the development of multicellular organisms. Although many genes in plants undergo alternative splicing, little is understood of its significance in regulating plant growth and development. In this study, alternative splicing of black cottonwood (Populus trichocarpa) wood-associated NAC domain transcription factor (PtrWNDs), PtrWND1B, is shown to occur exclusively in secondary xylem fiber cells. PtrWND1B is expressed with a normal short-transcript PtrWND1B-s as well as its alternative long-transcript PtrWND1B-l. The intron 2 structure of the PtrWND1B gene was identified as a critical sequence that causes PtrWND1B alternative splicing. Suppression of PtrWND1B expression specifically inhibited fiber cell wall thickening. The two PtrWND1B isoforms play antagonistic roles in regulating cell wall thickening during fiber cell differentiation in Populus spp. PtrWND1B-s overexpression enhanced fiber cell wall thickening, while overexpression of PtrWND1B-l repressed fiber cell wall thickening. Alternative splicing may enable more specific regulation of processes such as fiber cell wall thickening during wood formation. PMID:24394777

Mechanistic quantitative structure-activity relationship model for the photoinduced toxicity of polycyclic aromatic hydrocarbons. 2: An empirical model for the toxicity of 16 polycyclic aromatic hydrocarbons to the duckweed Lemna gibba L. G-3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, X.D.; Krylov, S.N.; Ren, L.

1997-11-01

Photoinduced toxicity of polycyclic aromatic hydrocarbons (PAHs) occurs via photosensitization reactions (e.g., generation of singlet-state oxygen) and by photomodification (photooxidation and/or photolysis) of the chemicals to more toxic species. The quantitative structure-activity relationship (QSAR) described in the companion paper predicted, in theory, that photosensitization and photomodification additively contribute to toxicity. To substantiate this QSAR modeling exercise it was necessary to show that toxicity can be described by empirically derived parameters. The toxicity of 16 PAHs to the duckweed Lemna gibba was measured as inhibition of leaf production in simulated solar radiation (a light source with a spectrum similar to thatmore » of sunlight). A predictive model for toxicity was generated based on the theoretical model developed in the companion paper. The photophysical descriptors required of each PAH for modeling were efficiency of photon absorbance, relative uptake, quantum yield for triplet-state formation, and the rate of photomodification. The photomodification rates of the PAHs showed a moderate correlation to toxicity, whereas a derived photosensitization factor (PSF; based on absorbance, triplet-state quantum yield, and uptake) for each PAH showed only a weak, complex correlation to toxicity. However, summing the rate of photomodification and the PSF resulted in a strong correlation to toxicity that had predictive value. When the PSF and a derived photomodification factor (PMF; based on the photomodification rate and toxicity of the photomodified PAHs) were summed, an excellent explanatory model of toxicity was produced, substantiating the additive contributions of the two factors.« less

Correction of dichroic mirror-induced PSF distortion in STED microscopy

NASA Astrophysics Data System (ADS)

Li, Yanghui; Zhou, Hui; Liu, Xiaoyu; Xia, Chengliang; Wang, Le

2018-07-01

Due to its spectral transmission/reflection properties, dichroic mirror (DM) is widely applied in nearly all microscopes to separate and combine beams with diverse wavelengths. However, its potential distortion effects to the point-spread function (PSF) have been ignored to a large extent. Here, we built a mathematical model to quantify these effects. Specifically, we focus on STED microscopy, whose performance is extremely sensitive to the systematic deficiencies. Both excitation and depletion beams are analysed, and the peak intensity and the shape of the effective PSF are calculated accordingly. More importantly, the strategy to compensate the DM-induced PSF distortion is also given in this paper.

The optimal on-source region size for detections with counting-type telescopes

NASA Astrophysics Data System (ADS)

Klepser, S.

2017-03-01

Source detection in counting type experiments such as Cherenkov telescopes often involves the application of the classical Eq. (17) from the paper of Li & Ma (1983) to discrete on- and off-source regions. The on-source region is typically a circular area with radius θ in which the signal is expected to appear with the shape of the instrument point spread function (PSF). This paper addresses the question of what is the θ that maximises the probability of detection for a given PSF width and background event density. In the high count number limit and assuming a Gaussian PSF profile, the optimum is found to be at ζ∞2 ≈ 2.51 times the squared PSF width σPSF392. While this number is shown to be a good choice in many cases, a dynamic formula for cases of lower count numbers, which favour larger on-source regions, is given. The recipe to get to this parametrisation can also be applied to cases with a non-Gaussian PSF. This result can standardise and simplify analysis procedures, reduce trials and eliminate the need for experience-based ad hoc cut definitions or expensive case-by-case Monte Carlo simulations.

Evaluating pond sand filter as sustainable drinking water supplier in the Southwest coastal region of Bangladesh

NASA Astrophysics Data System (ADS)

Harun, M. A. Y. A.; Kabir, G. M. M.

2013-03-01

This study investigates existing water supply scenario, and evaluates the performance of pond sand filter (PSF) in meeting drinking water demand of Dacope Upazila in southwest coastal Bangladesh. Questionnaire survey to the villagers reveals that PSF is the major drinking water sources (38 %) of the study area followed by tubewells (30.4 %), rainwater harvesting (RWH) systems (12.6 %), ponds (10.3 %) and others (8.7 %). The spot test and laboratory analysis show that odour, colour, pH, dissolved oxygen, hardness, calcium, magnesium, nitrate, sulphate and phosphate of the PSFs water meet Bangladesh standard. The efficiency of PSF in reducing total dissolved solids (TDS) (15 %) and potassium (8.2 %) is not enough to meet the standard of 20 % PSFs for TDS and one-third PSFs for potassium. The study proves that PSF is unable to remove coliform bacteria by 100 % from highly contaminated water. Hence, disinfection should be adopted before distribution to ensure safe drinking water. Majority of the PSF's users (80 %) are either partially satisfied or dissatisfied with the existing system. The beneficiary's willingness to pay for drinking water technologies seems that the combination of PSF and RWH could ensure sustainable drinking water in coastal region of Bangladesh.

Splice loss requirements in multi-mode fiber mode-division-multiplex transmission links.

PubMed

Warm, Stefan; Petermann, Klaus

2013-01-14

We investigate numerically the influence of fiber splices and fiber connectors to the statistics of mode dependent loss (MDL) and multiple-input multiple-output (MIMO) outage capacity in mode multiplexed multi-mode fiber links. Our results indicate required splice losses much lower than currently feasible to achieve a reasonable outage capacity in long-haul transmission systems. Splice losses as low as 0.03dB may effectively lead to an outage of MIMO channels after only a few hundred kilometers transmission length. In a first approximation, the relative capacity solely depends on the accumulated splice loss and should be less than ≈ 2dB to ensure a relative capacity of 90%. We also show that discrete mode permutation (mixing) within the transmission line may effectively increase the maximum transmission distance by a factor of 5 for conventional splice losses.

SKIP Is a Component of the Spliceosome Linking Alternative Splicing and the Circadian Clock in Arabidopsis[W

PubMed Central

Wang, Xiaoxue; Wu, Fangming; Xie, Qiguang; Wang, Huamei; Wang, Ying; Yue, Yanling; Gahura, Ondrej; Ma, Shuangshuang; Liu, Lei; Cao, Ying; Jiao, Yuling; Puta, Frantisek; McClung, C. Robertson; Xu, Xiaodong; Ma, Ligeng

2012-01-01

Circadian clocks generate endogenous rhythms in most organisms from cyanobacteria to humans and facilitate entrainment to environmental diurnal cycles, thus conferring a fitness advantage. Both transcriptional and posttranslational mechanisms are prominent in the basic network architecture of circadian systems. Posttranscriptional regulation, including mRNA processing, is emerging as a critical step for clock function. However, little is known about the molecular mechanisms linking RNA metabolism to the circadian clock network. Here, we report that a conserved SNW/Ski-interacting protein (SKIP) domain protein, SKIP, a splicing factor and component of the spliceosome, is involved in posttranscriptional regulation of circadian clock genes in Arabidopsis thaliana. Mutation in SKIP lengthens the circadian period in a temperature-sensitive manner and affects light input and the sensitivity of the clock to light resetting. SKIP physically interacts with the spliceosomal splicing factor Ser/Arg-rich protein45 and associates with the pre-mRNA of clock genes, such as PSEUDORESPONSE REGULATOR7 (PRR7) and PRR9, and is necessary for the regulation of their alternative splicing and mRNA maturation. Genome-wide investigations reveal that SKIP functions in regulating alternative splicing of many genes, presumably through modulating recognition or cleavage of 5′ and 3′ splice donor and acceptor sites. Our study addresses a fundamental question on how the mRNA splicing machinery contributes to circadian clock function at a posttranscriptional level. PMID:22942380

Identification of protein features encoded by alternative exons using Exon Ontology.

PubMed

Tranchevent, Léon-Charles; Aubé, Fabien; Dulaurier, Louis; Benoit-Pilven, Clara; Rey, Amandine; Poret, Arnaud; Chautard, Emilie; Mortada, Hussein; Desmet, François-Olivier; Chakrama, Fatima Zahra; Moreno-Garcia, Maira Alejandra; Goillot, Evelyne; Janczarski, Stéphane; Mortreux, Franck; Bourgeois, Cyril F; Auboeuf, Didier

2017-06-01

Transcriptomic genome-wide analyses demonstrate massive variation of alternative splicing in many physiological and pathological situations. One major challenge is now to establish the biological contribution of alternative splicing variation in physiological- or pathological-associated cellular phenotypes. Toward this end, we developed a computational approach, named "Exon Ontology," based on terms corresponding to well-characterized protein features organized in an ontology tree. Exon Ontology is conceptually similar to Gene Ontology-based approaches but focuses on exon-encoded protein features instead of gene level functional annotations. Exon Ontology describes the protein features encoded by a selected list of exons and looks for potential Exon Ontology term enrichment. By applying this strategy to exons that are differentially spliced between epithelial and mesenchymal cells and after extensive experimental validation, we demonstrate that Exon Ontology provides support to discover specific protein features regulated by alternative splicing. We also show that Exon Ontology helps to unravel biological processes that depend on suites of coregulated alternative exons, as we uncovered a role of epithelial cell-enriched splicing factors in the AKT signaling pathway and of mesenchymal cell-enriched splicing factors in driving splicing events impacting on autophagy. Freely available on the web, Exon Ontology is the first computational resource that allows getting a quick insight into the protein features encoded by alternative exons and investigating whether coregulated exons contain the same biological information. © 2017 Tranchevent et al.; Published by Cold Spring Harbor Laboratory Press.

[RNA polymerase II and pre-mRNA splicing factors in diplotene oocyte nuclei of the giant African gastropod Achatina fulica].

PubMed

Stepanova, I S; Bogoliubov, D S

2003-01-01

The nuclear distribution of pre-mRNA splicing factors (snRNPs and SR-protein SC35) and unphosphorylated from of RNA polymerase II (Pol II) was studied using fluorescent and immunoelectron cytochemistry in diplotene oocytes of the gastropod Achatina fulica. Association of Pol II and splicing factors with oocyte nuclear structures was analysed. The antibodies against splicing factors and Pol II were shown to label perichromatin fibrils at the periphery of condensed chromatin blocks as well as those in interchromatin regions of nucleoplasm. The revealed character of distribution of snRNPs, SC35 protein, and Pol II, together with the decondensed chromatin and absence of karyosphere, enable us to suggest that oocyte chromosomes maintain their transcriptional activity at the diplotene stage of oogenesis. In A. fulica oocytes, sparse nuclear bodies (NBs) of a complex morphological structure were revealed. These NBs contain snRNPs rather than SC35 protein. NBs are associated with a fibrogranular material (FGM), which contains SC35 protein. No snRNPs were revealed in this material. Homology of A. fulica oocyte nuclear structures to Cajal bodies and interchromatin granule clusters is discussed.

Science Based Human Reliability Analysis: Using Digital Nuclear Power Plant Simulators for Human Reliability Research

NASA Astrophysics Data System (ADS)

Shirley, Rachel Elizabeth

Nuclear power plant (NPP) simulators are proliferating in academic research institutions and national laboratories in response to the availability of affordable, digital simulator platforms. Accompanying the new research facilities is a renewed interest in using data collected in NPP simulators for Human Reliability Analysis (HRA) research. An experiment conducted in The Ohio State University (OSU) NPP Simulator Facility develops data collection methods and analytical tools to improve use of simulator data in HRA. In the pilot experiment, student operators respond to design basis accidents in the OSU NPP Simulator Facility. Thirty-three undergraduate and graduate engineering students participated in the research. Following each accident scenario, student operators completed a survey about perceived simulator biases and watched a video of the scenario. During the video, they periodically recorded their perceived strength of significant Performance Shaping Factors (PSFs) such as Stress. This dissertation reviews three aspects of simulator-based research using the data collected in the OSU NPP Simulator Facility: First, a qualitative comparison of student operator performance to computer simulations of expected operator performance generated by the Information Decision Action Crew (IDAC) HRA method. Areas of comparison include procedure steps, timing of operator actions, and PSFs. Second, development of a quantitative model of the simulator bias introduced by the simulator environment. Two types of bias are defined: Environmental Bias and Motivational Bias. This research examines Motivational Bias--that is, the effect of the simulator environment on an operator's motivations, goals, and priorities. A bias causal map is introduced to model motivational bias interactions in the OSU experiment. Data collected in the OSU NPP Simulator Facility are analyzed using Structural Equation Modeling (SEM). Data include crew characteristics, operator surveys, and time to recognize and diagnose the accident in the scenario. These models estimate how the effects of the scenario conditions are mediated by simulator bias, and demonstrate how to quantify the strength of the simulator bias. Third, development of a quantitative model of subjective PSFs based on objective data (plant parameters, alarms, etc.) and PSF values reported by student operators. The objective PSF model is based on the PSF network in the IDAC HRA method. The final model is a mixed effects Bayesian hierarchical linear regression model. The subjective PSF model includes three factors: The Environmental PSF, the simulator Bias, and the Context. The Environmental Bias is mediated by an operator sensitivity coefficient that captures the variation in operator reactions to plant conditions. The data collected in the pilot experiments are not expected to reflect professional NPP operator performance, because the students are still novice operators. However, the models used in this research and the methods developed to analyze them demonstrate how to consider simulator bias in experiment design and how to use simulator data to enhance the technical basis of a complex HRA method. The contributions of the research include a framework for discussing simulator bias, a quantitative method for estimating simulator bias, a method for obtaining operator-reported PSF values, and a quantitative method for incorporating the variability in operator perception into PSF models. The research demonstrates applications of Structural Equation Modeling and hierarchical Bayesian linear regression models in HRA. Finally, the research demonstrates the benefits of using student operators as a test platform for HRA research.

Improvement of blood compatibility on polysulfone-polyvinylpyrrolidone blend films as a model membrane of dialyzer by physical adsorption of recombinant soluble human thrombomodulin (ART-123).

PubMed

Omichi, Masaaki; Matsusaki, Michiya; Maruyama, Ikuro; Akashi, Mitsuru

2012-01-01

ART-123 is a recombinant soluble human thrombomodulin (hTM) with potent anticoagulant activity, and is available for developing antithrombogenic surfaces by immobilization. We focused on improving blood compatibility on the dialyzer surface by the physical adsorption of ART-123 as a safe yet simple method without using chemical reagents. The physical adsorption mechanism and anticoagulant activities of adsorbed hTM on the surface of a polysulfone (PSF) membrane containing polyvinylpyrrolidone (PVP) as a model dialyzer were investigated in detail. The PVP content of the PSF-PVP films was saturated at 20 wt% after immersion in Tris-HCl buffer, even with the addition of over 20 wt% PVP. The surface morphology of the PSF-PVP films was strongly influenced by the PVP content, because PVP covered the outermost surface of the PSF-PVP films. The adsorption speed of hTM slowed dramatically with increasing PVP content up to 10 wt%, but the maximum adsorption amount of hTM onto the PSF-PVP film surface was almost the same, regardless of the PVP content. The PSF-PVP film with the physically adsorbed hTM showed higher protein C activity as compared to the PSF film, it showed excellent blood compatibility due to the protein C activity and the inhibition properties of platelet adhesion. The physical adsorption of hTM can be useful as a safe yet simple method to improve the blood compatibility of a dialyzer surface.

Searching for Faint Companions to Nearby Stars with the Hubble Space Telescope

NASA Technical Reports Server (NTRS)

Schroeder, Daniel J.; Golimowski, David A.

1996-01-01

A search for faint companions (FC's) to selected stars within 5 pc of the Sun using the Hubble Space Telescope's Planetary Camera (PC) has been initiated. To assess the PC's ability to detect FCs, we have constructed both model and laboratory-simulated images and compared them to actual PC images. We find that the PC's point-spread function (PSF) is 3-4 times brighter over the angular range 2-5 sec than the PSF expected for a perfect optical system. Azimuthal variations of the PC's PSF are 10-20 times larger than expected for a perfect PSF. These variations suggest that light is scattered nonuniformly from the surface of the detector. Because the anomalies in the PC's PSF cannot be precisely simulated, subtracting a reference PSF from the PC image is problematic. We have developed a computer algorithm that identifies local brightness anomalies within the PSF as potential FCs. We find that this search algorithm will successfully locate FCs anywhere within the circumstellar field provided that the average pixel signal from the FC is at least 10 sigma above the local background. This detection limit suggests that a comprehensive search for extrasolar Jovian planets with the PC is impractical. However, the PC is useful for detecting other types of substellar objects. With a stellar signal of 10(exp 9) e(-), for example, we may detect brown dwarfs as faint as M(sub I) = 16.7 separated by 1 sec from alpha Cen A.

Point spread function based classification of regions for linear digital tomosynthesis

NASA Astrophysics Data System (ADS)

Israni, Kenny; Avinash, Gopal; Li, Baojun

2007-03-01

In digital tomosynthesis, one of the limitations is the presence of out-of-plane blur due to the limited angle acquisition. The point spread function (PSF) characterizes blur in the imaging volume, and is shift-variant in tomosynthesis. The purpose of this research is to classify the tomosynthesis imaging volume into four different categories based on PSF-driven focus criteria. We considered linear tomosynthesis geometry and simple back projection algorithm for reconstruction. The three-dimensional PSF at every pixel in the imaging volume was determined. Intensity profiles were computed for every pixel by integrating the PSF-weighted intensities contained within the line segment defined by the PSF, at each slice. Classification rules based on these intensity profiles were used to categorize image regions. At background and low-frequency pixels, the derived intensity profiles were flat curves with relatively low and high maximum intensities respectively. At in-focus pixels, the maximum intensity of the profiles coincided with the PSF-weighted intensity of the pixel. At out-of-focus pixels, the PSF-weighted intensity of the pixel was always less than the maximum intensity of the profile. We validated our method using human observer classified regions as gold standard. Based on the computed and manual classifications, the mean sensitivity and specificity of the algorithm were 77+/-8.44% and 91+/-4.13% respectively (t=-0.64, p=0.56, DF=4). Such a classification algorithm may assist in mitigating out-of-focus blur from tomosynthesis image slices.

Modeled cost-effectiveness of transforaminal lumbar interbody fusion compared with posterolateral fusion for spondylolisthesis using N(2)QOD data.

PubMed

Carreon, Leah Y; Glassman, Steven D; Ghogawala, Zoher; Mummaneni, Praveen V; McGirt, Matthew J; Asher, Anthony L

2016-06-01

OBJECTIVE Transforaminal lumbar interbody fusion (TLIF) has become the most commonly used fusion technique for lumbar degenerative disorders. This suggests an expectation of better clinical outcomes with this technique, but this has not been validated consistently. How surgical variables and choice of health utility measures drive the cost-effectiveness of TLIF relative to posterolateral fusion (PSF) has not been established. The authors used health utility values derived from Short Form-6D (SF-6D) and EQ-5D and different cost-effectiveness thresholds to evaluate the relative cost-effectiveness of TLIF compared with PSF. METHODS From the National Neurosurgery Quality and Outcomes Database (N(2)QOD), 101 patients with spondylolisthesis who underwent PSF were propensity matched to patients who underwent TLIF. Health-related quality of life measures and perioperative parameters were compared. Because health utility values derived from the SF-6D and EQ-5D questionnaires have been shown to vary in patients with low-back pain, quality-adjusted life years (QALYs) were derived from both measures. On the basis of these matched cases, a sensitivity analysis for the relative cost per QALY of TLIF versus PSF was performed in a series of cost-assumption models. RESULTS Operative time, blood loss, hospital stay, and 30-day and 90-day readmission rates were similar for the TLIF and PSF groups. Both TLIF and PSF significantly improved back and leg pain, Oswestry Disability Index (ODI) scores, and EQ-5D and SF-6D scores at 3 and 12 months postoperatively. At 12 months postoperatively, patients who had undergone TLIF had greater improvements in mean ODI scores (30.4 vs 21.1, p = 0.001) and mean SF-6D scores (0.16 vs 0.11, p = 0.001) but similar improvements in mean EQ-5D scores (0.25 vs 0.22, p = 0.415) as patients treated with PSF. At a cost per QALY threshold of $100,000 and using SF-6D-based QALYs, the authors found that TLIF would be cost-prohibitive compared with PSF at a surgical cost of $4830 above that of PSF. However, with EQ-5D-based QALYs, TLIF would become cost-prohibitive at an increased surgical cost of $2960 relative to that of PSF. With the 2014 US per capita gross domestic product of $53,042 as a more stringent cost-effectiveness threshold, TLIF would become cost-prohibitive at surgical costs $2562 above that of PSF with SF-6D-based QALYs or at a surgical cost exceeding that of PSF by $1570 with EQ-5D-derived QALYs. CONCLUSIONS As with all cost-effectiveness studies, cost per QALY depended on the measure of health utility selected, durability of the intervention, readmission rates, and the accuracy of the cost assumptions.

Vitamin D and alternative splicing of RNA

PubMed Central

Zhou, Rui; Chun, Rene F.; Lisse, Thomas S.; Garcia, Alejandro J.; Xu, Jianzhong; Adams, John S.; Hewison, Martin

2014-01-01

The active form of vitamin D (1α,25-dihydroxyvitamin D, 1,25(OH)2D) exerts its genomic effects via binding to a nuclear high-affinity vitamin D receptor (VDR). Recent deep sequencing analysis of VDR binding locations across the complete genome has significantly expanded our understanding of the actions of vitamin D and VDR on gene transcription. However, these studies have also promoted appreciation of the extra-transcriptional impact of vitamin D on gene expression. It is now clear that vitamin D interacts with the epigenome via effects on DNA methylation, histone acetylation, and microRNA generation to maintain normal biological functions. There is also increasing evidence that vitamin D can influence pre-mRNA constitutive splicing and alternative splicing, although the mechanism for this remains unclear. Pre-mRNA splicing has long been thought to be a post-transcription RNA processing event, but current data indicate that this occurs co-transcriptionally. Several steroid hormones have been recognized to coordinately control gene transcription and pre-mRNA splicing through the recruitment of nuclear receptor co-regulators that can both control gene transcription and splicing. The current review will discuss this concept with specific reference to vitamin D, and the potential role of heterogeneous nuclear ribonucleoprotein C (hnRNPC), a nuclear factor with an established function in RNA splicing. hnRNPC, has been shown to be involved in the VDR transcriptional complex as a vitamin D-response element-binding protein (VDRE-BP), and may act as a coupling factor linking VDR-directed gene transcription with RNA splicing. In this way hnRNPC may provide an additional mechanism for the fine-tuning of vitamin D-regulated target gene expression. PMID:25447737

FOX-2 Dependent Splicing of Ataxin-2 Transcript Is Affected by Ataxin-1 Overexpression

PubMed Central

Welzel, Franziska; Kaehler, Christian; Isau, Melanie; Hallen, Linda; Lehrach, Hans; Krobitsch, Sylvia

2012-01-01

Alternative splicing is a fundamental posttranscriptional mechanism for controlling gene expression, and splicing defects have been linked to various human disorders. The splicing factor FOX-2 is part of a main protein interaction hub in a network related to human inherited ataxias, however, its impact remains to be elucidated. Here, we focused on the reported interaction between FOX-2 and ataxin-1, the disease-causing protein in spinocerebellar ataxia type 1. In this line, we further evaluated this interaction by yeast-2-hybrid analyses and co-immunoprecipitation experiments in mammalian cells. Interestingly, we discovered that FOX-2 localization and splicing activity is affected in the presence of nuclear ataxin-1 inclusions. Moreover, we observed that FOX-2 directly interacts with ataxin-2, a protein modulating spinocerebellar ataxia type 1 pathogenesis. Finally, we provide evidence that splicing of pre-mRNA of ataxin-2 depends on FOX-2 activity, since reduction of FOX-2 levels led to increased skipping of exon 18 in ataxin-2 transcripts. Most striking, we observed that ataxin-1 overexpression has an effect on this splicing event as well. Thus, our results demonstrate that FOX-2 is involved in splicing of ataxin-2 transcripts and that this splicing event is altered by overexpression of ataxin-1. PMID:22666429

Image-based Modeling of PSF Deformation with Application to Limited Angle PET Data

PubMed Central

Matej, Samuel; Li, Yusheng; Panetta, Joseph; Karp, Joel S.; Surti, Suleman

2016-01-01

The point-spread-functions (PSFs) of reconstructed images can be deformed due to detector effects such as resolution blurring and parallax error, data acquisition geometry such as insufficient sampling or limited angular coverage in dual-panel PET systems, or reconstruction imperfections/simplifications. PSF deformation decreases quantitative accuracy and its spatial variation lowers consistency of lesion uptake measurement across the imaging field-of-view (FOV). This can be a significant problem with dual panel PET systems even when using TOF data and image reconstruction models of the detector and data acquisition process. To correct for the spatially variant reconstructed PSF distortions we propose to use an image-based resolution model (IRM) that includes such image PSF deformation effects. Originally the IRM was mostly used for approximating data resolution effects of standard PET systems with full angular coverage in a computationally efficient way, but recently it was also used to mitigate effects of simplified geometric projectors. Our work goes beyond this by including into the IRM reconstruction imperfections caused by combination of the limited angle, parallax errors, and any other (residual) deformation effects and testing it for challenging dual panel data with strongly asymmetric and variable PSF deformations. We applied and tested these concepts using simulated data based on our design for a dedicated breast imaging geometry (B-PET) consisting of dual-panel, time-of-flight (TOF) detectors. We compared two image-based resolution models; i) a simple spatially invariant approximation to PSF deformation, which captures only the general PSF shape through an elongated 3D Gaussian function, and ii) a spatially variant model using a Gaussian mixture model (GMM) to more accurately capture the asymmetric PSF shape in images reconstructed from data acquired with the B-PET scanner geometry. Results demonstrate that while both IRMs decrease the overall uptake bias in the reconstructed image, the second one with the spatially variant and accurate PSF shape model is also able to ameliorate the spatially variant deformation effects to provide consistent uptake results independent of the lesion location within the FOV. PMID:27812222

Vimentin and PSF Act in Concert to Regulate IbeA+ E. coli K1 Induced Activation and Nuclear Translocation of NF-κB in Human Brain Endothelial Cells

PubMed Central

Wu, Chun-Hua; Jong, Ambrose; Huang, Sheng-He

2012-01-01

Background IbeA-induced NF-κB signaling through its primary receptor vimentin as well as its co-receptor PSF is required for meningitic E. coli K1 penetration and leukocyte transmigration across the blood-brain barrier (BBB), which are the hallmarks of bacterial meningitis. However, it is unknown how vimentin and PSF cooperatively contribute to IbeA-induced cytoplasmic activation and nuclear translocation of NF-κB, which are required for bacteria-mediated pathogenicities. Methodology/Principal Findings IbeA-induced E. coli K1 invasion, polymorphonuclear leukocyte (PMN) transmigration and IKK/NF-κB activation are blocked by Caffeic acid phenethyl ester (CAPE), an inhibitor of NF-κB. IKKα/β phosphorylation is blocked by ERK inhibitors. Co-immunoprecipitation analysis shows that vimentin forms a complex with IκB, NF-κB and tubulins in the resting cells. A dissociation of this complex and a simultaneous association of PSF with NF-κB could be induced by IbeA in a time-dependent manner. The head domain of vimentin is required for the complex formation. Two cytoskeletal components, vimentin filaments and microtubules, contribute to the regulation of NF-κB. SiRNA-mediated knockdown studies demonstrate that IKKα/β phosphorylation is completely abolished in HBMECs lacking vimentin and PSF. Phosphorylation of ERK and nuclear translocation of NF-κB are entirely dependent on PSF. These findings suggest that vimentin and PSF cooperatively contribute to IbeA-induced cytoplasmic activation and nuclear translocation of NF-κB activation. PSF is essential for translocation of NF-κB and ERK to the nucleus. Conclusion/Significance These findings reveal previously unappreciated facets of the IbeA-binding proteins. Cooperative contributions of vimentin and PSF to IbeA-induced cytoplasmic activation and nuclear translocation of NF-κB may represent a new paradigm in pathogen-induced signal transduction and lead to the development of novel strategies for the prevention and treatment of bacterial meningitis. PMID:22536447

Estimation of a super-resolved PSF for the data reduction of undersampled stellar observations. Deriving an accurate model for fitting photometry with Corot space telescope

NASA Astrophysics Data System (ADS)

Pinheiro da Silva, L.; Auvergne, M.; Toublanc, D.; Rowe, J.; Kuschnig, R.; Matthews, J.

2006-06-01

Context: .Fitting photometry algorithms can be very effective provided that an accurate model of the instrumental point spread function (PSF) is available. When high-precision time-resolved photometry is required, however, the use of point-source star images as empirical PSF models can be unsatisfactory, due to the limits in their spatial resolution. Theoretically-derived models, on the other hand, are limited by the unavoidable assumption of simplifying hypothesis, while the use of analytical approximations is restricted to regularly-shaped PSFs. Aims: .This work investigates an innovative technique for space-based fitting photometry, based on the reconstruction of an empirical but properly-resolved PSF. The aim is the exploitation of arbitrary star images, including those produced under intentional defocus. The cases of both MOST and COROT, the first space telescopes dedicated to time-resolved stellar photometry, are considered in the evaluation of the effectiveness and performances of the proposed methodology. Methods: .PSF reconstruction is based on a set of star images, periodically acquired and presenting relative subpixel displacements due to motion of the acquisition system, in this case the jitter of the satellite attitude. Higher resolution is achieved through the solution of the inverse problem. The approach can be regarded as a special application of super-resolution techniques, though a specialised procedure is proposed to better meet the PSF determination problem specificities. The application of such a model to fitting photometry is illustrated by numerical simulations for COROT and on a complete set of observations from MOST. Results: .We verify that, in both scenarios, significantly better resolved PSFs can be estimated, leading to corresponding improvements in photometric results. For COROT, indeed, subpixel reconstruction enabled the successful use of fitting algorithms despite its rather complex PSF profile, which could hardly be modeled otherwise. For MOST, whose direct-imaging PSF is closer to the ordinary, comparison to other models or photometry techniques were carried out and confirmed the potential of PSF reconstruction in real observational conditions.

How do messenger RNA splicing alterations drive myelodysplasia?

PubMed Central

2017-01-01

Mutations in RNA splicing factors are the single most common class of genetic alterations in myelodysplastic syndrome (MDS) patients. Although much has been learned about how these mutations affect splicing at a global- and transcript-specific level, critical questions about the role of these mutations in MDS development and maintenance remain. Here we present the questions to be addressed in order to understand the unique enrichment of these mutations in MDS. PMID:28348147

Role of the DNA Damage Response in Human Papillomavirus RNA Splicing and Polyadenylation.

PubMed

Nilsson, Kersti; Wu, Chengjun; Schwartz, Stefan

2018-06-12

Human papillomaviruses (HPVs) have evolved to use the DNA repair machinery to replicate its DNA genome in differentiated cells. HPV activates the DNA damage response (DDR) in infected cells. Cellular DDR factors are recruited to the HPV DNA genome and position the cellular DNA polymerase on the HPV DNA and progeny genomes are synthesized. Following HPV DNA replication, HPV late gene expression is activated. Recent research has shown that the DDR factors also interact with RNA binding proteins and affects RNA processing. DDR factors activated by DNA damage and that associate with HPV DNA can recruit splicing factors and RNA binding proteins to the HPV DNA and induce HPV late gene expression. This induction is the result of altered alternative polyadenylation and splicing of HPV messenger RNA (mRNA). HPV uses the DDR machinery to replicate its DNA genome and to activate HPV late gene expression at the level of RNA processing.

RBFox1-mediated RNA splicing regulates cardiac hypertrophy and heart failure.

PubMed

Gao, Chen; Ren, Shuxun; Lee, Jae-Hyung; Qiu, Jinsong; Chapski, Douglas J; Rau, Christoph D; Zhou, Yu; Abdellatif, Maha; Nakano, Astushi; Vondriska, Thomas M; Xiao, Xinshu; Fu, Xiang-Dong; Chen, Jau-Nian; Wang, Yibin

2016-01-01

RNA splicing is a major contributor to total transcriptome complexity; however, the functional role and regulation of splicing in heart failure remain poorly understood. Here, we used a total transcriptome profiling and bioinformatic analysis approach and identified a muscle-specific isoform of an RNA splicing regulator, RBFox1 (also known as A2BP1), as a prominent regulator of alternative RNA splicing during heart failure. Evaluation of developing murine and zebrafish hearts revealed that RBFox1 is induced during postnatal cardiac maturation. However, we found that RBFox1 is markedly diminished in failing human and mouse hearts. In a mouse model, RBFox1 deficiency in the heart promoted pressure overload-induced heart failure. We determined that RBFox1 is a potent regulator of RNA splicing and is required for a conserved splicing process of transcription factor MEF2 family members that yields different MEF2 isoforms with differential effects on cardiac hypertrophic gene expression. Finally, induction of RBFox1 expression in murine pressure overload models substantially attenuated cardiac hypertrophy and pathological manifestations. Together, this study identifies regulation of RNA splicing by RBFox1 as an important player in transcriptome reprogramming during heart failure that influence pathogenesis of the disease.

Spatio-temporal regulations and functions of neuronal alternative RNA splicing in developing and adult brains.

PubMed

Iijima, Takatoshi; Hidaka, Chiharu; Iijima, Yoko

2016-08-01

Alternative pre-mRNA splicing is a fundamental mechanism that generates molecular diversity from a single gene. In the central nervous system (CNS), key neural developmental steps are thought to be controlled by alternative splicing decisions, including the molecular diversity underlying synaptic wiring, plasticity, and remodeling. Significant progress has been made in understanding the molecular mechanisms and functions of alternative pre-mRNA splicing in neurons through studies in invertebrate systems; however, recent studies have begun to uncover the potential role of neuronal alternative splicing in the mammalian CNS. This article provides an overview of recent findings regarding the regulation and function of neuronal alternative splicing. In particular, we focus on the spatio-temporal regulation of neurexin, a synaptic adhesion molecule, by neuronal cell type-specific factors and neuronal activity, which are thought to be especially important for characterizing neural development and function within the mammalian CNS. Notably, there is increasing evidence that implicates the dysregulation of neuronal splicing events in several neurological disorders. Therefore, understanding the detailed mechanisms of neuronal alternative splicing in the mammalian CNS may provide plausible treatment strategies for these diseases. Copyright © 2016 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.

RBFox1-mediated RNA splicing regulates cardiac hypertrophy and heart failure

PubMed Central

Gao, Chen; Ren, Shuxun; Lee, Jae-Hyung; Qiu, Jinsong; Chapski, Douglas J.; Rau, Christoph D.; Zhou, Yu; Abdellatif, Maha; Nakano, Astushi; Vondriska, Thomas M.; Xiao, Xinshu; Fu, Xiang-Dong; Chen, Jau-Nian; Wang, Yibin

2015-01-01

RNA splicing is a major contributor to total transcriptome complexity; however, the functional role and regulation of splicing in heart failure remain poorly understood. Here, we used a total transcriptome profiling and bioinformatic analysis approach and identified a muscle-specific isoform of an RNA splicing regulator, RBFox1 (also known as A2BP1), as a prominent regulator of alternative RNA splicing during heart failure. Evaluation of developing murine and zebrafish hearts revealed that RBFox1 is induced during postnatal cardiac maturation. However, we found that RBFox1 is markedly diminished in failing human and mouse hearts. In a mouse model, RBFox1 deficiency in the heart promoted pressure overload–induced heart failure. We determined that RBFox1 is a potent regulator of RNA splicing and is required for a conserved splicing process of transcription factor MEF2 family members that yields different MEF2 isoforms with differential effects on cardiac hypertrophic gene expression. Finally, induction of RBFox1 expression in murine pressure overload models substantially attenuated cardiac hypertrophy and pathological manifestations. Together, this study identifies regulation of RNA splicing by RBFox1 as an important player in transcriptome reprogramming during heart failure that influence pathogenesis of the disease. PMID:26619120

RBFOX and PTBP1 proteins regulate the alternative splicing of micro-exons in human brain transcripts

PubMed Central

Sanchez-Pulido, Luis; Haerty, Wilfried

2015-01-01

Ninety-four percent of mammalian protein-coding exons exceed 51 nucleotides (nt) in length. The paucity of micro-exons (≤ 51 nt) suggests that their recognition and correct processing by the splicing machinery present greater challenges than for longer exons. Yet, because thousands of human genes harbor processed micro-exons, specialized mechanisms may be in place to promote their splicing. Here, we survey deep genomic data sets to define 13,085 micro-exons and to study their splicing mechanisms and molecular functions. More than 60% of annotated human micro-exons exhibit a high level of sequence conservation, an indicator of functionality. While most human micro-exons require splicing-enhancing genomic features to be processed, the splicing of hundreds of micro-exons is enhanced by the adjacent binding of splice factors in the introns of pre-messenger RNAs. Notably, splicing of a significant number of micro-exons was found to be facilitated by the binding of RBFOX proteins, which promote their inclusion in the brain, muscle, and heart. Our analyses suggest that accurate regulation of micro-exon inclusion by RBFOX proteins and PTBP1 plays an important role in the maintenance of tissue-specific protein–protein interactions. PMID:25524026

Peptidic tools applied to redirect alternative splicing events.

PubMed

Nancy, Martínez-Montiel; Nora, Rosas-Murrieta; Rebeca, Martínez-Contreras

2015-05-01

Peptides are versatile and attractive biomolecules that can be applied to modulate genetic mechanisms like alternative splicing. In this process, a single transcript yields different mature RNAs leading to the production of protein isoforms with diverse or even antagonistic functions. During splicing events, errors can be caused either by mutations present in the genome or by defects or imbalances in regulatory protein factors. In any case, defects in alternative splicing have been related to several genetic diseases including muscular dystrophy, Alzheimer's disease and cancer from almost every origin. One of the most effective approaches to redirect alternative splicing events has been to attach cell-penetrating peptides to oligonucleotides that can modulate a single splicing event and restore correct gene expression. Here, we summarize how natural existing and bioengineered peptides have been applied over the last few years to regulate alternative splicing and genetic expression. Under different genetic and cellular backgrounds, peptides have been shown to function as potent vehicles for splice correction, and their therapeutic benefits have reached clinical trials and patenting stages, emphasizing the use of regulatory peptides as an exciting therapeutic tool for the treatment of different genetic diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

The combinatorial control of alternative splicing in C. elegans

PubMed Central

2017-01-01

Normal development requires the right splice variants to be made in the right tissues at the right time. The core splicing machinery is engaged in all splicing events, but which precise splice variant is made requires the choice between alternative splice sites—for this to occur, a set of splicing factors (SFs) must recognize and bind to short RNA motifs in the pre-mRNA. In C. elegans, there is known to be extensive variation in splicing patterns across development, but little is known about the targets of each SF or how multiple SFs combine to regulate splicing. Here we combine RNA-seq with in vitro binding assays to study how 4 different C. elegans SFs, ASD-1, FOX-1, MEC-8, and EXC-7, regulate splicing. The 4 SFs chosen all have well-characterised biology and well-studied loss-of-function genetic alleles, and all contain RRM domains. Intriguingly, while the SFs we examined have varied roles in C. elegans development, they show an unexpectedly high overlap in their targets. We also find that binding sites for these SFs occur on the same pre-mRNAs more frequently than expected suggesting extensive combinatorial control of splicing. We confirm that regulation of splicing by multiple SFs is often combinatorial and show that this is functionally significant. We also find that SFs appear to combine to affect splicing in two modes—they either bind in close proximity within the same intron or they appear to bind to separate regions of the intron in a conserved order. Finally, we find that the genes whose splicing are regulated by multiple SFs are highly enriched for genes involved in the cytoskeleton and in ion channels that are key for neurotransmission. Together, this shows that specific classes of genes have complex combinatorial regulation of splicing and that this combinatorial regulation is critical for normal development to occur. PMID:29121637

Multi-limit unsymmetrical MLIBD image restoration algorithm

NASA Astrophysics Data System (ADS)

Yang, Yang; Cheng, Yiping; Chen, Zai-wang; Bo, Chen

2012-11-01

A novel multi-limit unsymmetrical iterative blind deconvolution(MLIBD) algorithm was presented to enhance the performance of adaptive optics image restoration.The algorithm enhances the reliability of iterative blind deconvolution by introducing the bandwidth limit into the frequency domain of point spread(PSF),and adopts the PSF dynamic support region estimation to improve the convergence speed.The unsymmetrical factor is automatically computed to advance its adaptivity.Image deconvolution comparing experiments between Richardson-Lucy IBD and MLIBD were done,and the result indicates that the iteration number is reduced by 22.4% and the peak signal-to-noise ratio is improved by 10.18dB with MLIBD method. The performance of MLIBD algorithm is outstanding in the images restoration the FK5-857 adaptive optics and the double-star adaptive optics.

Tissue-specific alternative splicing of TCF7L2

PubMed Central

Prokunina-Olsson, Ludmila; Welch, Cullan; Hansson, Ola; Adhikari, Neeta; Scott, Laura J.; Usher, Nicolle; Tong, Maurine; Sprau, Andrew; Swift, Amy; Bonnycastle, Lori L.; Erdos, Michael R.; He, Zhi; Saxena, Richa; Harmon, Brennan; Kotova, Olga; Hoffman, Eric P.; Altshuler, David; Groop, Leif; Boehnke, Michael; Collins, Francis S.; Hall, Jennifer L.

2009-01-01

Common variants in the transcription factor 7-like 2 (TCF7L2) gene have been identified as the strongest genetic risk factors for type 2 diabetes (T2D). However, the mechanisms by which these non-coding variants increase risk for T2D are not well-established. We used 13 expression assays to survey mRNA expression of multiple TCF7L2 splicing forms in up to 380 samples from eight types of human tissue (pancreas, pancreatic islets, colon, liver, monocytes, skeletal muscle, subcutaneous adipose tissue and lymphoblastoid cell lines) and observed a tissue-specific pattern of alternative splicing. We tested whether the expression of TCF7L2 splicing forms was associated with single nucleotide polymorphisms (SNPs), rs7903146 and rs12255372, located within introns 3 and 4 of the gene and most strongly associated with T2D. Expression of two splicing forms was lower in pancreatic islets with increasing counts of T2D-associated alleles of the SNPs: a ubiquitous splicing form (P = 0.018 for rs7903146 and P = 0.020 for rs12255372) and a splicing form found in pancreatic islets, pancreas and colon but not in other tissues tested here (P = 0.009 for rs12255372 and P = 0.053 for rs7903146). Expression of this form in glucose-stimulated pancreatic islets correlated with expression of proinsulin (r2 = 0.84–0.90, P < 0.00063). In summary, we identified a tissue-specific pattern of alternative splicing of TCF7L2. After adjustment for multiple tests, no association between expression of TCF7L2 in eight types of human tissue samples and T2D-associated genetic variants remained significant. Alternative splicing of TCF7L2 in pancreatic islets warrants future studies. GenBank Accession Numbers: FJ010164–FJ010174. PMID:19602480

Clinical Impact of Time-of-Flight and Point Response Modeling in PET Reconstructions: A Lesion Detection Study

PubMed Central

Schaefferkoetter, Joshua; Casey, Michael; Townsend, David; Fakhri, Georges El

2013-01-01

Time-of-flight (TOF) and point spread function (PSF) modeling have been shown to improve PET reconstructions, but the impact on physicians in the clinical setting has not been thoroughly investigated. A lesion detection and localization study was performed using simulated lesions in real patient images. Four reconstruction schemes were considered: ordinary Poisson OSEM (OP) alone and combined with TOF, PSF, and TOF+PSF. The images were presented to physicians experienced in reading PET images, and the performance of each was quantified using localization receiver operating characteristic (LROC). Numerical observers (non-prewhitening and Hotelling) were used to identify optimal reconstruction parameters, and observer SNR was compared to the performance of the physicians. The numerical models showed good agreement with human performance, and best performance was achieved by both when using TOF+PSF. These findings suggest a large potential benefit of TOF+PSF for oncology PET studies, especially in the detection of small, low-intensity, focal disease in larger patients. PMID:23403399

The algorithm of motion blur image restoration based on PSF half-blind estimation

NASA Astrophysics Data System (ADS)

Chen, Da-Ke; Lin, Zhe

2011-08-01

A novel algorithm of motion blur image restoration based on PSF half-blind estimation with Hough transform was introduced on the basis of full analysis of the principle of TDICCD camera, with the problem that vertical uniform linear motion estimation used by IBD algorithm as the original value of PSF led to image restoration distortion. Firstly, the mathematical model of image degradation was established with the transcendental information of multi-frame images, and then two parameters (movement blur length and angle) that have crucial influence on PSF estimation was set accordingly. Finally, the ultimate restored image can be acquired through multiple iterative of the initial value of PSF estimation in Fourier domain, which the initial value was gained by the above method. Experimental results show that the proposal algorithm can not only effectively solve the image distortion problem caused by relative motion between TDICCD camera and movement objects, but also the details characteristics of original image are clearly restored.

Evaluation of the image quality of telescopes using the star test

NASA Astrophysics Data System (ADS)

Vazquez y Monteil, Sergio; Salazar Romero, Marcos A.; Gale, David M.

2004-10-01

The Point Spread Function (PSF) or star test is one of the main criteria to be considered in the quality of the image formed by a telescope. In a real system the distribution of irradiance in the image of a point source is given by the PSF, a function which is highly sensitive to aberrations. The PSF of a telescope may be determined by measuring the intensity distribution in the image of a star. Alternatively, if we already know the aberrations present in the optical system, then we may use diffraction theory to calculate the function. In this paper we propose a method for determining the wavefront aberrations from the PSF, using Genetic Algorithms to perform an optimization process starting from the PSF instead of the more traditional method of adjusting an aberration polynomial. We show that this method of phase recuperation is immune to noise-induced errors arising during image aquisition and registration. Some practical results are shown.

MapX: 2D XRF for Planetary Exploration - Image Formation and Optic Characterization

DOE PAGES

Sarrazin, P.; Blake, D.; Gailhanou, M.; ...

2018-04-01

Map-X is a planetary instrument concept for 2D X-Ray Fluorescence (XRF) spectroscopy. The instrument is placed directly on the surface of an object and held in a fixed position during the measurement. The formation of XRF images on the CCD detector relies on a multichannel optic configured for 1:1 imaging and can be analyzed through the point spread function (PSF) of the optic. The PSF can be directly measured using a micron-sized monochromatic X-ray source in place of the sample. Such PSF measurements were carried out at the Stanford Synchrotron and are compared with ray tracing simulations. It is shownmore » that artifacts are introduced by the periodicity of the PSF at the channel scale and the proximity of the CCD pixel size and the optic channel size. A strategy of sub-channel random moves was used to cancel out these artifacts and provide a clean experimental PSF directly usable for XRF image deconvolution.« less

MapX: 2D XRF for Planetary Exploration - Image Formation and Optic Characterization

NASA Astrophysics Data System (ADS)

Sarrazin, P.; Blake, D.; Gailhanou, M.; Marchis, F.; Chalumeau, C.; Webb, S.; Walter, P.; Schyns, E.; Thompson, K.; Bristow, T.

2018-04-01

Map-X is a planetary instrument concept for 2D X-Ray Fluorescence (XRF) spectroscopy. The instrument is placed directly on the surface of an object and held in a fixed position during the measurement. The formation of XRF images on the CCD detector relies on a multichannel optic configured for 1:1 imaging and can be analyzed through the point spread function (PSF) of the optic. The PSF can be directly measured using a micron-sized monochromatic X-ray source in place of the sample. Such PSF measurements were carried out at the Stanford Synchrotron and are compared with ray tracing simulations. It is shown that artifacts are introduced by the periodicity of the PSF at the channel scale and the proximity of the CCD pixel size and the optic channel size. A strategy of sub-channel random moves was used to cancel out these artifacts and provide a clean experimental PSF directly usable for XRF image deconvolution.

MapX: 2D XRF for Planetary Exploration - Image Formation and Optic Characterization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sarrazin, P.; Blake, D.; Gailhanou, M.

Map-X is a planetary instrument concept for 2D X-Ray Fluorescence (XRF) spectroscopy. The instrument is placed directly on the surface of an object and held in a fixed position during the measurement. The formation of XRF images on the CCD detector relies on a multichannel optic configured for 1:1 imaging and can be analyzed through the point spread function (PSF) of the optic. The PSF can be directly measured using a micron-sized monochromatic X-ray source in place of the sample. Such PSF measurements were carried out at the Stanford Synchrotron and are compared with ray tracing simulations. It is shownmore » that artifacts are introduced by the periodicity of the PSF at the channel scale and the proximity of the CCD pixel size and the optic channel size. A strategy of sub-channel random moves was used to cancel out these artifacts and provide a clean experimental PSF directly usable for XRF image deconvolution.« less

Clinical impact of time-of-flight and point response modeling in PET reconstructions: a lesion detection study

NASA Astrophysics Data System (ADS)

Schaefferkoetter, Joshua; Casey, Michael; Townsend, David; El Fakhri, Georges

2013-03-01

Time-of-flight (TOF) and point spread function (PSF) modeling have been shown to improve PET reconstructions, but the impact on physicians in the clinical setting has not been thoroughly investigated. A lesion detection and localization study was performed using simulated lesions in real patient images. Four reconstruction schemes were considered: ordinary Poisson OSEM (OP) alone and combined with TOF, PSF, and TOF + PSF. The images were presented to physicians experienced in reading PET images, and the performance of each was quantified using localization receiver operating characteristic. Numerical observers (non-prewhitening and Hotelling) were used to identify optimal reconstruction parameters, and observer SNR was compared to the performance of the physicians. The numerical models showed good agreement with human performance, and best performance was achieved by both when using TOF + PSF. These findings suggest a large potential benefit of TOF + PSF for oncology PET studies, especially in the detection of small, low-intensity, focal disease in larger patients.

Development of Neuroendocrine Prostate Cancers by the Ser/Arg Repetitive Matrix 4-Mediated RNA Splicing Network.

PubMed

Lee, Ahn R; Che, Nicole; Lovnicki, Jessica M; Dong, Xuesen

2018-01-01

While the use of next-generation androgen receptor pathway inhibition (ARPI) therapy has significantly increased the survival of patients with metastatic prostate adenocarcinoma (AdPC), several groups have reported a treatment-resistant mechanism, whereby cancer cells can become androgen receptor (AR) indifferent and gain a neuroendocrine (NE)-like phenotype. This subtype of castration-resistant prostate cancer has been termed "treatment-induced castration-resistant neuroendocrine prostate cancer" (CRPC-NE). Recent reports indicate that the overall genomic landscapes of castration-resistant tumors with AdPC phenotypes and CRPC-NE are not significantly altered. However, CRPC-NE tumors have been found to contain a NE-specific pattern throughout their epigenome and splicing transcriptome, which are significantly modified. The molecular mechanisms by which CRPC-NE develops remain unclear, but several factors have been implicated in the progression of the disease. Recently, Ser/Arg repetitive matrix 4 (SRRM4), a neuronal-specific RNA splicing factor that is upregulated in CRPC-NE tumors, has been shown to establish a CRPC-NE-unique splicing transcriptome, to induce a NE-like morphology in AdPC cells, and, most importantly, to transform AdPC cells into CRPC-NE xenografts under ARPI. Moreover, the SRRM4-targeted splicing genes are highly enriched in various neuronal processes, suggesting their roles in facilitating a CRPC-NE program. This article will address the importance of SRRM4-mediated alternative RNA splicing in reprogramming translated proteins to facilitate NE differentiation, survival, and proliferation of cells to establish CRPC-NE tumors. In addition, we will discuss the potential roles of SRRM4 in conjunction with other known pathways and factors important for CRPC-NE development, such as the AR pathway, TP53 and RB1 genes, the FOXA family of proteins, and environmental factors. This study aims to explore the multifaceted functions of SRRM4 and SRRM4-mediated splicing in driving a CRPC-NE program as a coping mechanism for therapy resistance, as well as define future SRRM4-targeted therapeutic approaches for treating CRPC-NE or mitigating its development.

Development of Neuroendocrine Prostate Cancers by the Ser/Arg Repetitive Matrix 4-Mediated RNA Splicing Network

PubMed Central

Lee, Ahn R.; Che, Nicole; Lovnicki, Jessica M.; Dong, Xuesen

2018-01-01

While the use of next-generation androgen receptor pathway inhibition (ARPI) therapy has significantly increased the survival of patients with metastatic prostate adenocarcinoma (AdPC), several groups have reported a treatment-resistant mechanism, whereby cancer cells can become androgen receptor (AR) indifferent and gain a neuroendocrine (NE)-like phenotype. This subtype of castration-resistant prostate cancer has been termed “treatment-induced castration-resistant neuroendocrine prostate cancer” (CRPC-NE). Recent reports indicate that the overall genomic landscapes of castration-resistant tumors with AdPC phenotypes and CRPC-NE are not significantly altered. However, CRPC-NE tumors have been found to contain a NE-specific pattern throughout their epigenome and splicing transcriptome, which are significantly modified. The molecular mechanisms by which CRPC-NE develops remain unclear, but several factors have been implicated in the progression of the disease. Recently, Ser/Arg repetitive matrix 4 (SRRM4), a neuronal-specific RNA splicing factor that is upregulated in CRPC-NE tumors, has been shown to establish a CRPC-NE-unique splicing transcriptome, to induce a NE-like morphology in AdPC cells, and, most importantly, to transform AdPC cells into CRPC-NE xenografts under ARPI. Moreover, the SRRM4-targeted splicing genes are highly enriched in various neuronal processes, suggesting their roles in facilitating a CRPC-NE program. This article will address the importance of SRRM4-mediated alternative RNA splicing in reprogramming translated proteins to facilitate NE differentiation, survival, and proliferation of cells to establish CRPC-NE tumors. In addition, we will discuss the potential roles of SRRM4 in conjunction with other known pathways and factors important for CRPC-NE development, such as the AR pathway, TP53 and RB1 genes, the FOXA family of proteins, and environmental factors. This study aims to explore the multifaceted functions of SRRM4 and SRRM4-mediated splicing in driving a CRPC-NE program as a coping mechanism for therapy resistance, as well as define future SRRM4-targeted therapeutic approaches for treating CRPC-NE or mitigating its development. PMID:29666783

Aberrant RNA splicing and mutations in spliceosome complex in acute myeloid leukemia.

PubMed

Zhou, Jianbiao; Chng, Wee-Joo

2017-01-01

The spliceosome, the cellular splicing machinery, regulates RNA splicing of messenger RNA precursors (pre-mRNAs) into maturation of protein coding RNAs. Recurrent mutations and copy number changes in genes encoding spliceosomal proteins and splicing regulatory factors have tumor promoting or suppressive functions in hematological malignancies, as well as some other cancers. Leukemia stem cell (LSC) populations, although rare, are essential contributors of treatment failure and relapse. Recent researches have provided the compelling evidence that link the erratic spicing activity to the LSC phenotype in acute myeloid leukemia (AML). In this article, we describe the diverse roles of aberrant splicing in hematological malignancies, particularly in AML and their contributions to the characteristics of LSC. We review these promising strategies to exploit the addiction of aberrant spliceosomal machinery for anti-leukemic therapy with aim to eradicate LSC. However, given the complexity and plasticity of spliceosome and not fully known functions of splicing in cancer, the challenges facing the development of the therapeutic strategies targeting RAN splicing are highlighted and future directions are discussed too.

Aberrant splicing in maize rough endosperm3 reveals a conserved role for U12 splicing in eukaryotic multicellular development

PubMed Central

Barbazuk, W. Brad

2017-01-01

RNA splicing of U12-type introns functions in human cell differentiation, but it is not known whether this class of introns has a similar role in plants. The maize ROUGH ENDOSPERM3 (RGH3) protein is orthologous to the human splicing factor, ZRSR2. ZRSR2 mutations are associated with myelodysplastic syndrome (MDS) and cause U12 splicing defects. Maize rgh3 mutants have aberrant endosperm cell differentiation and proliferation. We found that most U12-type introns are retained or misspliced in rgh3. Genes affected in rgh3 and ZRSR2 mutants identify cell cycle and protein glycosylation as common pathways disrupted. Transcripts with retained U12-type introns can be found in polysomes, suggesting that splicing efficiency can alter protein isoforms. The rgh3 mutant protein disrupts colocalization with a known ZRSR2-interacting protein, U2AF2. These results indicate conserved function for RGH3/ZRSR2 in U12 splicing and a deeply conserved role for the minor spliceosome to promote cell differentiation from stem cells to terminal fates. PMID:28242684

Prolyl hydroxylation regulates protein degradation, synthesis, and splicing in human induced pluripotent stem cell-derived cardiomyocytes.

PubMed

Stoehr, Andrea; Yang, Yanqin; Patel, Sajni; Evangelista, Alicia M; Aponte, Angel; Wang, Guanghui; Liu, Poching; Boylston, Jennifer; Kloner, Philip H; Lin, Yongshun; Gucek, Marjan; Zhu, Jun; Murphy, Elizabeth

2016-06-01

Protein hydroxylases are oxygen- and α-ketoglutarate-dependent enzymes that catalyse hydroxylation of amino acids such as proline, thus linking oxygen and metabolism to enzymatic activity. Prolyl hydroxylation is a dynamic post-translational modification that regulates protein stability and protein-protein interactions; however, the extent of this modification is largely uncharacterized. The goals of this study are to investigate the biological consequences of prolyl hydroxylation and to identify new targets that undergo prolyl hydroxylation in human cardiomyocytes. We used human induced pluripotent stem cell-derived cardiomyocytes in combination with pulse-chase amino acid labelling and proteomics to analyse the effects of prolyl hydroxylation on protein degradation and synthesis. We identified 167 proteins that exhibit differences in degradation with inhibition of prolyl hydroxylation by dimethyloxalylglycine (DMOG); 164 were stabilized. Proteins involved in RNA splicing such as serine/arginine-rich splicing factor 2 (SRSF2) and splicing factor and proline- and glutamine-rich (SFPQ) were stabilized with DMOG. DMOG also decreased protein translation of cytoskeletal and sarcomeric proteins such as α-cardiac actin. We searched the mass spectrometry data for proline hydroxylation and identified 134 high confidence peptides mapping to 78 unique proteins. We identified SRSF2, SFPQ, α-cardiac actin, and cardiac titin as prolyl hydroxylated. We identified 29 prolyl hydroxylated proteins that showed a significant difference in either protein degradation or synthesis. Additionally, we performed next-generation RNA sequencing and showed that the observed decrease in protein synthesis was not due to changes in mRNA levels. Because RNA splicing factors were prolyl hydroxylated, we investigated splicing ± inhibition of prolyl hydroxylation and detected 369 alternative splicing events, with a preponderance of exon skipping. This study provides the first extensive characterization of the cardiac prolyl hydroxylome and demonstrates that inhibition of α-ketoglutarate hydroxylases alters protein stability, translation, and splicing. Published by Oxford University Press on behalf of the European Society of Cardiology 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Enhancement of clover growth by inoculation of P-solubilizing fungi and arbuscular mycorrhizal fungi.

PubMed

Souchie, Edson L; Azcón, Rosario; Barea, Jose M; Silva, Eliane M R; Saggin-Júnior, Orivaldo J

2010-09-01

This study evaluated the synergism between several P-solubilizing fungi isolates and arbuscular mycorrhizal fungi to improve clover ( Trifolium pratense) growth in the presence of Araxá apatite. Clover was sown directly in plastic pots with 300g of sterilized washed sand, vermiculite and sepiolite 1:1:1 (v:v:v) as substrate, and grown in a controlled environment chamber. The substrate was fertilized with 3 g L(-1) of Araxá apatite. A completely randomized design, in 8×2 factorial scheme (eight P-solubilizing fungi treatments with or without arbuscular mycorrhizal fungi)and four replicates were used. The P-solubilizing fungi treatments consisted of five Brazilian P-solubilizing fungi isolates (PSF 7, 9, 20, 21 and 22), two Spanish isolates ( Aspergillus niger and the yeast Yarowia lipolytica) and control (non-inoculated treatment). The greatest clover growth rate was recorded when Aspergillus niger and PSF 21 were co-inoculated with arbuscular mycorrhizal fungi. Aspergillus niger, PSF 7 and PSF 21 were the most effective isolates on increasing clover growth in the presence of arbuscular mycorrhizal fungi. Greater mycorrhizal colonization resulted in greater clover growth rate in most PSF treatments. PSF 7 was the best isolate to improve the establishment of mycorrhizal and rhizobia symbiosis.

Spatial Specificity in Spatiotemporal Encoding and Fourier Imaging

PubMed Central

Goerke, Ute

2015-01-01

Purpose Ultrafast imaging techniques based on spatiotemporal-encoding (SPEN), such as RASER (rapid acquisition with sequential excitation and refocusing), is a promising new class of sequences since they are largely insensitive to magnetic field variations which cause signal loss and geometric distortion in EPI. So far, attempts to theoretically describe the point-spread-function (PSF) for the original SPEN-imaging techniques have yielded limited success. To fill this gap a novel definition for an apparent PSF is proposed. Theory Spatial resolution in SPEN-imaging is determined by the spatial phase dispersion imprinted on the acquired signal by a frequency-swept excitation or refocusing pulse. The resulting signal attenuation increases with larger distance from the vertex of the quadratic phase profile. Methods Bloch simulations and experiments were performed to validate theoretical derivations. Results The apparent PSF quantifies the fractional contribution of magnetization to a voxel’s signal as a function of distance to the voxel. In contrast, the conventional PSF represents the signal intensity at various locations. Conclusion The definition of the conventional PSF fails for SPEN-imaging since only the phase of isochromats, but not the amplitude of the signal varies. The concept of the apparent PSF is shown to be generalizable to conventional Fourier- imaging techniques. PMID:26712657

Elaboration, characterization and application of polysulfone and polyacrylic acid blends as ultrafiltration membranes for removal of some heavy metals from water.

PubMed

Mbareck, Chamekh; Nguyen, Quang Trong; Alaoui, Ouafa Tahiri; Barillier, Daniel

2009-11-15

Polysulfone (PSf)/polyacrylic acid ultrafiltration (PSf/PAA) membranes were prepared from a polymer blend in dimethylformamide by coagulation in water according to the wet phase inversion method. Immobilization of water-soluble PAA within the non-soluble PSf matrix was proven by the increase of ion exchange capacity and the intensity of the carboxyl groups' peak with the increase of PAA content as shown by Fourier transform infrared spectra. These results lead to consider that PSf and PAA form a semi-interpenetrating polymer networks. The obtained membranes showed a decrease of mean surface-pore sizes, the overall porosity and the hydraulic permeability with the increase in PAA content. Such results were imputed to the morphologic modifications of PSf film with the immobilization of increasing PAA amount. PSf/PAA membranes showed high lead, cadmium and chromium rejection which reaches 100% at pH superior to 5.7 and a low rejection at low pH. Moreover, the heavy metal rejection decreases with feed solution concentration and applied pressure increases. These behaviors were attributed to the role of carboxylic groups in ion exchange or complexation. As a matter of fact, the strong lead ion-PAA interactions were revealed by the scanning electron microscopy with energy dispersive X-rays (SEM-EDX).

Distortion correction of echo planar images applying the concept of finite rate of innovation to point spread function mapping (FRIP).

PubMed

Nunes, Rita G; Hajnal, Joseph V

2018-06-01

Point spread function (PSF) mapping enables estimating the displacement fields required for distortion correction of echo planar images. Recently, a highly accelerated approach was introduced for estimating displacements from the phase slope of under-sampled PSF mapping data. Sampling schemes with varying spacing were proposed requiring stepwise phase unwrapping. To avoid unwrapping errors, an alternative approach applying the concept of finite rate of innovation to PSF mapping (FRIP) is introduced, using a pattern search strategy to locate the PSF peak, and the two methods are compared. Fully sampled PSF data was acquired in six subjects at 3.0 T, and distortion maps were estimated after retrospective under-sampling. The two methods were compared for both previously published and newly optimized sampling patterns. Prospectively under-sampled data were also acquired. Shift maps were estimated and deviations relative to the fully sampled reference map were calculated. The best performance was achieved when using FRIP with a previously proposed sampling scheme. The two methods were comparable for the remaining schemes. The displacement field errors tended to be lower as the number of samples or their spacing increased. A robust method for estimating the position of the PSF peak has been introduced.

Stress field forming of sector array transducers for vibro-acoustography.

PubMed

Silva, Glauber T; Chen, Shigao; Frery, Alejandro C; Greenleaf, James F; Fatemi, Mostafa

2005-11-01

This paper presents a study of the stress field forming of sector array transducers for vibro-acoustography applications. The system point-spread function (PSF) is given in terms of the dynamic radiation stress exerted on a point target by a dual ultrasound beam with slightly different frequencies. The radiation stress is calculated by assuming that the resulting ultrasound beam is a plane wave. The stress is proportional to the product of the velocity potential of each incident ultrasound beam. The beamforming and stress field forming of sector array transducers are analyzed through linear acoustics. An expression for the velocity potential produced by sector array transducers is derived. The vibro-acoustography PSF is evaluated numerically. A comparison between the PSF of a sector array and a confocal transducers is presented. The compared characteristics of the PSF are sidelobe levels, transverse, and in-depth spatial resolution. Indeed, one motivation to study sector transducers is the fact the depth-of-field of these transducers should be smaller than that of same size confocal transducers. An experimental setup was used to validate the theoretical PSF of sector array transducers. Results show that the measured PSF is in good agreement with the theoretical predications. Vibro-acoustography images of a breast-phantom by both transducers are presented and discussed.

Drosophila Sld5 is essential for normal cell cycle progression and maintenance of genomic integrity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gouge, Catherine A.; Christensen, Tim W., E-mail: christensent@ecu.edu

2010-09-10

Research highlights: {yields} Drosophila Sld5 interacts with Psf1, PPsf2, and Mcm10. {yields} Haploinsufficiency of Sld5 leads to M-phase delay and genomic instability. {yields} Sld5 is also required for normal S phase progression. -- Abstract: Essential for the normal functioning of a cell is the maintenance of genomic integrity. Failure in this process is often catastrophic for the organism, leading to cell death or mis-proliferation. Central to genomic integrity is the faithful replication of DNA during S phase. The GINS complex has recently come to light as a critical player in DNA replication through stabilization of MCM2-7 and Cdc45 as amore » member of the CMG complex which is likely responsible for the processivity of helicase activity during S phase. The GINS complex is made up of 4 members in a 1:1:1:1 ratio: Psf1, Psf2, Psf3, And Sld5. Here we present the first analysis of the function of the Sld5 subunit in a multicellular organism. We show that Drosophila Sld5 interacts with Psf1, Psf2, and Mcm10 and that mutations in Sld5 lead to M and S phase delays with chromosomes exhibiting hallmarks of genomic instability.« less

Polysulfone hemodiafiltration membranes with enhanced anti-fouling and hemocompatibility modified by poly(vinyl pyrrolidone) via in situ cross-linked polymerization.

PubMed

Zhu, Lijing; Song, Haiming; Wang, Jiarong; Xue, Lixin

2017-05-01

Poly(vinyl pyrrolidone) (PVP) and its copolymers have been widely employed for the modification of hemodiafiltration membranes due to their excellent hydrophilicity, antifouling and hemocompatibility. However, challenges still remain to simplify the modification procedure and to improve the utilization efficiency. In this paper, antifouling and hemocompatibility polysulfone (PSf) hemodiafiltration membranes were fabricated via in situ cross-linked polymerization of vinyl pyrrolidone (VP) and vinyltriethoxysilane (VTEOS) in PSf solutions and non-solvent induced phase separation (NIPS) technique. The prepared membranes were characterized by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM), which suggested that VP and VTEOS have been cross-linked copolymerized in PSf membranes. The modified PSf membranes with high polymer content showed improved hydrophilicity, ultrafiltration and protein antifouling ability. In addition, the modified PSf membranes showed lower protein adsorption, inhibited platelet adhesion and deformation, prolonged the activated partial thromboplastin time (APTT), prothrombin time (PT), and decreased the content of fibrinogen (FIB) transferring to fibrin, indicating enhanced hemocompatibility. In a word, the present work provides a simple and effective one-step modification method to construct PSf membranes with improved hydrophilicity, antifouling and hemocompatibility. Copyright © 2017 Elsevier B.V. All rights reserved.

RNA splicing and its connection with other regulatory layers in somatic cell reprogramming.

PubMed

Zavolan, Mihaela; Kanitz, Alexander

2018-06-01

Understanding how cell identity is established and maintained is one of the most exciting challenges of molecular biology today. Recent work has added a conserved layer of RNA splicing and other post-transcriptional regulatory processes to the transcriptional and epigenetic networks already known to cooperate in the establishment and maintenance of cell identity. Here we summarize these findings, highlighting specifically the multitude of splicing factors that can modulate the efficiency of somatic cell reprogramming. Distinct patterns of gene expression dynamics of these factors during reprogramming suggest that further improvements in efficiency could be obtained through optimal timing of overexpression or knockdown of individual regulators. Copyright © 2017 Elsevier Ltd. All rights reserved.

Efectos combinados de la ampliación de la atención primaria de salud y de las transferencias condicionadas de dinero en efectivo sobre la mortalidad infantil en Brasil, 1998–2010*

PubMed Central

Guanais, Frederico C.

2015-01-01

Objetivos. Examiné los efectos combinados del acceso a la atención primaria mediante el Programa de Salud Familiar (PSF) y las transferencias condicionadas de dinero en efectivo del Programa Bolsa Familia (PBF) sobre la mortalidad infantil posneonatal (MIPN) en Brasil. Métodos. Empleé un análisis ecológico longitudinal usando datos en panel de 4 583 municipios brasileños de 1998 al 2010, con 54 253 observaciones en total. Estimé modelos de regresión de efectos fijos por mínimos cuadrados ordinarios, con la tasa de MIPN como la variable dependiente y el PSF, el PBF y sus interacciones como las principales variables independientes de interés. Resultados. La asociación de una mayor cobertura del PSF con una menor tasa de MIPN se volvió más fuerte conforme aumentaba la cobertura del PBF. En los promedios de todas las demás variables, cuando la cobertura de PBF era 25%, la MIPN predicha fue 5,24 (intervalo de confianza [IC] de 95% = 4,95, 5,53) para una cobertura del PSF de 0%, y de 3,54 (IC de 95% = 2,77, 4,31) para una cobertura del PSF de 100%. Cuando la cobertura del PBF era de 60%, la MIPN predicha fue 4,65 (IC de 95% = 4,36, 4,94) para una cobertura del PSF de 0%, y de 1,38 (IC de 95% = 0,88, 1,89) para una cobertura del PSF de 100%. Conclusiones. El efecto del PSF depende de la ampliación del PBF. Para las poblaciones empobrecidas y subatendidas, la combinación de intervenciones tanto del lado de la oferta como del lado de la demanda podría ser necesaria para mejorar los resultados en salud.

The Effect of Detector Nonlinearity on WFIRST PSF Profiles for Weak Gravitational Lensing Measurements

NASA Astrophysics Data System (ADS)

Plazas, A. A.; Shapiro, C.; Kannawadi, A.; Mandelbaum, R.; Rhodes, J.; Smith, R.

2016-10-01

Weak gravitational lensing (WL) is one of the most powerful techniques to learn about the dark sector of the universe. To extract the WL signal from astronomical observations, galaxy shapes must be measured and corrected for the point-spread function (PSF) of the imaging system with extreme accuracy. Future WL missions—such as NASA’s Wide-Field Infrared Survey Telescope (WFIRST)—will use a family of hybrid near-infrared complementary metal-oxide-semiconductor detectors (HAWAII-4RG) that are untested for accurate WL measurements. Like all image sensors, these devices are subject to conversion gain nonlinearities (voltage response to collected photo-charge) that bias the shape and size of bright objects such as reference stars that are used in PSF determination. We study this type of detector nonlinearity (NL) and show how to derive requirements on it from WFIRST PSF size and ellipticity requirements. We simulate the PSF optical profiles expected for WFIRST and measure the fractional error in the PSF size (ΔR/R) and the absolute error in the PSF ellipticity (Δe) as a function of star magnitude and the NL model. For our nominal NL model (a quadratic correction), we find that, uncalibrated, NL can induce an error of ΔR/R = 1 × 10-2 and Δe 2 = 1.75 × 10-3 in the H158 bandpass for the brightest unsaturated stars in WFIRST. In addition, our simulations show that to limit the bias of ΔR/R and Δe in the H158 band to ˜10% of the estimated WFIRST error budget, the quadratic NL model parameter β must be calibrated to ˜1% and ˜2.4%, respectively. We present a fitting formula that can be used to estimate WFIRST detector NL requirements once a true PSF error budget is established.

Evaluation of Alternative Splicing Regulators As Targets For Selective Therapy of Triple Negative (Basal) Breast Carcinoma

DTIC Science & Technology

2016-10-01

KHDRBS3 and SRSF12 on tumor progression and metastasis (Task1). We analyzed the effect of KHDRBS3 depletion on the growth and migration properties of the...tumor growth during the second year of this project. Continued analysis of the splicing factor expression in primary tumor samples further supports...depletion on tumor initiation, growth and metastasis. Keywords Pre-mRNA splicing, breast cancer, KHDRBS3, SRPK1, SRSF12, metastasis

Role of Bmznf-2, a Bombyx mori CCCH zinc finger gene, in masculinisation and differential splicing of Bmtra-2.

PubMed

Gopinath, Gajula; Arunkumar, Kallare P; Mita, Kazuei; Nagaraju, Javaregowda

2016-08-01

Deciphering the regulatory factors involved in Bombyx mori sex determination has been a puzzle, challenging researchers for nearly a century now. The pre-mRNA of B. mori doublesex (Bmdsx), a master regulator gene of sexual differentiation, is differentially spliced, producing Bmdsxm and Bmdsxf transcripts in males and females respectively. The putative proteins encoded by these differential transcripts orchestrate antagonistic functions, which lead to sexual differentiation. A recent study in B. mori illustrated the role of a W-derived fem piRNA in conferring femaleness. In females, the fem piRNA was shown to suppress the activity of a Z-linked CCCH type zinc finger (znf) gene, Masculiniser (masc), which indirectly promotes the Bmdsxm type of splicing. In this study, we report a novel autosomal (Chr 25) CCCH type znf motif encoding gene Bmznf-2 as one of the potential factors in the Bmdsx sex specific differential splicing, and we also provide insights into its role in the alternative splicing of Bmtra2 by using ovary derived BmN cells. Over-expression of Bmznf-2 induced Bmdsxm type of splicing (masculinisation) with a correspondingly reduced expression of Bmdsxf type isoform in BmN cells. Further, the site-directed mutational studies targeting the tandem CCCH znf motifs revealed their indispensability in the observed phenotype of masculinisation. Additionally, the dual luciferase assays in BmN cells using 5' UTR region of the Bmznf-2 strongly implied the existence of a translational repression over this gene. From these findings, we propose Bmznf-2 to be one of the potential factors of masculinisation similar to Masc. From the growing number of Bmdsx splicing regulators, we assume that the sex determination cascade of B. mori is quite intricate in nature; hence, it has to be further investigated for its comprehensive understanding. Copyright © 2016 Elsevier Ltd. All rights reserved.

The determinants of alternative RNA splicing in human cells.

PubMed

Ramanouskaya, Tatsiana V; Grinev, Vasily V

2017-12-01

Alternative splicing represents an important level of the regulation of gene function in eukaryotic organisms. It plays a critical role in virtually every biological process within an organism, including regulation of cell division and cell death, differentiation of tissues in the embryo and the adult organism, as well as in cellular response to diverse environmental factors. In turn, studies of the last decade have shown that alternative splicing itself is controlled by different mechanisms. Unfortunately, there is no clear understanding of how these diverse mechanisms, or determinants, regulate and constrain the set of alternative RNA species produced from any particular gene in every cell of the human body. Here, we provide a consolidated overview of alternative splicing determinants including RNA-protein interactions, epigenetic regulation via chromatin remodeling, coupling of transcription-to-alternative splicing, effect of secondary structures in pre-RNA, and function of the RNA quality control systems. We also extensively and critically discuss some mechanistic insights on coordinated inclusion/exclusion of exons during the formation of mature RNA molecules. We conclude that the final structure of RNA is pre-determined by a complex interplay between cis- and trans-acting factors. Altogether, currently available empirical data significantly expand our understanding of the functioning of the alternative splicing machinery of cells in normal and pathological conditions. On the other hand, there are still many blind spots that require further deep investigations.

7SK snRNP/P-TEFb couples transcription elongation with alternative splicing and is essential for vertebrate development

PubMed Central

Barboric, Matjaz; Lenasi, Tina; Chen, Hui; Johansen, Eric B.; Guo, Su; Peterlin, B. Matija

2009-01-01

Eukaryotic gene expression is commonly controlled at the level of RNA polymerase II (RNAPII) pausing subsequent to transcription initiation. Transcription elongation is stimulated by the positive transcription elongation factor b (P-TEFb) kinase, which is suppressed within the 7SK small nuclear ribonucleoprotein (7SK snRNP). However, the biogenesis and functional significance of 7SK snRNP remain poorly understood. Here, we report that LARP7, BCDIN3, and the noncoding 7SK small nuclear RNA (7SK) are vital for the formation and stability of a cell stress-resistant core 7SK snRNP. Our functional studies demonstrate that 7SK snRNP is not only critical for controlling transcription elongation, but also for regulating alternative splicing of pre-mRNAs. Using a transient expression splicing assay, we find that 7SK snRNP disintegration promotes inclusion of an alternative exon via the increased occupancy of P-TEFb, Ser2-phosphorylated (Ser2-P) RNAPII, and the splicing factor SF2/ASF at the minigene. Importantly, knockdown of larp7 or bcdin3 orthologues in zebrafish embryos destabilizes 7SK and causes severe developmental defects and aberrant splicing of analyzed transcripts. These findings reveal a key role for P-TEFb in coupling transcription elongation with alternative splicing, and suggest that maintaining core 7SK snRNP is essential for vertebrate development. PMID:19416841

Genome-Wide Analysis of Alternative Splicing Landscapes Modulated during Plant-Virus Interactions in Brachypodium distachyon

PubMed Central

Scholthof, Karen-Beth G.

2015-01-01

In eukaryotes, alternative splicing (AS) promotes transcriptome and proteome diversity. The extent of genome-wide AS changes occurring during a plant-microbe interaction is largely unknown. Here, using high-throughput, paired-end RNA sequencing, we generated an isoform-level spliceome map of Brachypodium distachyon infected with Panicum mosaic virus and its satellite virus. Overall, we detected ∼44,443 transcripts in B. distachyon, ∼30% more than those annotated in the reference genome. Expression of ∼28,900 transcripts was ≥2 fragments per kilobase of transcript per million mapped fragments, and ∼42% of multi-exonic genes were alternatively spliced. Comparative analysis of AS patterns in B. distachyon, rice (Oryza sativa), maize (Zea mays), sorghum (Sorghum bicolor), Arabidopsis thaliana, potato (Solanum tuberosum), Medicago truncatula, and poplar (Populus trichocarpa) revealed conserved ratios of the AS types between monocots and dicots. Virus infection quantitatively altered AS events in Brachypodium with little effect on the AS ratios. We discovered AS events for >100 immune-related genes encoding receptor-like kinases, NB-LRR resistance proteins, transcription factors, RNA silencing, and splicing-associated proteins. Cloning and molecular characterization of SCL33, a serine/arginine-rich splicing factor, identified multiple novel intron-retaining splice variants that are developmentally regulated and modulated during virus infection. B. distachyon SCL33 splicing patterns are also strikingly conserved compared with a distant Arabidopsis SCL33 ortholog. This analysis provides new insights into AS landscapes conserved among monocots and dicots and uncovered AS events in plant defense-related genes. PMID:25634987

RNA structure in splicing: An evolutionary perspective.

PubMed

Lin, Chien-Ling; Taggart, Allison J; Fairbrother, William G

2016-09-01

Pre-mRNA splicing is a key post-transcriptional regulation process in which introns are excised and exons are ligated together. A novel class of structured intron was recently discovered in fish. Simple expansions of complementary AC and GT dimers at opposite boundaries of an intron were found to form a bridging structure, thereby enforcing correct splice site pairing across the intron. In some fish introns, the RNA structures are strong enough to bypass the need of regulatory protein factors for splicing. Here, we discuss the prevalence and potential functions of highly structured introns. In humans, structured introns usually arise through the co-occurrence of C and G-rich repeats at intron boundaries. We explore the potentially instructive example of the HLA receptor genes. In HLA pre-mRNA, structured introns flank the exons that encode the highly polymorphic β sheet cleft, making the processing of the transcript robust to variants that disrupt splicing factor binding. While selective forces that have shaped HLA receptor are fairly atypical, numerous other highly polymorphic genes that encode receptors contain structured introns. Finally, we discuss how the elevated mutation rate associated with the simple repeats that often compose structured intron can make structured introns themselves rapidly evolving elements.

The RNA helicase DDX39B and its paralog DDX39A regulate androgen receptor splice variant AR-V7 generation.

PubMed

Nakata, Daisuke; Nakao, Shoichi; Nakayama, Kazuhide; Araki, Shinsuke; Nakayama, Yusuke; Aparicio, Samuel; Hara, Takahito; Nakanishi, Atsushi

2017-01-29

Mounting evidence suggests that constitutively active androgen receptor (AR) splice variants, typified by AR-V7, are associated with poor prognosis and resistance to androgen deprivation therapy in prostate cancer patients. However, mechanisms governing the generation of AR splice variants are not fully understood. In this study, we aimed to investigate the dynamics of AR splice variant generation using the JDCaP prostate cancer model that expresses AR splice variants under androgen depletion. Microarray analysis of JDCaP xenografts before and after expression of AR splice variants suggested that dysregulation of RNA processing pathways is likely involved in AR splice variant generation. To explore factors contributing to generation of AR-V7 mRNA, we conducted a focused RNA interference screen in AR-V7-positive JDCaP-hr cells using an shRNA library targeting spliceosome-related genes. This screen identified DDX39B as a regulator of AR-V7 mRNA expression. Simultaneous knockdown of DDX39B and its paralog DDX39A drastically and selectively downregulated AR-V7 mRNA expression in multiple AR-V7-positive prostate cancer cell lines. DDX39B was upregulated in relapsed JDCaP xenografts expressing AR splice variants, suggesting its role in expression of AR splice variants. Taken together, our findings offer insight into the mechanisms of AR splice variant generation and identify DDX39 as a potential drug target for the treatment of AR splice variant-positive prostate cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

Novel splice mutation in microthalmia-associated transcription factor in Waardenburg Syndrome.

PubMed

Brenner, Laura; Burke, Kelly; Leduc, Charles A; Guha, Saurav; Guo, Jiancheng; Chung, Wendy K

2011-01-01

Waardenburg Syndrome (WS) is a syndromic form of hearing loss associated with mutations in six different genes. We identified a large family with WS that had previously undergone clinical testing, with no reported pathogenic mutation. Using linkage analysis, a region on 3p14.1 with an LOD score of 6.6 was identified. Microthalmia-Associated Transcription Factor, a gene known to cause WS, is located within this region of linkage. Sequencing of Microthalmia-Associated Transcription Factor demonstrated a c.1212 G>A synonymous variant that segregated with the WS in the family and was predicted to cause a novel splicing site that was confirmed with expression analysis of the mRNA. This case illustrates the need to computationally analyze novel synonymous sequence variants for possible effects on splicing to maximize the clinical sensitivity of sequence-based genetic testing.

Oncogenes and RNA splicing of human tumor viruses

PubMed Central

Ajiro, Masahiko; Zheng, Zhi-Ming

2014-01-01

Approximately 10.8% of human cancers are associated with infection by an oncogenic virus. These viruses include human papillomavirus (HPV), Epstein–Barr virus (EBV), Merkel cell polyomavirus (MCV), human T-cell leukemia virus 1 (HTLV-1), Kaposi's sarcoma-associated herpesvirus (KSHV), hepatitis C virus (HCV) and hepatitis B virus (HBV). These oncogenic viruses, with the exception of HCV, require the host RNA splicing machinery in order to exercise their oncogenic activities, a strategy that allows the viruses to efficiently export and stabilize viral RNA and to produce spliced RNA isoforms from a bicistronic or polycistronic RNA transcript for efficient protein translation. Infection with a tumor virus affects the expression of host genes, including host RNA splicing factors, which play a key role in regulating viral RNA splicing of oncogene transcripts. A current prospective focus is to explore how alternative RNA splicing and the expression of viral oncogenes take place in a cell- or tissue-specific manner in virus-induced human carcinogenesis. PMID:26038756

From General Aberrant Alternative Splicing in Cancers and Its Therapeutic Application to the Discovery of an Oncogenic DMTF1 Isoform

PubMed Central

Tian, Na; Li, Jialiang; Shi, Jinming; Sui, Guangchao

2017-01-01

Alternative pre-mRNA splicing is a crucial process that allows the generation of diversified RNA and protein products from a multi-exon gene. In tumor cells, this mechanism can facilitate cancer development and progression through both creating oncogenic isoforms and reducing the expression of normal or controllable protein species. We recently demonstrated that an alternative cyclin D-binding myb-like transcription factor 1 (DMTF1) pre-mRNA splicing isoform, DMTF1β, is increasingly expressed in breast cancer and promotes mammary tumorigenesis in a transgenic mouse model. Aberrant pre-mRNA splicing is a typical event occurring for many cancer-related functional proteins. In this review, we introduce general aberrant pre-mRNA splicing in cancers and discuss its therapeutic application using our recent discovery of the oncogenic DMTF1 isoform as an example. We also summarize new insights in designing novel targeting strategies of cancer therapies based on the understanding of deregulated pre-mRNA splicing mechanisms. PMID:28257090

Point spread function and depth-invariant focal sweep point spread function for plenoptic camera 2.0.

PubMed

Jin, Xin; Liu, Li; Chen, Yanqin; Dai, Qionghai

2017-05-01

This paper derives a mathematical point spread function (PSF) and a depth-invariant focal sweep point spread function (FSPSF) for plenoptic camera 2.0. Derivation of PSF is based on the Fresnel diffraction equation and image formation analysis of a self-built imaging system which is divided into two sub-systems to reflect the relay imaging properties of plenoptic camera 2.0. The variations in PSF, which are caused by changes of object's depth and sensor position variation, are analyzed. A mathematical model of FSPSF is further derived, which is verified to be depth-invariant. Experiments on the real imaging systems demonstrate the consistency between the proposed PSF and the actual imaging results.

An intronic mutation c.6430-3C>G in the F8 gene causes splicing efficiency and premature termination in hemophilia A.

PubMed

Xia, Zunjing; Lin, Jie; Lu, Lingping; Kim, Chol; Yu, Ping; Qi, Ming

2018-06-01

: Hemophilia A is a bleeding disorder caused by coagulation factor VIII protein deficiency or dysfunction, which is classified into severe, moderate, and mild according to factor clotting activity. An overwhelming majority of missense and nonsense mutations occur in exons of F8 gene, whereas mutations in introns can also be pathogenic. This study aimed to investigate the effect of an intronic mutation, c.6430-3C>G (IVS22-3C>G), on pre-mRNA splicing of the F8 gene. We applied DNA and cDNA sequencing in a Chinese boy with hemophilia A to search if any pathogenic mutation in the F8 gene. Functional analysis was performed to investigate the effect of an intronic mutation at the transcriptional level. Human Splicing Finder and PyMol were also used to predict its effect. We found the mutation c.6430-3C>G (IVS22-3C>G) in the F8 gene in the affected boy, with his mother being a carrier. cDNA from the mother and pSPL3 splicing assay showed that the mutation IVS22-3C>G results in a two-nucleotide AG inclusion at the 3' end of intron 22 and leads to a truncated coagulation factor VIII protein, with partial loss of the C1 domain and complete loss of the C2 domain. The in-silico tool predicted that the mutation induces altered pre-mRNA splicing by using a cryptic acceptor site in intron 22. The IVS22-3C>G mutation was confirmed to affect pre-mRNA splicing and produce a truncated protein, which reduces the stability of binding between the F8 protein and von Willebrand factor carrier protein due to the loss of an interaction domain.

An Accurate Centroiding Algorithm for PSF Reconstruction

NASA Astrophysics Data System (ADS)

Lu, Tianhuan; Luo, Wentao; Zhang, Jun; Zhang, Jiajun; Li, Hekun; Dong, Fuyu; Li, Yingke; Liu, Dezi; Fu, Liping; Li, Guoliang; Fan, Zuhui

2018-07-01

In this work, we present a novel centroiding method based on Fourier space Phase Fitting (FPF) for Point Spread Function (PSF) reconstruction. We generate two sets of simulations to test our method. The first set is generated by GalSim with an elliptical Moffat profile and strong anisotropy that shifts the center of the PSF. The second set of simulations is drawn from CFHT i band stellar imaging data. We find non-negligible anisotropy from CFHT stellar images, which leads to ∼0.08 scatter in units of pixels using a polynomial fitting method (Vakili & Hogg). When we apply the FPF method to estimate the centroid in real space, the scatter reduces to ∼0.04 in S/N = 200 CFHT-like sample. In low signal-to-noise ratio (S/N; 50 and 100) CFHT-like samples, the background noise dominates the shifting of the centroid; therefore, the scatter estimated from different methods is similar. We compare polynomial fitting and FPF using GalSim simulation with optical anisotropy. We find that in all S/N (50, 100, and 200) samples, FPF performs better than polynomial fitting by a factor of ∼3. In general, we suggest that in real observations there exists anisotropy that shifts the centroid, and thus, the FPF method provides a better way to accurately locate it.

A Plant 5S Ribosomal RNA Mimic Regulates Alternative Splicing of Transcription Factor IIIA Pre-mRNAs

PubMed Central

Hammond, Ming C.; Wachter, Andreas; Breaker, Ronald R.

2009-01-01

Transcription factor IIIA (TFIIIA) is required for eukaryotic synthesis of 5S ribosomal RNA by RNA polymerase III. Here we report the discovery of a structured RNA element with striking resemblance to 5S rRNA that is conserved within TFIIIA precursor mRNAs (pre-mRNAs) from diverse plant lineages. TFIIIA protein expression is controlled by alternative splicing of the exon containing the plant 5S rRNA mimic (P5SM). P5SM triggers exon skipping upon binding of ribosomal protein L5, a natural partner of 5S rRNA, which demonstrates the functional adaptation of its structural mimicry. Since the exon-skipped splice product encodes full-length TFIIIA protein, these results reveal a ribosomal protein-mRNA interaction that is involved in 5S rRNA synthesis and has implications for cross-coordination of ribosomal components. This study also provides insight into the origin and function of a newfound class of structured RNA that regulates alternative splicing. PMID:19377483

Alternative splicing regulation in tumor necrosis factor-mediated inflammation.

PubMed

López-Urrutia, Eduardo; Campos-Parra, Alma; Herrera, Luis Alonso; Pérez-Plasencia, Carlos

2017-11-01

It is generally accepted that alternative splicing has an effect on disease when it leads to conspicuous changes in relevant proteins, but that the combinatorial effect of several small modifications can have marked outcomes as well. Inflammation is a complex process involving numerous signaling pathways, among which the tumor necrosis factor (TNF) pathway is one of the most studied. Signaling pathways are commonly represented as intricate cascades of molecular interactions that eventually lead to the activation of one or several genes. Alternative splicing is a common means of controlling protein expression in time and space; therefore, it can modulate the outcome of signaling pathways through small changes in their elements. Notably, the overall process is tightly regulated, which is easily overlooked when analyzing the pathway as a whole. The present review summarizes recent studies of the alternative splicing of key players of the TNF pathway leading to inflammation, and hypothesizes on the cumulative results of those modifications and the impact on cancer development.

Alternative splicing regulation in tumor necrosis factor-mediated inflammation

PubMed Central

López-Urrutia, Eduardo; Campos-Parra, Alma; Herrera, Luis Alonso; Pérez-Plasencia, Carlos

2017-01-01

It is generally accepted that alternative splicing has an effect on disease when it leads to conspicuous changes in relevant proteins, but that the combinatorial effect of several small modifications can have marked outcomes as well. Inflammation is a complex process involving numerous signaling pathways, among which the tumor necrosis factor (TNF) pathway is one of the most studied. Signaling pathways are commonly represented as intricate cascades of molecular interactions that eventually lead to the activation of one or several genes. Alternative splicing is a common means of controlling protein expression in time and space; therefore, it can modulate the outcome of signaling pathways through small changes in their elements. Notably, the overall process is tightly regulated, which is easily overlooked when analyzing the pathway as a whole. The present review summarizes recent studies of the alternative splicing of key players of the TNF pathway leading to inflammation, and hypothesizes on the cumulative results of those modifications and the impact on cancer development. PMID:29113151

A plant 5S ribosomal RNA mimic regulates alternative splicing of transcription factor IIIA pre-mRNAs.

PubMed

Hammond, Ming C; Wachter, Andreas; Breaker, Ronald R

2009-05-01

Transcription factor IIIA (TFIIIA) is required for eukaryotic synthesis of 5S ribosomal RNA by RNA polymerase III. Here we report the discovery of a structured RNA element with clear resemblance to 5S rRNA that is conserved within TFIIIA precursor mRNAs from diverse plant lineages. TFIIIA protein expression is controlled by alternative splicing of the exon containing the plant 5S rRNA mimic (P5SM). P5SM triggers exon skipping upon binding of ribosomal protein L5, a natural partner of 5S rRNA, which demonstrates the functional adaptation of its structural mimicry. As the exon-skipped splice product encodes full-length TFIIIA protein, these results reveal a ribosomal protein-mRNA interaction that is involved in 5S rRNA synthesis and has implications for cross-coordination of ribosomal components. This study also provides insight into the origin and function of a newfound class of structured RNA that regulates alternative splicing.

Low-loss, robust fusion splicing of silica to chalcogenide fiber for integrated mid-infrared laser technology development.

PubMed

Thapa, Rajesh; Gattass, Rafael R; Nguyen, Vinh; Chin, Geoff; Gibson, Dan; Kim, Woohong; Shaw, L Brandon; Sanghera, Jasbinder S

2015-11-01

We demonstrate a low-loss, repeatable, and robust splice between single-mode silica fiber and single-mode chalcogenide (CHG) fiber. These splices are particularly difficult to create because of the significant difference in the two fibers' glass transition temperatures (∼1000°C) as well as the large difference in the coefficients of thermal expansion between the fibers (∼20×10(-6)/°C). With 90% light coupled through the silica-CHG fiber splice, predominantly in the fundamental circular-symmetric mode, into the core of the CHG fiber and with 0.5 dB of splice loss measured around the wavelength of 2.5 μm, after correcting only for the Fresnel loss, the silica-CHG splice offers excellent beam quality and coupling efficiency. The tensile strength of the splice is greater than 12 kpsi, and the laser damage threshold is greater than 2 W (CW) and was limited by the available laser pump power. We also utilized this splicing technique to demonstrate 2 to 4.5 μm ultrabroadband supercontinuum generation in a monolithic all-fiber system comprising a CHG fiber and a high peak power 2 μm pulsed Raman-shifted thulium fiber laser. This is a major development toward compact form factor commercial applications of soft-glass mid-IR fibers.

RBFOX and PTBP1 proteins regulate the alternative splicing of micro-exons in human brain transcripts.

PubMed

Li, Yang I; Sanchez-Pulido, Luis; Haerty, Wilfried; Ponting, Chris P

2015-01-01

Ninety-four percent of mammalian protein-coding exons exceed 51 nucleotides (nt) in length. The paucity of micro-exons (≤ 51 nt) suggests that their recognition and correct processing by the splicing machinery present greater challenges than for longer exons. Yet, because thousands of human genes harbor processed micro-exons, specialized mechanisms may be in place to promote their splicing. Here, we survey deep genomic data sets to define 13,085 micro-exons and to study their splicing mechanisms and molecular functions. More than 60% of annotated human micro-exons exhibit a high level of sequence conservation, an indicator of functionality. While most human micro-exons require splicing-enhancing genomic features to be processed, the splicing of hundreds of micro-exons is enhanced by the adjacent binding of splice factors in the introns of pre-messenger RNAs. Notably, splicing of a significant number of micro-exons was found to be facilitated by the binding of RBFOX proteins, which promote their inclusion in the brain, muscle, and heart. Our analyses suggest that accurate regulation of micro-exon inclusion by RBFOX proteins and PTBP1 plays an important role in the maintenance of tissue-specific protein-protein interactions. © 2015 Li et al.; Published by Cold Spring Harbor Laboratory Press.

Evolution of the anti-truncated stellar profiles of S0 galaxies since z = 0.6 in the SHARDS survey. I. Sample and methods

NASA Astrophysics Data System (ADS)

Borlaff, Alejandro; Eliche-Moral, M. Carmen; Beckman, John E.; Ciambur, Bogdan C.; Pérez-González, Pablo G.; Barro, Guillermo; Cava, Antonio; Cardiel, Nicolas

2017-08-01

Context. The controversy about the origin of the structure of early-type S0-E/S0 galaxies may be due to the difficulty of comparing surface brightness profiles with different depths, photometric corrections and point spread function (PSF) effects (which are almost always ignored). Aims: We aim to quantify the properties of Type-III (anti-truncated) discs in a sample of S0 galaxies at 0.2

Direct measurement and calibration of the Kepler CCD Pixel Response Function for improved photometry and astrometry

NASA Astrophysics Data System (ADS)

Ninkov, Zoran

Stellar images taken with telescopes and detectors in space are usually undersampled, and to correct for this, an accurate pixel response function is required. The standard approach for HST and KEPLER has been to measure the telescope PSF combined ("convolved") with the actual pixel response function, super-sampled by taking into account dithered or offset observed images of many stars (Lauer [1999]). This combined response function has been called the "PRF" (Bryson et al. [2011]). However, using such results has not allowed astrometry from KEPLER to reach its full potential (Monet et al. [2010], [2014]). Given the precision of KEPLER photometry, it should be feasible to use a pre-determined detector pixel response function (PRF) and an optical point spread function (PSF) as separable quantities to more accurately correct photometry and astrometry for undersampling. Wavelength (i.e. stellar color) and instrumental temperature should be affecting each of these differently. Discussion of the PRF in the "KEPLER Instrument Handbook" is limited to an ad-hoc extension of earlier measurements on a quite different CCD. It is known that the KEPLER PSF typically has a sharp spike in the middle, and the main bulk of the PSF is still small enough to be undersampled, so that any substructure in the pixel may interact significantly with the optical PSF. Both the PSF and PRF are probably asymmetric. We propose to measure the PRF for an example of the CCD sensors used on KEPLER at sufficient sampling resolution to allow significant improvement of KEPLER photometry and astrometry, in particular allowing PSF fitting techniques to be used on the data archive.

Degradation of Malaysian peatlands decreases levels of phenolics in soil and in leaves of Macaranga pruinosa

NASA Astrophysics Data System (ADS)

Yule, Catherine; Lim, Yau; Lim, Tse

2016-04-01

Indo-Malaysian tropical peat swamp forests (PSF) sequester enormous stores of carbon in the form of phenolic compounds, particularly lignin as well as tannins. These phenolic compounds are crucial for ecosystem functioning in PSF through their inter-related roles in peat formation and plant defenses. Disturbance of PSF causes destruction of the peat substrate, but the specific impact of disturbance on phenolic compounds in peat and its associated vegetation has not previously been examined. A scale was developed to score peatland degradation based on the three major human impacts that affect tropical PSF - logging, drainage and fire. The objectives of this study were to compare the amount of phenolic compounds in Macaranga pruinosa, a common PSF tree, and in the peat substrate along a gradient of peatland degradation from pristine peat swamp forest to cleared, drained and burnt peatlands. We examined phenolic compounds in M. pruinosa and in peat and found that levels of total phenolic compounds and total tannins decrease in the leaves of M.pruinosa and also in the surface peat layers with an increase in peatland degradation. We conclude that waterlogged conditions preserve the concentration of phenolic compounds in peat, and that even PSF that has been previously logged but which has recovered a full canopy cover will have high levels of total phenolic content (TPC) in peat. High levels of TPC in peat and in the flora are vital for the inhibition of decomposition of organic matter and this is crucial for the accretion of peat and the sequestration of carbon. Thus regional PSF flourish despite the phenolic rich, toxic, waterlogged, nutrient poor, conditions, and reversal of such conditions is a sign of degradation.

Xp11.2 translocation renal cell carcinoma with PSF-TFE3 rearrangement.

PubMed

Zhong, Minghao; Weisman, Paul; Zhu, Bing; Brassesco, Maria; Yang, Youfeng; Linehan, W Marston; Merino, Maria J; Zhang, David; Rohan, Stephen; Cai, Dongming; Yang, Ximing

2013-06-01

Xp11.2 translocation renal cell carcinoma (Xp11.2 RCC) is a subtype of RCC characterized by translocations involving a breakpoint at the TFE3 gene (Xp11.2). Moderate to strong nuclear TFE3 immunoreactivity has been recognized as a specific diagnostic marker for this type of tumor. However, exclusive cytoplasmic localization of a TFE3 fusion protein was reported in UOK 145 cells, a cell line derived from an Xp11.2 RCC harboring the PSF-TFE3 translocation. If reproducible using immunohistochemistry (IHC), this finding would have important implications for pathologists in the diagnosis of Xp11.2 RCC, calling into question the specificity of nuclear immunoreactivity for TFE3 in these tumors. The purpose of this study was to determine whether the above-noted cytoplasmic localization of the TFE3 fusion protein could be reproduced using IHC. UOK 145 cells and fresh frozen tissue from 2 clinical cases of Xp11.2 RCC found to harbor the PSF-TFE3 gene rearrangement (by cytogenetic testing) were collected. All samples were subjected to histopathologic evaluation by board-certified pathologists, TFE3 IHC, reverse transcription polymerase chain reaction, and Sanger sequencing analysis. A strong nuclear TFE3 immunoreactivity was demonstrated in all samples including the UOK 145 cell line. No cytoplasmic immunoreactivity was seen. Reverse transcription polymerase chain reaction and Sanger sequencing confirmed the previously reported PSF-TFE3 gene fusion between exon 9 of PSF and exon 6 of TFE3 in the UOK 145 cell line and in one of 2 clinical cases of Xp11.2 RCC. A novel PSF-TFE3 gene fusion between exon 9 of PSF and exon 5 of TFE3 was detected in the second clinical case of Xp11.2 RCC.

A short conserved motif in ALYREF directs cap- and EJC-dependent assembly of export complexes on spliced mRNAs

PubMed Central

Gromadzka, Agnieszka M.; Steckelberg, Anna-Lena; Singh, Kusum K.; Hofmann, Kay; Gehring, Niels H.

2016-01-01

The export of messenger RNAs (mRNAs) is the final of several nuclear posttranscriptional steps of gene expression. The formation of export-competent mRNPs involves the recruitment of export factors that are assumed to facilitate transport of the mature mRNAs. Using in vitro splicing assays, we show that a core set of export factors, including ALYREF, UAP56 and DDX39, readily associate with the spliced RNAs in an EJC (exon junction complex)- and cap-dependent manner. In order to elucidate how ALYREF and other export adaptors mediate mRNA export, we conducted a computational analysis and discovered four short, conserved, linear motifs present in RNA-binding proteins. We show that mutation in one of the new motifs (WxHD) in an unstructured region of ALYREF reduced RNA binding and abolished the interaction with eIF4A3 and CBP80. Additionally, the mutation impaired proper localization to nuclear speckles and export of a spliced reporter mRNA. Our results reveal important details of the orchestrated recruitment of export factors during the formation of export competent mRNPs. PMID:26773052

Sources of image degradation in fundamental and harmonic ultrasound imaging using nonlinear, full-wave simulations.

PubMed

Pinton, Gianmarco F; Trahey, Gregg E; Dahl, Jeremy J

2011-04-01

A full-wave equation that describes nonlinear propagation in a heterogeneous attenuating medium is solved numerically with finite differences in the time domain (FDTD). This numerical method is used to simulate propagation of a diagnostic ultrasound pulse through a measured representation of the human abdomen with heterogeneities in speed of sound, attenuation, density, and nonlinearity. Conventional delay-andsum beamforming is used to generate point spread functions (PSF) that display the effects of these heterogeneities. For the particular imaging configuration that is modeled, these PSFs reveal that the primary source of degradation in fundamental imaging is reverberation from near-field structures. Reverberation clutter in the harmonic PSF is 26 dB higher than the fundamental PSF. An artificial medium with uniform velocity but unchanged impedance characteristics indicates that for the fundamental PSF, the primary source of degradation is phase aberration. An ultrasound image is created in silico using the same physical and algorithmic process used in an ultrasound scanner: a series of pulses are transmitted through heterogeneous scattering tissue and the received echoes are used in a delay-and-sum beamforming algorithm to generate images. These beamformed images are compared with images obtained from convolution of the PSF with a scatterer field to demonstrate that a very large portion of the PSF must be used to accurately represent the clutter observed in conventional imaging. © 2011 IEEE

The efficacy of fluorocarbon, surfactant, and their combination for improving acute lung injury induced by intratracheal acidified infant formula.

PubMed

Nishina, Kahoru; Mikawa, Katsuya; Takao, Yumiko; Obara, Hidefumi

2005-04-01

We conducted the current study to compare the efficacy of partial liquid ventilation (PLV), pulmonary surfactant (PSF), and their combination in ameliorating the acidified infant-formula-induced acute lung injury (ALI). In the Part I study, 42 rabbits receiving volume-controlled ventilation with positive end-expiratory pressure 10 cm H(2)O were randomly divided into 6 groups (groups noninjuryI, gas ventilation [GVi], PLVi, PSFi, PLVi-->PSFi, and PSFi-->PLVi). ALI was induced by intratracheal acidified infant formula (2 mL/kg, pH 1.8). Group GVi received neither PLV nor PSF therapy. Groups PLV and PSF received intratracheal fluorocarbon 15 mL/kg or surfactant 100 mg/kg, respectively, 30 min after acidified infant formula. Groups PLVi-->PSFi and PSFi-->PLVi received both treatments at 30-min intervals. In Part II, 42 rabbits (in 6 groups) undergoing pressure-controlled ventilation received the same drug therapies as in Part I. The lungs were excised to assess biochemical and histological damage 150 min after induction of ALI. In Parts I and II, PSF, fluorocarbon, and their combination attenuated lung leukosequestration and edema and superoxide production of neutrophils, consequently improving oxygenation, lung mechanics, and pathological changes. Independent of ventilation mode, PSF followed by fluorocarbon provided the most beneficial effects and fluorocarbon followed by PSF produced the least efficacy.

Effect of Evaporation Time on Separation Performance of Polysulfone/Cellulose Acetate (PSF/CA) Membrane

NASA Astrophysics Data System (ADS)

Syahbanu, Intan; Piluharto, Bambang; Khairi, Syahrul; Sudarko

2018-01-01

Polysulfone and cellulose acetate are common material in separation. In this research, polysulfone/cellulose actetate (PSF/CA) blend membrane was prepared. The aim of this research was to study effect of evaporation time in casting of PSF/CA membrane and its performance in filtration. CA was obtained by acetylation process of bacterial cellulose (BC) from fermentation of coconut water. Fourier Transform Infra Red (FTIR) Spectroscopy was used to examine functional groups of BC, CA and commercial cellulose acetate. Subtitution of acetyl groups determined by titration method. Blend membranes were prepared through phase inversion technique in which composition of PSF/PEG/CA/NMP(%w) was 15/5/5/75. Polyethyleneglycol (PEG) and N-methyl-2-pyrrolidone (NMP) were act as pore forming agent and solvent, respectively. Variation of evaporation times were used as parameter to examine water uptake, flux, and morphology of PSF/CA blend membranes. FTIR spectra of CA show characteristic peak of acetyl group at 1220 cm-1 indicated that BC was acetylated succesfully. Degree of subtitution of BCA was found at 2.62. Highest water flux was performed at 2 bar obtained at 106.31 L.m-2.h-1 at 0 minute variation, and decrease as increasing evaporation time. Morphology of PSF/BCA blend membranes were investigated by Scanning Electron Microscopy (SEM) showed that porous asymetric membrane were formed.

Characterization of Endogenous Plasmids from Lactobacillus salivarius UCC118▿ †

PubMed Central

Fang, Fang; Flynn, Sarah; Li, Yin; Claesson, Marcus J.; van Pijkeren, Jan-Peter; Collins, J. Kevin; van Sinderen, Douwe; O'Toole, Paul W.

2008-01-01

The genome of Lactobacillus salivarius UCC118 comprises a 1.83-Mb chromosome, a 242-kb megaplasmid (pMP118), and two smaller plasmids of 20 kb (pSF118-20) and 44 kb (pSF118-44). Annotation and bioinformatic analyses suggest that both of the smaller plasmids replicate by a theta replication mechanism. Furthermore, it appears that they are transmissible, although neither possesses a complete set of conjugation genes. Plasmid pSF118-20 encodes a toxin-antitoxin system composed of pemI and pemK homologs, and this plasmid could be cured when PemI was produced in trans. The minimal replicon of pSF118-20 was determined by deletion analysis. Shuttle vector derivatives of pSF118-20 were generated that included the replication region (pLS203) and the replication region plus mobilization genes (pLS208). The plasmid pLS203 was stably maintained without selection in Lactobacillus plantarum, Lactobacillus fermentum, and the pSF118-20-cured derivative strain of L. salivarius UCC118 (strain LS201). Cloning in pLS203 of genes encoding luciferase and green fluorescent protein, and expression from a constitutive L. salivarius promoter, demonstrated the utility of this vector for the expression of heterologous genes in Lactobacillus. This study thus expands the knowledge base and vector repertoire of probiotic lactobacilli. PMID:18390685

Splicing regulation and dysregulation of cholinergic genes expressed at the neuromuscular junction.

PubMed

Ohno, Kinji; Rahman, Mohammad Alinoor; Nazim, Mohammad; Nasrin, Farhana; Lin, Yingni; Takeda, Jun-Ichi; Masuda, Akio

2017-08-01

We humans have evolved by acquiring diversity of alternative RNA metabolisms including alternative means of splicing and transcribing non-coding genes, and not by acquiring new coding genes. Tissue-specific and developmental stage-specific alternative RNA splicing is achieved by tightly regulated spatiotemporal regulation of expressions and activations of RNA-binding proteins that recognize their cognate splicing cis-elements on nascent RNA transcripts. Genes expressed at the neuromuscular junction are also alternatively spliced. In addition, germline mutations provoke aberrant splicing by compromising binding of RNA-binding proteins, and cause congenital myasthenic syndromes (CMS). We present physiological splicing mechanisms of genes for agrin (AGRN), acetylcholinesterase (ACHE), MuSK (MUSK), acetylcholine receptor (AChR) α1 subunit (CHRNA1), and collagen Q (COLQ) in human, and their aberration in diseases. Splicing isoforms of AChE T , AChE H , and AChE R are generated by hnRNP H/F. Skipping of MUSK exon 10 makes a Wnt-insensitive MuSK isoform, which is unique to human. Skipping of exon 10 is achieved by coordinated binding of hnRNP C, YB-1, and hnRNP L to exon 10. Exon P3A of CHRNA1 is alternatively included to generate a non-functional AChR α1 subunit in human. Molecular dissection of splicing mutations in patients with CMS reveals that exon P3A is alternatively skipped by hnRNP H, polypyrimidine tract-binding protein 1, and hnRNP L. Similarly, analysis of an exonic mutation in COLQ exon 16 in a CMS patient discloses that constitutive splicing of exon 16 requires binding of serine arginine-rich splicing factor 1. Intronic and exonic splicing mutations in CMS enable us to dissect molecular mechanisms underlying alternative and constitutive splicing of genes expressed at the neuromuscular junction. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms. © 2017 International Society for Neurochemistry.

The Mitochondrial Genome of the Prasinophyte Prasinoderma coloniale Reveals Two Trans-Spliced Group I Introns in the Large Subunit rRNA Gene

PubMed Central

Pombert, Jean-François; Otis, Christian; Turmel, Monique; Lemieux, Claude

2013-01-01

Organelle genes are often interrupted by group I and or group II introns. Splicing of these mobile genetic occurs at the RNA level via serial transesterification steps catalyzed by the introns'own tertiary structures and, sometimes, with the help of external factors. These catalytic ribozymes can be found in cis or trans configuration, and although trans-arrayed group II introns have been known for decades, trans-spliced group I introns have been reported only recently. In the course of sequencing the complete mitochondrial genome of the prasinophyte picoplanktonic green alga Prasinoderma coloniale CCMP 1220 (Prasinococcales, clade VI), we uncovered two additional cases of trans-spliced group I introns. Here, we describe these introns and compare the 54,546 bp-long mitochondrial genome of Prasinoderma with those of four other prasinophytes (clades II, III and V). This comparison underscores the highly variable mitochondrial genome architecture in these ancient chlorophyte lineages. Both Prasinoderma trans-spliced introns reside within the large subunit rRNA gene (rnl) at positions where cis-spliced relatives, often containing homing endonuclease genes, have been found in other organelles. In contrast, all previously reported trans-spliced group I introns occur in different mitochondrial genes (rns or coxI). Each Prasinoderma intron is fragmented into two pieces, forming at the RNA level a secondary structure that resembles those of its cis-spliced counterparts. As observed for other trans-spliced group I introns, the breakpoint of the first intron maps to the variable loop L8, whereas that of the second is uniquely located downstream of P9.1. The breakpoint In each Prasinoderma intron corresponds to the same region where the open reading frame (ORF) occurs when present in cis-spliced orthologs. This correlation between the intron breakpoint and the ORF location in cis-spliced orthologs also holds for other trans-spliced introns; we discuss the possible implications of this interesting observation for trans-splicing of group I introns. PMID:24386369

[Identification of C(2)M interacting proteins by yeast two-hybrid screening].

PubMed

Yue, Shan-shan; Xia, Lai-xin

2015-11-01

The synaptonemal complex (SC) is a huge structure which assembles between the homologous chromosomes during meiotic prophase I. Drosophila germ cell-specific nucleoprotein C(2)M clustering at chromosomes can induce SC formation. To further study the molecular function and mechanism of C(2)M in meiosis, we constructed a bait vector for C(2)M and used the yeast two-hybrid system to identify C(2)M interacting proteins. Forty interacting proteins were obtained, including many DNA and histone binding proteins, ATP synthases and transcription factors. Gene silencing assays in Drosophila showed that two genes, wech and Psf1, may delay the disappearance of SC. These results indicate that Wech and Psf1 may form a complex with C(2)M to participate in the formation or stabilization of the SC complex.

Correcting spherical aberrations induced by an unknown medium through determination of its refractive index and thickness.

PubMed

Iwaniuk, Daniel; Rastogi, Pramod; Hack, Erwin

2011-09-26

In imaging and focusing applications, spherical aberration induces axial broadening of the point spread function (PSF). A transparent medium between lens and object of interest induces spherical aberration. We propose a method that first obtains both the physical thickness and the refractive index of the aberration inducing medium in situ by measuring the induced focal shifts for paraxial and large angle rays. Then, the fourth order angle dependence of the optical path difference inside the medium is used to correct the spherical aberration using a phase-only spatial light modulator. The obtained measurement accuracy of 3% is sufficient for a complete compensation as demonstrated in a model microscope with NA 0.3 with glass plate induced axial broadening of the PSF by a factor of 5. © 2011 Optical Society of America

Preparation and Characterization of Chitosan Thin Films on Mixed-Matrix Membranes for Complete Removal of Chromium.

PubMed

Nayak, Vignesh; Jyothi, Mannekote Shivanna; Balakrishna, R Geetha; Padaki, Mahesh; Ismail, Ahmad Fauzi

2015-06-01

Herein we present a new approach for the complete removal of Cr(VI) species, through reduction of Cr(VI) to Cr(III), followed by adsorption of Cr(III). Reduction of chromium from water is an important challenge, as Cr(IV) is one of the most toxic substances emitted from industrial processes. Chitosan (CS) thin films were developed on plain polysulfone (PSf) and PSf/TiO2 membrane substrates by a temperature-induced technique using polyvinyl alcohol as a binder. Structure property elucidation was carried out by X-ray diffraction, microscopy, spectroscopy, contact angle measurement, and water uptake studies. The increase in hydrophilicity followed the order: PSf < PSf/TiO2 < PSf/TiO2/CS membranes. Use of this thin-film composite membrane for chromium removal was investigated with regards to the effects of light and pH. The observations reveal 100 % reduction of Cr(VI) to Cr(III) through electrons and protons donated from OH and NH2 groups of the CS layer; the reduced Cr(III) species are adsorbed onto the CS layer via complexation to give chromium-free water.

Point spread functions and deconvolution of ultrasonic images.

PubMed

Dalitz, Christoph; Pohle-Fröhlich, Regina; Michalk, Thorsten

2015-03-01

This article investigates the restoration of ultrasonic pulse-echo C-scan images by means of deconvolution with a point spread function (PSF). The deconvolution concept from linear system theory (LST) is linked to the wave equation formulation of the imaging process, and an analytic formula for the PSF of planar transducers is derived. For this analytic expression, different numerical and analytic approximation schemes for evaluating the PSF are presented. By comparing simulated images with measured C-scan images, we demonstrate that the assumptions of LST in combination with our formula for the PSF are a good model for the pulse-echo imaging process. To reconstruct the object from a C-scan image, we compare different deconvolution schemes: the Wiener filter, the ForWaRD algorithm, and the Richardson-Lucy algorithm. The best results are obtained with the Richardson-Lucy algorithm with total variation regularization. For distances greater or equal twice the near field distance, our experiments show that the numerically computed PSF can be replaced with a simple closed analytic term based on a far field approximation.

Tuning a Parallel Segmented Flow Column and Enabling Multiplexed Detection.

PubMed

Pravadali-Cekic, Sercan; Kocic, Danijela; Hua, Stanley; Jones, Andrew; Dennis, Gary R; Shalliker, R Andrew

2015-12-15

Active flow technology (AFT) is new form of column technology that was designed to overcome flow heterogeneity to increase separation performance in terms of efficiency and sensitivity and to enable multiplexed detection. This form of AFT uses a parallel segmented flow (PSF) column. A PSF column outlet end-fitting consists of 2 or 4 ports, which can be multiplexed to connect up to 4 detectors. The PSF column not only allows a platform for multiplexed detection but also the combination of both destructive and non-destructive detectors, without additional dead volume tubing, simultaneously. The amount of flow through each port can also be adjusted through pressure management to suit the requirements of a specific detector(s). To achieve multiplexed detection using a PSF column there are a number of parameters which can be controlled to ensure optimal separation performance and quality of results; that is tube dimensions for each port, choice of port for each type of detector and flow adjustment. This protocol is intended to show how to use and tune a PSF column functioning in a multiplexed mode of detection.

Mammalian splicing factor SF1 interacts with SURP domains of U2 snRNP-associated proteins

PubMed Central

Crisci, Angela; Raleff, Flore; Bagdiul, Ivona; Raabe, Monika; Urlaub, Henning; Rain, Jean-Christophe; Krämer, Angela

2015-01-01

Splicing factor 1 (SF1) recognizes the branch point sequence (BPS) at the 3′ splice site during the formation of early complex E, thereby pre-bulging the BPS adenosine, thought to facilitate subsequent base-pairing of the U2 snRNA with the BPS. The 65-kDa subunit of U2 snRNP auxiliary factor (U2AF65) interacts with SF1 and was shown to recruit the U2 snRNP to the spliceosome. Co-immunoprecipitation experiments of SF1-interacting proteins from HeLa cell extracts shown here are consistent with the presence of SF1 in early splicing complexes. Surprisingly almost all U2 snRNP proteins were found associated with SF1. Yeast two-hybrid screens identified two SURP domain-containing U2 snRNP proteins as partners of SF1. A short, evolutionarily conserved region of SF1 interacts with the SURP domains, stressing their role in protein–protein interactions. A reduction of A complex formation in SF1-depleted extracts could be rescued with recombinant SF1 containing the SURP-interaction domain, but only partial rescue was observed with SF1 lacking this sequence. Thus, SF1 can initially recruit the U2 snRNP to the spliceosome during E complex formation, whereas U2AF65 may stabilize the association of the U2 snRNP with the spliceosome at later times. In addition, these findings may have implications for alternative splicing decisions. PMID:26420826

Rearrangement of competing U2 RNA helices within the spliceosome promotes multiple steps in splicing

PubMed Central

Perriman, Rhonda J.; Ares, Manuel

2007-01-01

Nuclear pre-messenger RNA (pre-mRNA) splicing requires multiple spliceosomal small nuclear RNA (snRNA) and pre-mRNA rearrangements. Here we reveal a new snRNA conformational switch in which successive roles for two competing U2 helices, stem IIa and stem IIc, promote distinct splicing steps. When stem IIa is stabilized by loss of stem IIc, rapid ATP-independent and Cus2p-insensitive prespliceosome formation occurs. In contrast, hyperstabilized stem IIc improves the first splicing step on aberrant branchpoint pre-mRNAs and rescues temperature-sensitive U6–U57C, a U6 mutation that also suppresses first-step splicing defects of branchpoint mutations. A second, later role for stem IIa is revealed by its suppression of a cold-sensitive allele of the second-step splicing factor PRP16. Our data expose a spliceosomal progression cycle of U2 stem IIa formation, disruption by stem IIc, and then reformation of stem IIa before the second catalytic step. We propose that the competing stem IIa and stem IIc helices are key spliceosomal RNA elements that optimize juxtaposition of the proper reactive sites during splicing. PMID:17403781

HuB/C/D, nPTB, REST4, and miR-124 regulators of neuronal cell identity are also utilized in the lens.

PubMed

Bitel, Claudine L; Perrone-Bizzozero, Nora I; Frederikse, Peter H

2010-11-04

An interlocking network of transcription factors, RNA binding proteins, and miRNAs globally regulates gene expression and alternative splicing throughout development, and ensures the coordinated mutually exclusive expression of non-neural and neuronal forms of these factors during neurogenesis. Striking similarities between lens fiber cell and neuron cell morphology led us to determine if these factors are also used in the lens. HuR and polypyrimidine tract binding protein (PTB) have been described as 'global regulators' of RNA alternative splicing, stability, and translation in non-neuronal (including ectodermal) tissues examined to date in diverse species, and REST/NRSF (RE-1 Silencing Transcription Factor/Neuron Restrictive Silencing Factor) represses>2,000 neuronal genes in all non-neuronal tissues examined to date, but has not included the lens. During neurogenesis these factors are replaced by what has been considered neuron-specific HuB/C/D, nPTB, and alternatively spliced REST (REST4), which work with miR-124 to activate this battery of genes, comprehensively reprogram neuronal alternative splicing, and maintain their exclusive expression in post-mitotic neurons. Immunoprecipitation, western blot, immunofluorescence, and immunohistochemistry were used to determine the expression and distribution of proteins in mouse and rat lenses. Mobility shift assays were used to examine lenses for REST/NRSF DNA binding activity, and RT-PCR, DNA sequencing, and northern blots were used to identify RNA expression and alternative splicing events in lenses from mouse, rat, and goldfish (N. crassa). We demonstrated that REST, HuR, and PTB proteins are expressed predominantly in epithelial cells in mouse and rat lenses, and showed these factors are also replaced by the predominant expression of REST4, HuB/C/D and nPTB in post-mitotic fiber cells, together with miR-124 expression in vertebrate lenses. REST-regulated gene products were found to be restricted to fiber cells where REST is decreased. These findings predicted nPTB- and HuB/C/D-dependent splicing reactions can also occur in lenses, and we showed Neuronal C-src and Type 1 Neurofibromatosis 1 splicing as well as calcitonin gene related peptide (CGRP) and neural cell adhesion molecule (NCAM-180) alternative transcripts in lenses. Transgenic mice with increased HuD in lens also showed increased growth associated protein 43 (GAP43) and Ca++/Calmodulin dependent kinase IIα (CamKIIα) HuD target gene expression in the lens, similar to brain. The present study provides the first evidence this fundamental set of regulatory factors, previously considered to have a unique role in governing neurogenesis are also used in the lens, and raises questions about the origins of these developmental factors and mechanisms in lens and neuronal cells that also have a basic role in determining the neuronal phenotype.

HuB/C/D, nPTB, REST4, and miR-124 regulators of neuronal cell identity are also utilized in the lens

PubMed Central

Bitel, Claudine L.; Perrone-Bizzozero, Nora I.

2010-01-01

Purpose An interlocking network of transcription factors, RNA binding proteins, and miRNAs globally regulates gene expression and alternative splicing throughout development, and ensures the coordinated mutually exclusive expression of non-neural and neuronal forms of these factors during neurogenesis. Striking similarities between lens fiber cell and neuron cell morphology led us to determine if these factors are also used in the lens. HuR and polypyrimidine tract binding protein (PTB) have been described as ‘global regulators’ of RNA alternative splicing, stability, and translation in non-neuronal (including ectodermal) tissues examined to date in diverse species, and REST/NRSF (RE-1 Silencing Transcription Factor/Neuron Restrictive Silencing Factor) represses >2,000 neuronal genes in all non-neuronal tissues examined to date, but has not included the lens. During neurogenesis these factors are replaced by what has been considered neuron-specific HuB/C/D, nPTB, and alternatively spliced REST (REST4), which work with miR-124 to activate this battery of genes, comprehensively reprogram neuronal alternative splicing, and maintain their exclusive expression in post-mitotic neurons. Methods Immunoprecipitation, western blot, immunofluorescence, and immunohistochemistry were used to determine the expression and distribution of proteins in mouse and rat lenses. Mobility shift assays were used to examine lenses for REST/NRSF DNA binding activity, and RT–PCR, DNA sequencing, and northern blots were used to identify RNA expression and alternative splicing events in lenses from mouse, rat, and goldfish (N. crassa). Results We demonstrated that REST, HuR, and PTB proteins are expressed predominantly in epithelial cells in mouse and rat lenses, and showed these factors are also replaced by the predominant expression of REST4, HuB/C/D and nPTB in post-mitotic fiber cells, together with miR-124 expression in vertebrate lenses. REST-regulated gene products were found to be restricted to fiber cells where REST is decreased. These findings predicted nPTB- and HuB/C/D-dependent splicing reactions can also occur in lenses, and we showed Neuronal C-src and Type 1 Neurofibromatosis 1 splicing as well as calcitonin gene related peptide (CGRP) and neural cell adhesion molecule (NCAM-180) alternative transcripts in lenses. Transgenic mice with increased HuD in lens also showed increased growth associated protein 43 (GAP43) and Ca++/Calmodulin dependent kinase IIα (CamKIIα) HuD target gene expression in the lens, similar to brain. Conclusions The present study provides the first evidence this fundamental set of regulatory factors, previously considered to have a unique role in governing neurogenesis are also used in the lens, and raises questions about the origins of these developmental factors and mechanisms in lens and neuronal cells that also have a basic role in determining the neuronal phenotype. PMID:21139978

4Pi microscopy deconvolution with a variable point-spread function.

PubMed

Baddeley, David; Carl, Christian; Cremer, Christoph

2006-09-20

To remove the axial sidelobes from 4Pi images, deconvolution forms an integral part of 4Pi microscopy. As a result of its high axial resolution, the 4Pi point spread function (PSF) is particularly susceptible to imperfect optical conditions within the sample. This is typically observed as a shift in the position of the maxima under the PSF envelope. A significantly varying phase shift renders deconvolution procedures based on a spatially invariant PSF essentially useless. We present a technique for computing the forward transformation in the case of a varying phase at a computational expense of the same order of magnitude as that of the shift invariant case, a method for the estimation of PSF phase from an acquired image, and a deconvolution procedure built on these techniques.

Extending the Li&Ma method to include PSF information

NASA Astrophysics Data System (ADS)

Nievas-Rosillo, M.; Contreras, J. L.

2016-02-01

The so called Li&Ma formula is still the most frequently used method for estimating the significance of observations carried out by Imaging Atmospheric Cherenkov Telescopes. In this work a straightforward extension of the method for point sources that profits from the good imaging capabilities of current instruments is proposed. It is based on a likelihood ratio under the assumption of a well-known PSF and a smooth background. Its performance is tested with Monte Carlo simulations based on real observations and its sensitivity is compared to standard methods which do not incorporate PSF information. The gain of significance that can be attributed to the inclusion of the PSF is around 10% and can be boosted if a background model is assumed or a finer binning is used.

Wild-Type U2AF1 Antagonizes the Splicing Program Characteristic of U2AF1-Mutant Tumors and Is Required for Cell Survival

PubMed Central

Fei, Dennis Liang; Motowski, Hayley; Chatrikhi, Rakesh; Gao, Shaojian; Kielkopf, Clara L.; Varmus, Harold

2016-01-01

We have asked how the common S34F mutation in the splicing factor U2AF1 regulates alternative splicing in lung cancer, and why wild-type U2AF1 is retained in cancers with this mutation. A human lung epithelial cell line was genetically modified so that U2AF1S34F is expressed from one of the two endogenous U2AF1 loci. By altering levels of mutant or wild-type U2AF1 in this cell line and by analyzing published data on human lung adenocarcinomas, we show that S34F-associated changes in alternative splicing are proportional to the ratio of S34F:wild-type gene products and not to absolute levels of either the mutant or wild-type factor. Preferential recognition of specific 3′ splice sites in S34F-expressing cells is largely explained by differential in vitro RNA-binding affinities of mutant versus wild-type U2AF1 for those same 3′ splice sites. Finally, we show that lung adenocarcinoma cell lines bearing U2AF1 mutations do not require the mutant protein for growth in vitro or in vivo. In contrast, wild-type U2AF1 is required for survival, regardless of whether cells carry the U2AF1S34F allele. Our results provide mechanistic explanations of the magnitude of splicing changes observed in U2AF1-mutant cells and why tumors harboring U2AF1 mutations always retain an expressed copy of the wild-type allele. PMID:27776121

Modulation of KCNQ1 alternative splicing regulates cardiac IKs and action potential repolarization.

PubMed

Lee, Hsiang-Chun; Rudy, Yoram; Po-Yuan, Phd; Sheu, Sheng-Hsiung; Chang, Jan-Gowth; Cui, Jianmin

2013-08-01

Slow delayed-rectifier potassium current (IKs) channels, made of the pore-forming KCNQ1 and auxiliary KCNE1 subunits, play a key role in determining action potential duration (APD) in cardiac myocytes. The consequences of drug-induced KCNQ1 splice alteration remain unknown. To study the modulation of KCNQ1 alternative splicing by amiloride and the consequent changes in IKs and action potentials (APs) in ventricular myocytes. Canine endocardial, midmyocardial, and epicardial ventricular myocytes were isolated. Levels of KCNQ1a and KCNQ1b as well as a series of splicing factors were quantified by using the reverse transcriptase-polymerase chain reaction and Western blot. The effect of amiloride-induced changes in the KCNQ1b/total KCNQ1 ratio on AP was measured by using whole-cell patch clamp with and without isoproterenol. With 50 μmol/L of amiloride for 6 hours, KCNQ1a at transcriptional and translational levels increased in midmyocardial myocytes but decreased in endo- and epicardial myocytes. Likewise, changes in splicing factors in midmyocardial were opposite to that in endo- and epicardial myocytes. In midmyocardial myocytes amiloride shortened APD and decreased isoproterenol-induced early afterdepolarizations significantly. The same amiloride-induced effects were demonstrated by using human ventricular myocyte model for AP simulations under beta-adrenergic stimulation. Moreover, amiloride reduced the transmural dispersion of repolarization in pseudo-electrocardiogram. Amiloride regulates IKs and APs with transmural differences and reduces arrhythmogenicity through the modulation of KCNQ1 splicing. We suggested that the modulation of KCNQ1 splicing may help prevent arrhythmia. Copyright © 2013 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

Characterization of an apparently synonymous F5 mutation causing aberrant splicing and factor V deficiency.

PubMed

Nuzzo, F; Bulato, C; Nielsen, B I; Lee, K; Wielders, S J; Simioni, P; Key, N S; Castoldi, E

2015-03-01

Coagulation factor V (FV) deficiency is a rare autosomal recessive bleeding disorder. We investigated a patient with severe FV deficiency (FV:C < 3%) and moderate bleeding symptoms. Thrombin generation experiments showed residual FV expression in the patient's plasma, which was quantified as 0.7 ± 0.3% by a sensitive prothrombinase-based assay. F5 gene sequencing identified a novel missense mutation in exon 4 (c.578G>C, p.Cys193Ser), predicting the abolition of a conserved disulphide bridge, and an apparently synonymous variant in exon 8 (c.1281C>G). The observation that half of the patient's F5 mRNA lacked the last 18 nucleotides of exon 8 prompted us to re-evaluate the c.1281C>G variant for its possible effects on splicing. Bioinformatics sequence analysis predicted that this transversion would activate a cryptic donor splice site and abolish an exonic splicing enhancer. Characterization in a F5 minigene model confirmed that the c.1281C>G variant was responsible for the patient's splicing defect, which could be partially corrected by a mutation-specific morpholino antisense oligonucleotide. The aberrantly spliced F5 mRNA, whose stability was similar to that of the normal mRNA, encoded a putative FV mutant lacking amino acids 427-432. Expression in COS-1 cells indicated that the mutant protein is poorly secreted and not functional. In conclusion, the c.1281C>G mutation, which was predicted to be translationally silent and hence neutral, causes FV deficiency by impairing pre-mRNA splicing. This finding underscores the importance of cDNA analysis for the correct assessment of exonic mutations. © 2014 John Wiley & Sons Ltd.

Transcriptome instability as a molecular pan-cancer characteristic of carcinomas.

PubMed

Sveen, Anita; Johannessen, Bjarne; Teixeira, Manuel R; Lothe, Ragnhild A; Skotheim, Rolf I

2014-08-10

We have previously proposed transcriptome instability as a genome-wide, pre-mRNA splicing-related characteristic of colorectal cancer. Here, we explore the hypothesis of transcriptome instability being a general characteristic of cancer. Exon-level microarray expression data from ten cancer datasets were analyzed, including breast cancer, cervical cancer, colorectal cancer, gastric cancer, lung cancer, neuroblastoma, and prostate cancer (555 samples), as well as paired normal tissue samples from the colon, lung, prostate, and stomach (93 samples). Based on alternative splicing scores across the genomes, we calculated sample-wise relative amounts of aberrant exon skipping and inclusion. Strong and non-random (P < 0.001) correlations between these estimates and the expression levels of splicing factor genes (n = 280) were found in most cancer types analyzed (breast-, cervical-, colorectal-, lung- and prostate cancer). This suggests a biological explanation for the splicing variation. Surprisingly, these associations prevailed in pan-cancer analyses. This is in contrast to the tissue and cancer specific patterns observed in comparisons across healthy tissue samples from the colon, lung, prostate, and stomach, and between paired cancer-normal samples from the same four tissue types. Based on exon-level expression profiling and computational analyses of alternative splicing, we propose transcriptome instability as a molecular pan-cancer characteristic. The affected cancers show strong and non-random associations between low expression levels of splicing factor genes, and high amounts of aberrant exon skipping and inclusion, and vice versa, on a genome-wide scale.

Genetic and physical interactions between factors involved in both cell cycle progression and pre-mRNA splicing in Saccharomyces cerevisiae.

PubMed Central

Ben-Yehuda, S; Dix, I; Russell, C S; McGarvey, M; Beggs, J D; Kupiec, M

2000-01-01

The PRP17/CDC40 gene of Saccharomyces cerevisiae functions in two different cellular processes: pre-mRNA splicing and cell cycle progression. The Prp17/Cdc40 protein participates in the second step of the splicing reaction and, in addition, prp17/cdc40 mutant cells held at the restrictive temperature arrest in the G2 phase of the cell cycle. Here we describe the identification of nine genes that, when mutated, show synthetic lethality with the prp17/cdc40Delta allele. Six of these encode known splicing factors: Prp8p, Slu7p, Prp16p, Prp22p, Slt11p, and U2 snRNA. The other three, SYF1, SYF2, and SYF3, represent genes also involved in cell cycle progression and in pre-mRNA splicing. Syf1p and Syf3p are highly conserved proteins containing several copies of a repeated motif, which we term RTPR. This newly defined motif is shared by proteins involved in RNA processing and represents a subfamily of the known TPR (tetratricopeptide repeat) motif. Using two-hybrid interaction screens and biochemical analysis, we show that the SYF gene products interact with each other and with four other proteins: Isy1p, Cef1p, Prp22p, and Ntc20p. We discuss the role played by these proteins in splicing and cell cycle progression. PMID:11102353

Genetic and physical interactions between factors involved in both cell cycle progression and pre-mRNA splicing in Saccharomyces cerevisiae.

PubMed

Ben-Yehuda, S; Dix, I; Russell, C S; McGarvey, M; Beggs, J D; Kupiec, M

2000-12-01

The PRP17/CDC40 gene of Saccharomyces cerevisiae functions in two different cellular processes: pre-mRNA splicing and cell cycle progression. The Prp17/Cdc40 protein participates in the second step of the splicing reaction and, in addition, prp17/cdc40 mutant cells held at the restrictive temperature arrest in the G2 phase of the cell cycle. Here we describe the identification of nine genes that, when mutated, show synthetic lethality with the prp17/cdc40Delta allele. Six of these encode known splicing factors: Prp8p, Slu7p, Prp16p, Prp22p, Slt11p, and U2 snRNA. The other three, SYF1, SYF2, and SYF3, represent genes also involved in cell cycle progression and in pre-mRNA splicing. Syf1p and Syf3p are highly conserved proteins containing several copies of a repeated motif, which we term RTPR. This newly defined motif is shared by proteins involved in RNA processing and represents a subfamily of the known TPR (tetratricopeptide repeat) motif. Using two-hybrid interaction screens and biochemical analysis, we show that the SYF gene products interact with each other and with four other proteins: Isy1p, Cef1p, Prp22p, and Ntc20p. We discuss the role played by these proteins in splicing and cell cycle progression.

Cwf16p Associating with the Nineteen Complex Ensures Ordered Exon Joining in Constitutive Pre-mRNA Splicing in Fission Yeast

PubMed Central

Sasaki-Haraguchi, Noriko; Ikuyama, Takeshi; Yoshii, Shogo; Takeuchi-Andoh, Tomoko; Frendewey, David; Tani, Tokio

2015-01-01

Exons are ligated in an ordered manner without the skipping of exons in the constitutive splicing of pre-mRNAs with multiple introns. To identify factors ensuring ordered exon joining in constitutive pre-mRNA splicing, we previously screened for exon skipping mutants in Schizosaccharomyces pombe using a reporter plasmid, and characterized three exon skipping mutants named ods1 (ordered splicing 1), ods2, and ods3, the responsible genes of which encode Prp2/U2AF59, U2AF23, and SF1, respectively. They form an SF1-U2AF59-U2AF23 complex involved in recognition of the branch and 3′ splice sites in pre-mRNA. In the present study, we identified a fourth ods mutant, ods4, which was isolated in an exon-skipping screen. The ods4 + gene encodes Cwf16p, which interacts with the NineTeen Complex (NTC), a complex thought to be involved in the first catalytic step of the splicing reaction. We isolated two multi-copy suppressors for the ods4-1 mutation, Srp2p, an SR protein essential for pre-mRNA splicing, and Tif213p, a translation initiation factor, in S. pombe. The overexpression of Srp2p suppressed the exon-skipping phenotype of all ods mutants, whereas Tif213p suppressed only ods4-1, which has a mutation in the translational start codon of the cwf16 gene. We also showed that the decrease in the transcriptional elongation rate induced by drug treatment suppressed exon skipping in ods4-1. We propose that Cwf16p/NTC participates in the early recognition of the branch and 3′ splice sites and cooperates with the SF1-U2AF59-U2AF23 complex to maintain ordered exon joining. PMID:26302002

Underwater Facilities Inspections and Assessments at Philadelphia Naval Shipyard, Philadelphia, Pennsylvania. Volume 2.

DTIC Science & Technology

1983-10-01

broken piles in a concentrated area . As a result, the relieving platform is unsupported and in this are loading should be restricted to 50 psf until...concentration of damaged piles occurs on Section A near Drydock No. 4 and until repairs are made, loading should be restricted to 100 psf in this area . The...a concentrated area . As a result, the relieving platform is unsup- * ported and in this area loading should be restricted to 50 psf until repairs

Genome-wide association between DNA methylation and alternative splicing in an invertebrate

PubMed Central

2012-01-01

Background Gene bodies are the most evolutionarily conserved targets of DNA methylation in eukaryotes. However, the regulatory functions of gene body DNA methylation remain largely unknown. DNA methylation in insects appears to be primarily confined to exons. Two recent studies in Apis mellifera (honeybee) and Nasonia vitripennis (jewel wasp) analyzed transcription and DNA methylation data for one gene in each species to demonstrate that exon-specific DNA methylation may be associated with alternative splicing events. In this study we investigated the relationship between DNA methylation, alternative splicing, and cross-species gene conservation on a genome-wide scale using genome-wide transcription and DNA methylation data. Results We generated RNA deep sequencing data (RNA-seq) to measure genome-wide mRNA expression at the exon- and gene-level. We produced a de novo transcriptome from this RNA-seq data and computationally predicted splice variants for the honeybee genome. We found that exons that are included in transcription are higher methylated than exons that are skipped during transcription. We detected enrichment for alternative splicing among methylated genes compared to unmethylated genes using fisher’s exact test. We performed a statistical analysis to reveal that the presence of DNA methylation or alternative splicing are both factors associated with a longer gene length and a greater number of exons in genes. In concordance with this observation, a conservation analysis using BLAST revealed that each of these factors is also associated with higher cross-species gene conservation. Conclusions This study constitutes the first genome-wide analysis exhibiting a positive relationship between exon-level DNA methylation and mRNA expression in the honeybee. Our finding that methylated genes are enriched for alternative splicing suggests that, in invertebrates, exon-level DNA methylation may play a role in the construction of splice variants by positively influencing exon inclusion during transcription. The results from our cross-species homology analysis suggest that DNA methylation and alternative splicing are genetic mechanisms whose utilization could contribute to a longer gene length and a slower rate of gene evolution. PMID:22978521

RNA splicing, cell signaling, and response to therapies.

PubMed

Abou Faycal, Cherine; Gazzeri, Sylvie; Eymin, Beatrice

2016-01-01

PremRNA alternative splicing is more a rule than an exception as it affects more than 90% of multiexons genes and plays a key role in proteome diversity. Here, we discuss some recent studies published in the extensively growing field linking RNA splicing and cancer. These last years, the development of high-throughput studies together with appropriate bioinformatic tools have led to the identification of new cancer-specific splicing patterns that allow to distinguish various cancer types, and provide new prognosis biomarkers. In addition, the functional consequences of hot spot mutations affecting various components of the spliceosome machinery in cancers have been described. As an example, missplicing of the enhancer of zeste homolog 2 histone methyltransferase premRNA in response to hot spot mutation of the splicing factor SRSF2 was found to participate to the pathogenesis of myelodysplastic syndrome. Moreover, proofs of principle that targeting the RNA splicing machinery can be used to correct aberrant missplicing, kill oncogene-driven cancer cells, or reverse resistance of tumor cells to targeted therapies have been done. As another example, the core spliceosomal function was recently found to be critical for the survival of Myc-driven breast cancer cells, rendering them hypersensitive to spliceosome inhibitors. Dysregulation of premRNA alternative splicing appears to be one of the hallmarks of cancer. The characterization of novel splicing signatures in cancer as well as the identification of original signaling networks involving RNA splicing regulators should allow to decipher novel oncogenic mechanisms and to develop new therapeutic strategies.

Histone H3K36 methylation regulates pre-mRNA splicing in Saccharomyces cerevisiae

PubMed Central

Sorenson, Matthew R.; Jha, Deepak K.; Ucles, Stefanie A.; Flood, Danielle M.; Strahl, Brian D.; Stevens, Scott W.; Kress, Tracy L.

2016-01-01

ABSTRACT Co-transcriptional splicing takes place in the context of a highly dynamic chromatin architecture, yet the role of chromatin restructuring in coordinating transcription with RNA splicing has not been fully resolved. To further define the contribution of histone modifications to pre-mRNA splicing in Saccharomyces cerevisiae, we probed a library of histone point mutants using a reporter to monitor pre-mRNA splicing. We found that mutation of H3 lysine 36 (H3K36) – a residue methylated by Set2 during transcription elongation – exhibited phenotypes similar to those of pre-mRNA splicing mutants. We identified genetic interactions between genes encoding RNA splicing factors and genes encoding the H3K36 methyltransferase Set2 and the demethylase Jhd1 as well as point mutations of H3K36 that block methylation. Consistent with the genetic interactions, deletion of SET2, mutations modifying the catalytic activity of Set2 or H3K36 point mutations significantly altered expression of our reporter and reduced splicing of endogenous introns. These effects were dependent on the association of Set2 with RNA polymerase II and H3K36 dimethylation. Additionally, we found that deletion of SET2 reduces the association of the U2 and U5 snRNPs with chromatin. Thus, our study provides the first evidence that H3K36 methylation plays a role in co-transcriptional RNA splicing in yeast. PMID:26821844

Control of alternative splicing by forskolin through hnRNP K during neuronal differentiation.

PubMed

Cao, Wenguang; Razanau, Aleh; Feng, Dairong; Lobo, Vincent G; Xie, Jiuyong

2012-09-01

The molecular basis of cell signal-regulated alternative splicing at the 3' splice site remains largely unknown. We isolated a protein kinase A-responsive ribonucleic acid (RNA) element from a 3' splice site of the synaptosomal-associated protein 25 (Snap25) gene for forskolin-inhibited splicing during neuronal differentiation of rat pheochromocytoma PC12 cells. The element binds specifically to heterogeneous nuclear ribonucleo protein (hnRNP) K in a phosphatase-sensitive way, which directly competes with the U2 auxiliary factor U2AF65, an essential component of early spliceosomes. Transcripts with similarly localized hnRNP K target motifs upstream of alternative exons are enriched in genes often associated with neurological diseases. We show that such motifs upstream of the Runx1 exon 6 also bind hnRNP K, and importantly, hnRNP K is required for forskolin-induced repression of the exon. Interestingly, this exon encodes the peptide domain that determines the switch of the transcriptional repressor/activator activity of Runx1, a change known to be critical in specifying neuron lineages. Consistent with an important role of the target genes in neurons, knocking down hnRNP K severely disrupts forskolin-induced neurite growth. Thus, through hnRNP K, the neuronal differentiation stimulus forskolin targets a critical 3' splice site component of the splicing machinery to control alternative splicing of crucial genes. This also provides a regulated direct competitor of U2AF65 for cell signal control of 3' splice site usage.

Profiling the array of Ca(v)3.1 variants from the human T-type calcium channel gene CACNA1G: alternative structures, developmental expression, and biophysical variations.

PubMed

Emerick, Mark C; Stein, Rebecca; Kunze, Robin; McNulty, Megan M; Regan, Melissa R; Hanck, Dorothy A; Agnew, William S

2006-08-01

We describe the regulated transcriptome of CACNA1G, a human gene for T-type Ca(v)3.1 calcium channels that is subject to extensive alternative RNA splicing. Fifteen sites of transcript variation include 2 alternative 5'-UTR promoter sites, 2 alternative 3'-UTR polyadenylation sites, and 11 sites of alternative splicing within the open reading frame. A survey of 1580 fetal and adult human brain full-length complementary DNAs reveals a family of 30 distinct transcripts, including multiple functional forms that vary in expression with development. Statistical analyses of fetal and adult transcript populations reveal patterns of linkages among intramolecular splice site configurations that change dramatically with development. A shift from nearly independent, biased splicing in fetal transcripts to strongly concerted splicing in adult transcripts suggests progressive activation of multiple "programs" of splicing regulation that reorganize molecular structures in differentiating cells. Patch-clamp studies of nine selected variants help relate splicing regulation to permutations of the gating parameters most likely to modify T-channel physiology in expressing neurons. Gating behavior reflects combinatorial interactions between variable domains so that molecular phenotype depends on ensembles of coselected domains, consistent with the observed emergence of concerted splicing during development. We conclude that the structural gene and networks of splicing regulatory factors define an integrated system for the phenotypic variation of Ca(v)3.1 biophysics during nervous system development. Copyright 2006 Wiley-Liss, Inc.

Integration of a splicing regulatory network within the meiotic gene expression program of Saccharomyces cerevisiae

PubMed Central

Munding, Elizabeth M.; Igel, A. Haller; Shiue, Lily; Dorighi, Kristel M.; Treviño, Lisa R.; Ares, Manuel

2010-01-01

Splicing regulatory networks are essential components of eukaryotic gene expression programs, yet little is known about how they are integrated with transcriptional regulatory networks into coherent gene expression programs. Here we define the MER1 splicing regulatory network and examine its role in the gene expression program during meiosis in budding yeast. Mer1p splicing factor promotes splicing of just four pre-mRNAs. All four Mer1p-responsive genes also require Nam8p for splicing activation by Mer1p; however, other genes require Nam8p but not Mer1p, exposing an overlapping meiotic splicing network controlled by Nam8p. MER1 mRNA and three of the four Mer1p substrate pre-mRNAs are induced by the transcriptional regulator Ume6p. This unusual arrangement delays expression of Mer1p-responsive genes relative to other genes under Ume6p control. Products of Mer1p-responsive genes are required for initiating and completing recombination and for activation of Ndt80p, the activator of the transcriptional network required for subsequent steps in the program. Thus, the MER1 splicing regulatory network mediates the dependent relationship between the UME6 and NDT80 transcriptional regulatory networks in the meiotic gene expression program. This study reveals how splicing regulatory networks can be interlaced with transcriptional regulatory networks in eukaryotic gene expression programs. PMID:21123654

Identification of Splicing Quantitative Trait Loci (sQTL) in Drosophila melanogaster with Developmental Lead (Pb2+) Exposure.

PubMed

Qu, Wen; Gurdziel, Katherine; Pique-Regi, Roger; Ruden, Douglas M

2017-01-01

Lead (Pb) poisoning has been a major public health issue globally and the recent Flint water crisis has drawn nation-wide attention to its effects. To better understand how lead plays a role as a neurotoxin, we utilized the Drosophila melanogaster model to study the genetic effects of lead exposure during development and identified lead-responsive genes. In our previous studies, we have successfully identified hundreds of lead-responsive expression QTLs (eQTLs) by using RNA-seq analysis on heads collected from the Drosophila Synthetic Population Resource. Cis -eQTLs, also known as allele-specific expression (ASE) polymorphisms, are generally single-nucleotide polymorphisms in the promoter regions of genes that affect expression of the gene, such as by inhibiting the binding of transcription factors. Trans -eQTLs are genes that regulate mRNA levels for many genes, and are generally thought to be SNPs in trans -acting transcription or translation factors. In this study, we focused our attention on alternative splicing events that are affected by lead exposure. Splicing QTLs (sQTLs), which can be caused by SNPs that alter splicing or alternative splicing (AS), such as by changing the sequence-specific binding affinity of splicing factors to the pre-mRNA. We applied two methods in search for sQTLs by using RNA-seq data from control and lead-exposed w 1118 Drosophila heads. First, we used the fraction of reads in a gene that falls in each exon as the phenotype. Second, we directly compared the transcript counts among the various splicing isoforms as the phenotype. Among the 1,236 potential Pb-responsive sQTLs ( p < 0.0001, FDR < 0.39), mostly cis -sQTLs, one of the most distinct genes is Dscam1 (Down Syndrome Cell Adhesion Molecule), which has over 30,000 potential alternative splicing isoforms. We have also identified a candidate Pb-responsive trans -sQTL hotspot that appears to regulate 129 genes that are enriched in the "cation channel" gene ontology category, suggesting a model in which alternative splicing of these channels might lead to an increase in the elimination of Pb 2+ from the neurons encoding these channels. To our knowledge, this is the first paper that uses sQTL analyses to understand the neurotoxicology of an environmental toxin in any organism, and the first reported discovery of a candidate trans -sQTL hotspot.

Identification of Splicing Quantitative Trait Loci (sQTL) in Drosophila melanogaster with Developmental Lead (Pb2+) Exposure

PubMed Central

Qu, Wen; Gurdziel, Katherine; Pique-Regi, Roger; Ruden, Douglas M.

2017-01-01

Lead (Pb) poisoning has been a major public health issue globally and the recent Flint water crisis has drawn nation-wide attention to its effects. To better understand how lead plays a role as a neurotoxin, we utilized the Drosophila melanogaster model to study the genetic effects of lead exposure during development and identified lead-responsive genes. In our previous studies, we have successfully identified hundreds of lead-responsive expression QTLs (eQTLs) by using RNA-seq analysis on heads collected from the Drosophila Synthetic Population Resource. Cis-eQTLs, also known as allele-specific expression (ASE) polymorphisms, are generally single-nucleotide polymorphisms in the promoter regions of genes that affect expression of the gene, such as by inhibiting the binding of transcription factors. Trans-eQTLs are genes that regulate mRNA levels for many genes, and are generally thought to be SNPs in trans-acting transcription or translation factors. In this study, we focused our attention on alternative splicing events that are affected by lead exposure. Splicing QTLs (sQTLs), which can be caused by SNPs that alter splicing or alternative splicing (AS), such as by changing the sequence-specific binding affinity of splicing factors to the pre-mRNA. We applied two methods in search for sQTLs by using RNA-seq data from control and lead-exposed w1118 Drosophila heads. First, we used the fraction of reads in a gene that falls in each exon as the phenotype. Second, we directly compared the transcript counts among the various splicing isoforms as the phenotype. Among the 1,236 potential Pb-responsive sQTLs (p < 0.0001, FDR < 0.39), mostly cis-sQTLs, one of the most distinct genes is Dscam1 (Down Syndrome Cell Adhesion Molecule), which has over 30,000 potential alternative splicing isoforms. We have also identified a candidate Pb-responsive trans-sQTL hotspot that appears to regulate 129 genes that are enriched in the “cation channel” gene ontology category, suggesting a model in which alternative splicing of these channels might lead to an increase in the elimination of Pb2+ from the neurons encoding these channels. To our knowledge, this is the first paper that uses sQTL analyses to understand the neurotoxicology of an environmental toxin in any organism, and the first reported discovery of a candidate trans-sQTL hotspot. PMID:29114259

Visualizing Mixed Variable-Type Multidimensional Data Using Tree Distances

DTIC Science & Technology

2015-09-01

24 1. The Regression Method .................................................. 25 2. Maximum Deviance Ratio Method... Deviance Change as a Factor of the Amount of Correlation? ................................................... 50 B. A PROPOSED SOLUTION...Splice mapping using d1 ...................... 36 Figure 18. Reduction in 2R analog ( deviance ratio-DevRat) per variable for the Splice data

The edge artifact in the point-spread function-based PET reconstruction at different sphere-to-background ratios of radioactivity.

PubMed

Kidera, Daisuke; Kihara, Ken; Akamatsu, Go; Mikasa, Shohei; Taniguchi, Takafumi; Tsutsui, Yuji; Takeshita, Toshiki; Maebatake, Akira; Miwa, Kenta; Sasaki, Masayuki

2016-02-01

The aim of this study was to quantitatively evaluate the edge artifacts in PET images reconstructed using the point-spread function (PSF) algorithm at different sphere-to-background ratios of radioactivity (SBRs). We used a NEMA IEC body phantom consisting of six spheres with 37, 28, 22, 17, 13 and 10 mm in inner diameter. The background was filled with (18)F solution with a radioactivity concentration of 2.65 kBq/mL. We prepared three sets of phantoms with SBRs of 16, 8, 4 and 2. The PET data were acquired for 20 min using a Biograph mCT scanner. The images were reconstructed with the baseline ordered subsets expectation maximization (OSEM) algorithm, and with the OSEM + PSF correction model (PSF). For the image reconstruction, the number of iterations ranged from one to 10. The phantom PET image analyses were performed by a visual assessment of the PET images and profiles, a contrast recovery coefficient (CRC), which is the ratio of SBR in the images to the true SBR, and the percent change in the maximum count between the OSEM and PSF images (Δ % counts). In the PSF images, the spheres with a diameter of 17 mm or larger were surrounded by a dense edge in comparison with the OSEM images. In the spheres with a diameter of 22 mm or smaller, an overshoot appeared in the center of the spheres as a sharp peak in the PSF images in low SBR. These edge artifacts were clearly observed in relation to the increase of the SBR. The overestimation of the CRC was observed in 13 mm spheres in the PSF images. In the spheres with a diameter of 17 mm or smaller, the Δ % counts increased with an increasing SBR. The Δ % counts increased to 91 % in the 10-mm sphere at the SBR of 16. The edge artifacts in the PET images reconstructed using the PSF algorithm increased with an increasing SBR. In the small spheres, the edge artifact was observed as a sharp peak at the center of spheres and could result in overestimation.

Performance measurement of PSF modeling reconstruction (True X) on Siemens Biograph TruePoint TrueV PET/CT.

PubMed

Lee, Young Sub; Kim, Jin Su; Kim, Kyeong Min; Kang, Joo Hyun; Lim, Sang Moo; Kim, Hee-Joung

2014-05-01

The Siemens Biograph TruePoint TrueV (B-TPTV) positron emission tomography (PET) scanner performs 3D PET reconstruction using a system matrix with point spread function (PSF) modeling (called the True X reconstruction). PET resolution was dramatically improved with the True X method. In this study, we assessed the spatial resolution and image quality on a B-TPTV PET scanner. In addition, we assessed the feasibility of animal imaging with a B-TPTV PET and compared it with a microPET R4 scanner. Spatial resolution was measured at center and at 8 cm offset from the center in transverse plane with warm background activity. True X, ordered subset expectation maximization (OSEM) without PSF modeling, and filtered back-projection (FBP) reconstruction methods were used. Percent contrast (% contrast) and percent background variability (% BV) were assessed according to NEMA NU2-2007. The recovery coefficient (RC), non-uniformity, spill-over ratio (SOR), and PET imaging of the Micro Deluxe Phantom were assessed to compare image quality of B-TPTV PET with that of the microPET R4. When True X reconstruction was used, spatial resolution was <3.65 mm with warm background activity. % contrast and % BV with True X reconstruction were higher than those with the OSEM reconstruction algorithm without PSF modeling. In addition, the RC with True X reconstruction was higher than that with the FBP method and the OSEM without PSF modeling method on the microPET R4. The non-uniformity with True X reconstruction was higher than that with FBP and OSEM without PSF modeling on microPET R4. SOR with True X reconstruction was better than that with FBP or OSEM without PSF modeling on the microPET R4. This study assessed the performance of the True X reconstruction. Spatial resolution with True X reconstruction was improved by 45 % and its % contrast was significantly improved compared to those with the conventional OSEM without PSF modeling reconstruction algorithm. The noise level was higher than that with the other reconstruction algorithm. Therefore, True X reconstruction should be used with caution when quantifying PET data.

Long noncoding RNA Saf and splicing factor 45 increase soluble Fas and resistance to apoptosis

PubMed Central

Riberdy, Janice M.; Persons, Derek A.; Wilber, Andrew

2016-01-01

In multicellular organisms, cell growth and differentiation is controlled in part by programmed cell death or apoptosis. One major apoptotic pathway is triggered by Fas receptor (Fas)-Fas ligand (FasL) interaction. Neoplastic cells are frequently resistant to Fas-mediated apoptosis, evade Fas signals through down regulation of Fas and produce soluble Fas proteins that bind FasL thereby blocking apoptosis. Soluble Fas (sFas) is an alternative splice product of Fas pre-mRNA, commonly created by exclusion of transmembrane spanning sequences encoded within exon 6 (FasΔEx6). Long non-coding RNAs (lncRNAs) interact with other RNAs, DNA, and proteins to regulate gene expression. One lncRNA, Fas-antisense or Saf, was shown to participate in alternative splicing of Fas pre-mRNA through unknown mechanisms. We show that Saf is localized in the nucleus where it interacts with Fas receptor pre-mRNA and human splicing factor 45 (SPF45) to facilitate alternative splicing and exclusion of exon 6. The product is a soluble Fas protein that protects cells against FasL-induced apoptosis. Collectively, these studies reveal a novel mechanism to modulate this critical cell death program by an lncRNA and its protein partner. PMID:26885613

Association of polymorphisms in genes of factors involved in regulation of splicing of cystic fibrosis transmembrane conductance regulator mRNA with acute respiratory distress syndrome in children with pneumonia.

PubMed

Perez-Marques, Francesca; Simpson, Pippa; Yan, Ke; Quasney, Michael W; Halligan, Nadine; Merchant, Daniel; Dahmer, Mary K

2016-09-05

Previous work has demonstrated a strong association between lung injury in African American children with pneumonia and a polymorphic (TG)mTn region in cystic fibrosis transmembrane conductance (CFTR) involved in the generation of a nonfunctional CFTR protein lacking exon 9. A number of splicing factors that regulate the inclusion/exclusion of exon 9 have been identified. The objective of this study was to determine whether genetic variants in these splicing factors were associated with acute respiratory distress syndrome (ARDS) in children with pneumonia. This is a prospective cohort genetic association study of lung injury in African American and non-Hispanic Caucasian children with community-acquired pneumonia evaluated in the emergency department or admitted to the hospital. Linkage-disequilibrium-tag single nucleotide polymorphisms (LD-tag SNPs) in genes of the following splicing factors (followed by gene name) involved in exon 9 skipping PTB1 (PTBP1), SRp40 (SFRS1), SR2/ASF (SFRS5), TDP-43 (TARDBP), TIA-1 (TIA1), and U2AF(65) (U2AF2) were genotyped. SNPs in the gene of the splicing factor CELF2 (CELF2) were selected by conservation score. Multivariable analysis was used to examine association between genotypes and ARDS. The African American cohort (n = 474) had 29 children with ARDS and the non-Hispanic Caucasian cohort (n = 304) had 32 children with ARDS. In the African American group multivariable analysis indicated that three variants in CELF2, rs7068124 (p = 0.004), rs3814634 (p = 0.032) and rs10905928 (p = 0.044), and two in TIA1, rs2592178 (p = 0.005) and rs13402990 (p = 0.018) were independently associated with ARDS. In the non-Hispanic Caucasian group, a single variant in CELF2, rs2277212 (p = 0.014), was associated with increased risk of developing ARDS. The data indicate that SNPs in CELF2 may be associated with the risk of developing ARDS in both African American and non-Hispanic Caucasian children with pneumonia and suggest that the potential role of the splicing factor CELF2 in ARDS should be explored further.

Mutations in PRPF31 Inhibit Pre-mRNA Splicing of Rhodopsin Gene and Cause Apoptosis of Retinal Cells

PubMed Central

Yuan, Liya; Kawada, Mariko; Havlioglu, Necat; Tang, Hao; Wu, Jane Y.

2007-01-01

Mutations in human PRPF31 gene have been identified in patients with autosomal dominant retinitis pigmentosa (adRP). To begin to understand mechanisms by which defects in this general splicing factor cause retinal degeneration, we examined the relationship between PRPF31 and pre-mRNA splicing of photoreceptor-specific genes. We used a specific anti-PRPF31 antibody to immunoprecipitate splicing complexes from retinal cells and identified the transcript of rhodopsin gene (RHO) among RNA species associated with PRPF31-containing complexes. Mutant PRPF31 proteins significantly inhibited pre-mRNA splicing of intron 3 in RHO gene. In primary retinal cell cultures, expression of the mutant PRPF31 proteins reduced rhodopsin expression and caused apoptosis of rhodopsin-positive retinal cells. This primary retinal culture assay provides an in vitro model to study photoreceptor cell death caused by PRPF31 mutations. Our results demonstrate that mutations in PRPF31 gene affect RHO pre-mRNA splicing and reveal a link between PRPF31 and RHO, two major adRP genes. PMID:15659613

Emerging roles of the spliceosomal machinery in myelodysplastic syndromes and other hematological disorders.

PubMed

Visconte, V; Makishima, H; Maciejewski, J P; Tiu, R V

2012-12-01

In humans, the majority of all protein-coding transcripts contain introns that are removed by mRNA splicing carried out by spliceosomes. Mutations in the spliceosome machinery have recently been identified using whole-exome/genome technologies in myelodysplastic syndromes (MDS) and in other hematological disorders. Alterations in splicing factor 3 subunit b1 (SF3b1) were the first spliceosomal mutations described, immediately followed by identification of other splicing factor mutations, including U2 small nuclear RNA auxillary factor 1 (U2AF1) and serine arginine-rich splicing factor 2 (SRSF2). SF3b1/U2AF1/SRSF2 mutations occur at varying frequencies in different disease subtypes, each contributing to differences in survival outcomes. However, the exact functional consequences of these spliceosomal mutations in the pathogenesis of MDS and other hematological malignancies remain largely unknown and subject to intense investigation. For SF3b1, a gain of function mutation may offer the promise of new targeted therapies for diseases that carry this molecular abnormality that can potentially lead to cure. This review aims to provide a comprehensive overview of the emerging role of the spliceosome machinery in the biology of MDS/hematological disorders with an emphasis on the functional consequences of mutations, their clinical significance, and perspectives on how they may influence our understanding and management of diseases affected by these mutations.

Emerging roles of the spliceosomal machinery in myelodysplastic syndromes and other hematologic disorders

PubMed Central

Visconte, V; Makishima, H; Maciejewski, JP; Tiu, RV

2013-01-01

In humans, the majority of all protein-coding transcripts contain introns that are removed by mRNA splicing carried out by spliceosomes. Mutations in the spliceosome machinery have recently been identified using whole exome/genome technologies in myelodysplastic syndromes (MDS) and in other hematologic disorders. Alterations in Splicing Factor 3 Subunit b1 (SF3b1) were the first spliceosomal mutations described, immediately followed by identification of other splicing factor mutations, including U2 Small Nuclear RNA Auxillary Factor 1 (U2AF1) and Serine Arginine Rich Splicing Factor 2 (SRSF2). SF3b1/U2AF1/SRSF2 mutations occur at varying frequencies in different disease subtypes, each contributing to differences in survival outcomes. However, the exact functional consequences of these spliceosomal mutations in the pathogenesis of MDS and other hematologic malignancies remain largely unknown and subject to intense investigation. For SF3b1, a gain of function mutation may offer the promise of new targeted therapies for diseases that carry this molecular abnormality that can potentially lead to cure. This review aims to provide a comprehensive overview of the emerging role of the spliceosome machinery in the biology of MDS/hematologic disorders with an emphasis on the functional consequences of mutations, their clinical significance, and perspectives on how they may influence our understanding and management of diseases affected by these mutations. PMID:22678168

Analysis of alternative splicing associated with aging and neurodegeneration in the human brain

PubMed Central

Tollervey, James R.; Wang, Zhen; Hortobágyi, Tibor; Witten, Joshua T.; Zarnack, Kathi; Kayikci, Melis; Clark, Tyson A.; Schweitzer, Anthony C.; Rot, Gregor; Curk, Tomaž; Zupan, Blaž; Rogelj, Boris; Shaw, Christopher E.; Ule, Jernej

2011-01-01

Age is the most important risk factor for neurodegeneration; however, the effects of aging and neurodegeneration on gene expression in the human brain have most often been studied separately. Here, we analyzed changes in transcript levels and alternative splicing in the temporal cortex of individuals of different ages who were cognitively normal, affected by frontotemporal lobar degeneration (FTLD), or affected by Alzheimer's disease (AD). We identified age-related splicing changes in cognitively normal individuals and found that these were present also in 95% of individuals with FTLD or AD, independent of their age. These changes were consistent with increased polypyrimidine tract binding protein (PTB)–dependent splicing activity. We also identified disease-specific splicing changes that were present in individuals with FTLD or AD, but not in cognitively normal individuals. These changes were consistent with the decreased neuro-oncological ventral antigen (NOVA)–dependent splicing regulation, and the decreased nuclear abundance of NOVA proteins. As expected, a dramatic down-regulation of neuronal genes was associated with disease, whereas a modest down-regulation of glial and neuronal genes was associated with aging. Whereas our data indicated that the age-related splicing changes are regulated independently of transcript-level changes, these two regulatory mechanisms affected expression of genes with similar functions, including metabolism and DNA repair. In conclusion, the alternative splicing changes identified in this study provide a new link between aging and neurodegeneration. PMID:21846794

A fringe projector-based study of the Brighter-Fatter Effect in LSST CCDs

DOE PAGES

Gilbertson, W.; Nomerotski, A.; Takacs, P.

2017-09-07

In order to achieve the goals of the Large Synoptic Survey Telescope for Dark Energy science requires a detailed understanding of CCD sensor effects. One such sensor effect is the Point Spread Function (PSF) increasing with flux, alternatively called the `Brighter-Fatter Effect.' Here a novel approach was tested to perform the PSF measurements in the context of the Brighter-Fatter Effect employing a Michelson interferometer to project a sinusoidal fringe pattern onto the CCD. The Brighter-Fatter effect predicts that the fringe pattern should become asymmetric in the intensity pattern as the brighter peaks corresponding to a larger flux are smeared bymore » a larger PSF. By fitting the data with a model that allows for a changing PSF, the strength of the Brighter-Fatter effect can be evaluated.« less

A fringe projector-based study of the Brighter-Fatter Effect in LSST CCDs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gilbertson, W.; Nomerotski, A.; Takacs, P.

In order to achieve the goals of the Large Synoptic Survey Telescope for Dark Energy science requires a detailed understanding of CCD sensor effects. One such sensor effect is the Point Spread Function (PSF) increasing with flux, alternatively called the `Brighter-Fatter Effect.' Here a novel approach was tested to perform the PSF measurements in the context of the Brighter-Fatter Effect employing a Michelson interferometer to project a sinusoidal fringe pattern onto the CCD. The Brighter-Fatter effect predicts that the fringe pattern should become asymmetric in the intensity pattern as the brighter peaks corresponding to a larger flux are smeared bymore » a larger PSF. By fitting the data with a model that allows for a changing PSF, the strength of the Brighter-Fatter effect can be evaluated.« less

The M 4 Core Project with HST - V. Characterizing the PSFs of WFC3/UVIS by focus★

NASA Astrophysics Data System (ADS)

Anderson, J.; Bedin, L. R.

2017-09-01

As part of the astrometric Hubble Space Telescope (HST) large program GO-12911, we conduct an in-depth study to characterize the point spread function (PSF) of the Uv-VISual channel of the Wide Field Camera 3 (WFC3), as a necessary step to achieve the astrometric goals of the program. We extracted a PSF from each of the 589 deep exposures taken through the F467M filter over the course of a year and find that the vast majority of the PSFs lie along a 1-D locus that stretches continuously from one side of focus, through optimal focus, to the other side of focus. We constructed a focus-diverse set of PSFs and find that with only five medium-bright stars in an exposure it is possible to pin down the focus level of that exposure. We show that the focus-optimized PSF does a considerably better job fitting stars than the average 'library' PSF, especially when the PSF is out of focus. The fluxes and positions are significantly improved over the 'library' PSF treatment. These results are beneficial for a much broader range of scientific applications than simply the program at hand, but the immediate use of these PSFs will enable us to search for astrometric wobble in the bright stars in the core of the globular cluster M 4, which would indicate a dark, high-mass companion, such as a white dwarf, neutron star or black hole.

Synovial Fluid Response to Extensional Flow: Effects of Dilution and Intermolecular Interactions

PubMed Central

Haward, Simon J.

2014-01-01

In this study, a microfluidic cross-slot device is used to examine the extensional flow response of diluted porcine synovial fluid (PSF) samples using flow-induced birefringence (FIB) measurements. The PSF sample is diluted to 10× 20× and 30× its original mass in a phosphate-buffered saline and its FIB response measured as a function of the strain rate at the stagnation point of the cross-slots. Equivalent experiments are also carried out using trypsin-treated PSF (t-PSF) in which the protein content is digested away using an enzyme. The results show that, at the synovial fluid concentrations tested, the protein content plays a negligible role in either the fluid's bulk shear or extensional flow behaviour. This helps support the validity of the analysis of synovial fluid HA content, either by microfluidic or by other techniques where the synovial fluid is first diluted, and suggests that the HA and protein content in synovial fluid must be higher than a certain minimum threshold concentration before HA-protein or protein-protein interactions become significant. However a systematic shift in the FIB response as the PSF and t-PSF samples are progressively diluted indicates that HA-HA interactions remain significant at the concentrations tested. These interactions influence FIB-derived macromolecular parameters such as the relaxation time and the molecular weight distribution and therefore must be minimized for the best validity of this method as an analytical technique, in which non-interaction between molecules is assumed. PMID:24651529

Synovial fluid response to extensional flow: effects of dilution and intermolecular interactions.

PubMed

Haward, Simon J

2014-01-01

In this study, a microfluidic cross-slot device is used to examine the extensional flow response of diluted porcine synovial fluid (PSF) samples using flow-induced birefringence (FIB) measurements. The PSF sample is diluted to 10× 20× and 30× its original mass in a phosphate-buffered saline and its FIB response measured as a function of the strain rate at the stagnation point of the cross-slots. Equivalent experiments are also carried out using trypsin-treated PSF (t-PSF) in which the protein content is digested away using an enzyme. The results show that, at the synovial fluid concentrations tested, the protein content plays a negligible role in either the fluid's bulk shear or extensional flow behaviour. This helps support the validity of the analysis of synovial fluid HA content, either by microfluidic or by other techniques where the synovial fluid is first diluted, and suggests that the HA and protein content in synovial fluid must be higher than a certain minimum threshold concentration before HA-protein or protein-protein interactions become significant. However a systematic shift in the FIB response as the PSF and t-PSF samples are progressively diluted indicates that HA-HA interactions remain significant at the concentrations tested. These interactions influence FIB-derived macromolecular parameters such as the relaxation time and the molecular weight distribution and therefore must be minimized for the best validity of this method as an analytical technique, in which non-interaction between molecules is assumed.

Impact of plant-soil feedback on plant traits at field scale, testing the use of UAV-based optical sensors

NASA Astrophysics Data System (ADS)

De Deyn, Gerlinde B.; van der Meij, Bob; Barel, Janna M.; Suomalainen, Juha; Kooistra, Lammert

2017-04-01

Plant responses to biotic and abiotic legacies left in soil by preceding plants is known as plant-soil feedback (PSF). PSF is an important mechanism to explain plant community dynamics and plant performance in natural and agricultural systems. However, most PSF studies are short-term and small-scale due to practical constraints for field scale quantification of PSF effects, yet field experiments are warranted to asses actual PSF effects under less controlled conditions. Here we used Unmanned Aerial Vehicle (UAV)-based optical sensors to test whether PSF effects on plant traits can be quantified remotely. We established a randomized agro-ecological field experiment in which six different cover crop species and species combinations from three different plant families (Poaceae, Fabaceae, Brassicaceae) were grown. The feedback effects on plant traits were tested in oat (Avena sativa) by quantifying the cover crop legacy effects on key plant traits: height, fresh biomass, nitrogen content and leaf chlorophyll content. Prior to destructive sampling, hyperspectral data was acquired and used for calibration and independent validation of regression models to retrieve plant traits from optical data. Subsequently, for each trait the model with highest precision and accuracy was selected. We used the hyperspectral analyses to predict the directly measured plant height (RMSE= 5.12 cm, R2= 0.79), chlorophyll content (RMSE= 0.11 g m-2, R2= 0.80), N-content (RMSE= 1.94 g m-2, R2= 0.68), and fresh biomass (RMSE= 0.72 kg m-2, R2=0.56). Overall the PSF effects of the different cover crop treatments based on the remote sensing data matched the results based on in situ measurements. The average oat canopy was tallest and its leaf chlorophyll content highest in response to legacy of Vicia sativa monocultures (100 cm, 0.95 g m-2, respectively) and in mixture with Raphanus sativus (100 cm, 1.09 g m-2, respectively), while the lowest values (76 cm, 0.41 g m-2, respectively) were found in response to legacy of Lolium perenne monoculture, and intermediate responses to the legacy of the other treatments. We show that PSF effects in the field occur and alter several important plant traits that can be sensed remotely and quantified in a non-destructive way using UAV-based optical sensors; these can be repeated over the growing season to increase temporal resolution. Remote sensing thereby offers great potential for studying PSF effects at field scale and relevant spatial-temporal resolutions which will facilitate the elucidation of the underlying mechanisms.

Remote sensing of plant trait responses to field-based plant-soil feedback using UAV-based optical sensors

NASA Astrophysics Data System (ADS)

van der Meij, Bob; Kooistra, Lammert; Suomalainen, Juha; Barel, Janna M.; De Deyn, Gerlinde B.

2017-02-01

Plant responses to biotic and abiotic legacies left in soil by preceding plants is known as plant-soil feedback (PSF). PSF is an important mechanism to explain plant community dynamics and plant performance in natural and agricultural systems. However, most PSF studies are short-term and small-scale due to practical constraints for field-scale quantification of PSF effects, yet field experiments are warranted to assess actual PSF effects under less controlled conditions. Here we used unmanned aerial vehicle (UAV)-based optical sensors to test whether PSF effects on plant traits can be quantified remotely. We established a randomized agro-ecological field experiment in which six different cover crop species and species combinations from three different plant families (Poaceae, Fabaceae, Brassicaceae) were grown. The feedback effects on plant traits were tested in oat (Avena sativa) by quantifying the cover crop legacy effects on key plant traits: height, fresh biomass, nitrogen content, and leaf chlorophyll content. Prior to destructive sampling, hyperspectral data were acquired and used for calibration and independent validation of regression models to retrieve plant traits from optical data. Subsequently, for each trait the model with highest precision and accuracy was selected. We used the hyperspectral analyses to predict the directly measured plant height (RMSE = 5.12 cm, R2 = 0.79), chlorophyll content (RMSE = 0.11 g m-2, R2 = 0.80), N-content (RMSE = 1.94 g m-2, R2 = 0.68), and fresh biomass (RMSE = 0.72 kg m-2, R2 = 0.56). Overall the PSF effects of the different cover crop treatments based on the remote sensing data matched the results based on in situ measurements. The average oat canopy was tallest and its leaf chlorophyll content highest in response to legacy of Vicia sativa monocultures (100 cm, 0.95 g m-2, respectively) and in mixture with Raphanus sativus (100 cm, 1.09 g m-2, respectively), while the lowest values (76 cm, 0.41 g m-2, respectively) were found in response to legacy of Lolium perenne monoculture, and intermediate responses to the legacy of the other treatments. We show that PSF effects in the field occur and alter several important plant traits that can be sensed remotely and quantified in a non-destructive way using UAV-based optical sensors; these can be repeated over the growing season to increase temporal resolution. Remote sensing thereby offers great potential for studying PSF effects at field scale and relevant spatial-temporal resolutions which will facilitate the elucidation of the underlying mechanisms.

Spliced integrated retrotransposed element (SpIRE) formation in the human genome.

PubMed

Larson, Peter A; Moldovan, John B; Jasti, Naveen; Kidd, Jeffrey M; Beck, Christine R; Moran, John V

2018-03-01

Human Long interspersed element-1 (L1) retrotransposons contain an internal RNA polymerase II promoter within their 5' untranslated region (UTR) and encode two proteins, (ORF1p and ORF2p) required for their mobilization (i.e., retrotransposition). The evolutionary success of L1 relies on the continuous retrotransposition of full-length L1 mRNAs. Previous studies identified functional splice donor (SD), splice acceptor (SA), and polyadenylation sequences in L1 mRNA and provided evidence that a small number of spliced L1 mRNAs retrotransposed in the human genome. Here, we demonstrate that the retrotransposition of intra-5'UTR or 5'UTR/ORF1 spliced L1 mRNAs leads to the generation of spliced integrated retrotransposed elements (SpIREs). We identified a new intra-5'UTR SpIRE that is ten times more abundant than previously identified SpIREs. Functional analyses demonstrated that both intra-5'UTR and 5'UTR/ORF1 SpIREs lack Cis-acting transcription factor binding sites and exhibit reduced promoter activity. The 5'UTR/ORF1 SpIREs also produce nonfunctional ORF1p variants. Finally, we demonstrate that sequence changes within the L1 5'UTR over evolutionary time, which permitted L1 to evade the repressive effects of a host protein, can lead to the generation of new L1 splicing events, which, upon retrotransposition, generates a new SpIRE subfamily. We conclude that splicing inhibits L1 retrotransposition, SpIREs generally represent evolutionary "dead-ends" in the L1 retrotransposition process, mutations within the L1 5'UTR alter L1 splicing dynamics, and that retrotransposition of the resultant spliced transcripts can generate interindividual genomic variation.

Spliced integrated retrotransposed element (SpIRE) formation in the human genome

PubMed Central

Larson, Peter A.; Moldovan, John B.; Jasti, Naveen; Kidd, Jeffrey M.; Beck, Christine R.; Moran, John V.

2018-01-01

Human Long interspersed element-1 (L1) retrotransposons contain an internal RNA polymerase II promoter within their 5′ untranslated region (UTR) and encode two proteins, (ORF1p and ORF2p) required for their mobilization (i.e., retrotransposition). The evolutionary success of L1 relies on the continuous retrotransposition of full-length L1 mRNAs. Previous studies identified functional splice donor (SD), splice acceptor (SA), and polyadenylation sequences in L1 mRNA and provided evidence that a small number of spliced L1 mRNAs retrotransposed in the human genome. Here, we demonstrate that the retrotransposition of intra-5′UTR or 5′UTR/ORF1 spliced L1 mRNAs leads to the generation of spliced integrated retrotransposed elements (SpIREs). We identified a new intra-5′UTR SpIRE that is ten times more abundant than previously identified SpIREs. Functional analyses demonstrated that both intra-5′UTR and 5′UTR/ORF1 SpIREs lack Cis-acting transcription factor binding sites and exhibit reduced promoter activity. The 5′UTR/ORF1 SpIREs also produce nonfunctional ORF1p variants. Finally, we demonstrate that sequence changes within the L1 5′UTR over evolutionary time, which permitted L1 to evade the repressive effects of a host protein, can lead to the generation of new L1 splicing events, which, upon retrotransposition, generates a new SpIRE subfamily. We conclude that splicing inhibits L1 retrotransposition, SpIREs generally represent evolutionary “dead-ends” in the L1 retrotransposition process, mutations within the L1 5′UTR alter L1 splicing dynamics, and that retrotransposition of the resultant spliced transcripts can generate interindividual genomic variation. PMID:29505568

Therapeutic strategies based on modified U1 snRNAs and chaperones for Sanfilippo C splicing mutations.

PubMed

Matos, Liliana; Canals, Isaac; Dridi, Larbi; Choi, Yoo; Prata, Maria João; Jordan, Peter; Desviat, Lourdes R; Pérez, Belén; Pshezhetsky, Alexey V; Grinberg, Daniel; Alves, Sandra; Vilageliu, Lluïsa

2014-12-10

Mutations affecting RNA splicing represent more than 20% of the mutant alleles in Sanfilippo syndrome type C, a rare lysosomal storage disorder that causes severe neurodegeneration. Many of these mutations are localized in the conserved donor or acceptor splice sites, while few are found in the nearby nucleotides. In this study we tested several therapeutic approaches specifically designed for different splicing mutations depending on how the mutations affect mRNA processing. For three mutations that affect the donor site (c.234 + 1G > A, c.633 + 1G > A and c.1542 + 4dupA), different modified U1 snRNAs recognizing the mutated donor sites, have been developed in an attempt to rescue the normal splicing process. For another mutation that affects an acceptor splice site (c.372-2A > G) and gives rise to a protein lacking four amino acids, a competitive inhibitor of the HGSNAT protein, glucosamine, was tested as a pharmacological chaperone to correct the aberrant folding and to restore the normal trafficking of the protein to the lysosome. Partial correction of c.234 + 1G > A mutation was achieved with a modified U1 snRNA that completely matches the splice donor site suggesting that these molecules may have a therapeutic potential for some splicing mutations. Furthermore, the importance of the splice site sequence context is highlighted as a key factor in the success of this type of therapy. Additionally, glucosamine treatment resulted in an increase in the enzymatic activity, indicating a partial recovery of the correct folding. We have assayed two therapeutic strategies for different splicing mutations with promising results for the future applications.

Hemocompatibility and oxygenation performance of polysulfone membranes grafted with polyethylene glycol and heparin by plasma-induced surface modification.

PubMed

Wang, Weiping; Zheng, Zhi; Huang, Xin; Fan, Wenling; Yu, Wenkui; Zhang, Zhibing; Li, Lei; Mao, Chun

2017-10-01

Polyethylene glycol (PEG) and heparin (Hep) were grafted onto polysulfone (PSF) membrane by plasma-induced surface modification to prepare PSF-PEG-Hep membranes used for artificial lung. The effects of plasma treatment parameters, including power, gas type, gas flow rate, and treatment time, were investigated, and different PEG chains were bonded covalently onto the surface in the postplasma grafting process. Membrane surfaces were characterized by water contact angle, PEG grafting degree, attenuated total reflectance-Fourier transform infrared spectroscopy, ultraviolet-visible spectrophotometry, X-ray photoelectron spectroscopy, critical water permeability pressure, and scanning electron microscopy. Protein adsorption, platelet adhesion, and coagulation tests showed significant improvement in the hemocompatibility of PSF-PEG-Hep membranes compared to pristine PSF membrane. Gas exchange tests through PSF-PEG6000-Hep membrane showed that when the flow rate of porcine blood reached 5.0 L/min, the permeation fluxes of O 2 and CO 2 reached 192.6 and 166.9 mL/min, respectively, which were close to the gas exchange capacity of a commercial membrane oxygenator. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1737-1746, 2017. © 2016 Wiley Periodicals, Inc.

Effect of rotational diffusion in an orientational potential well on the point spread function of electric dipole emitters.

PubMed

Stallinga, Sjoerd

2015-02-01

A study is presented of the point spread function (PSF) of electric dipole emitters that go through a series of absorption-emission cycles while the dipole orientation is changing due to rotational diffusion within the constraint of an orientational potential well. An analytical expression for the PSF is derived valid for arbitrary orientational potential wells in the limit of image acquisition times much larger than the rotational relaxation time. This framework is used to study the effects of the direction of incidence, polarization, and degree of coherence of the illumination. In the limit of fast rotational diffusion on the scale of the fluorescence lifetime the illumination influences only the PSF height, not its shape. In the limit of slow rotational diffusion on the scale of the fluorescence lifetime there is a significant effect on the PSF shape as well, provided the illumination is (partially) coherent. For oblique incidence, illumination asymmetries can arise in the PSF that give rise to position offsets in localization based on Gaussian spot fitting. These asymmetries persist in the limit of free diffusion in a zero orientational potential well.

[Costs with personnel and productivity analysis of family health program teams in Fortaleza, Ceará].

PubMed

Rocha Filho, Fernando dos Santos; da Silva, Marcelo Gurgel Carlos

2009-01-01

The research verified the productivity and the operational personnel costs of eight teams of the Family Health Program (PSF) of two Basic Units of Family Health (UBASF) in Fortaleza, Ceará, through the methodology of costs by absorption, in 2004. The largest expenses were with personnel (75%), mainly with the PSF teams, and medicines (18%). The federal allocations received in September, 2004, by each PSF team, were R$ 9,543.33. The total cost by team was R$ 15,719.00. Some professionals of various PSF teams showed productivity above the set objectives, but the average productivity by doctor and nurse was below fifty per cent of objectives, with high idle time making the work onerous. The unity cost of home visits by doctor and prenatal attendance by nurses were checked, and, would be less expensive if the idle time were reduced. Various alternatives of cost reduction were observed, such as scale savings of some resources and services and re-negotiation of contracts with suppliers and cooperatives. The data obtained will contribute to more accurate planning for the installation and maintenance of PSF teams as well as alternatives of cost reductions, higher productivity, and better quality.

A Point Spread Function for the EPOXI Mission

NASA Technical Reports Server (NTRS)

Barry, Richard K.

2010-01-01

The Extrasolar Planet Observation Characterization and the Deep Impact Extended Investigation missions (EPOXI) are currently observing the transits of exoplanets, two comet nuclei at short range, and the Earth and Mars using the High Resolution Instrument (HRI) - a 0.3 m f/35 telescope on the Deep Impact probe. The HRI is in a permanently defocused state with the instrument pOint of focus about 0.6 cm before the focal plane due to the use of a reference flat mirror that took a power during ground thermal-vacuum testing. Consequently, the point spread function (PSF) covers approximately nine pixels FWHM and is characterized by a patch with three-fold symmetry due to the three-point support structures of the primary and secondary mirrors. The PSF is also strongly color dependent varying in shape and size with change in filtration and target color. While defocus is highly desirable for exoplanet transit observations to limit sensitivity to intra-pixel variation, it is suboptimal for observations of spatially resolved targets. Consequently, all images used in our analysis of such objects were deconvolved with an instrument PSF. The instrument PSF is also being used to optimize transit analysis. We discuss development and usage of an instrument PSF for these observations.

Preparation and Characterization of Chitosan Thin Films on Mixed-Matrix Membranes for Complete Removal of Chromium

PubMed Central

Nayak, Vignesh; Jyothi, Mannekote Shivanna; Balakrishna, R Geetha; Padaki, Mahesh; Ismail, Ahmad Fauzi

2015-01-01

Herein we present a new approach for the complete removal of CrVI species, through reduction of CrVI to CrIII, followed by adsorption of CrIII. Reduction of chromium from water is an important challenge, as CrIV is one of the most toxic substances emitted from industrial processes. Chitosan (CS) thin films were developed on plain polysulfone (PSf) and PSf/TiO2 membrane substrates by a temperature-induced technique using polyvinyl alcohol as a binder. Structure property elucidation was carried out by X-ray diffraction, microscopy, spectroscopy, contact angle measurement, and water uptake studies. The increase in hydrophilicity followed the order: PSf < PSf/TiO2 < PSf/TiO2/CS membranes. Use of this thin-film composite membrane for chromium removal was investigated with regards to the effects of light and pH. The observations reveal 100 % reduction of CrVI to CrIII through electrons and protons donated from OH and NH2 groups of the CS layer; the reduced CrIII species are adsorbed onto the CS layer via complexation to give chromium-free water. PMID:26246989

[Primary health care--the "apple of the eye" of SUS: about social representations of the protagonists of the Unified Health System].

PubMed

Gomes, Karine de Oliveira; Cotta, Rosângela Minardi Mitre; Araújo, Raquel Maria Amaral; Cherchiglia, Mariângela Leal; Martins, Tatiana de Castro Pereira

2011-01-01

The Primary Health Care (PHC) is the first contact level with the health system. In Brazil, the Family Health Program (PSF) is the main implementation and organization strategy of the PHC. The objective of this study is to evaluate the actions and services of health offered by the PSF, starting from the social representations of the interviewee on the exclusive dimensions of PHC - attention to the first contact, longitudinality, integrality and coordination. It is a quali-quantitative research, accomplished in Cajuri, Minas Gerais State. Municipal managers, PSF professionals and pregnant women assisted by PSF were interviewed. Regarding social representations on SUS, it was observed an inadequate level of apprehension and knowledge of their principles and guidelines. As for PSF, several positive connotations were expressed and the set of perceptions of the protagonists identified it as a restructuring strategy of PHC in the municipality. In spite of this, strong influences of the biomedical model and the challenge of the integration with the other levels of attention were noticed, indicating the need of investments in the professional's training and in the organization of the other levels of attention to health.

Adult psychosocial outcome of prepubertal major depressive disorder.

PubMed

Geller, B; Zimerman, B; Williams, M; Bolhofner, K; Craney, J L

2001-06-01

To compare adult psychosocial functioning (PSF) of subjects with prepubertal major depressive disorder (PMDD) to a normal comparison (NC) group. PSF of subjects with PMDD (n = 72) and of NC subjects (n = 28) was compared after prospective follow-up to adulthood. These 100 subjects were 90.9% of the baseline 110 subjects who participated in the "Nortriptyline in Childhood Depression: Follow-up Study." Research nurses who were blind to group status conducted telephone interviews using the Longitudinal Interval Follow-up Evaluation (LIFE) to obtain PSF data. At follow-up, the PMDD group was 20.7+/-2.0 and the NC subjects were 20.9+/-2.2 years old. The PMDD subjects were 10.3+/-1.5 years old at baseline. Time between baseline and follow-up was 9.9+/-1.5 years. In the PMDD group, subjects with MDD, bipolar disorder, or substance use disorders during the previous 5 years had significantly worse PSF than NC subjects. These PSF impairments included significantly worse relationships with parents, siblings, and friends; significantly worse functioning in household, school, and work settings; and worse overall quality of life and global social adjustment. Although combined treatments for PMDD have little scientific basis, multimodality regimens seem prudent until definitive treatment data become available.

Elucidation of the 14-3-3ζ interactome reveals critical roles of RNA-splicing factors during adipogenesis.

PubMed

Mugabo, Yves; Sadeghi, Mina; Fang, Nancy N; Mayor, Thibault; Lim, Gareth E

2018-05-04

Adipogenesis involves a complex signaling network requiring strict temporal and spatial organization of effector molecules. Molecular scaffolds, such as 14-3-3 proteins, facilitate such organization, and we have previously identified 14-3-3ζ as an essential scaffold in adipocyte differentiation. The interactome of 14-3-3ζ is large and diverse, and it is possible that novel adipogenic factors may be present within it, but this possibility has not yet been tested. Herein, we generated mouse embryonic fibroblasts from mice overexpressing a tandem affinity purification (TAP) epitope-tagged 14-3-3ζ molecule. After inducing adipogenesis, TAP-14-3-3ζ complexes were purified, followed by MS analysis to determine the 14-3-3ζ interactome. We observed more than 100 proteins that were unique to adipocyte differentiation, 56 of which were novel interacting partners. Among these, we were able to identify previously established regulators of adipogenesis ( i.e. Ptrf/Cavin1) within the 14-3-3ζ interactome, confirming the utility of this approach to detect adipogenic factors. We found that proteins related to RNA metabolism, processing, and splicing were enriched in the interactome. Analysis of transcriptomic data revealed that 14-3-3ζ depletion in 3T3-L1 cells affected alternative splicing of mRNA during adipocyte differentiation. siRNA-mediated depletion of RNA-splicing factors within the 14-3-3ζ interactome, that is, of Hnrpf, Hnrpk, Ddx6, and Sfpq, revealed that they have essential roles in adipogenesis and in the alternative splicing of Pparg and the adipogenesis-associated gene Lpin1 In summary, we have identified novel adipogenic factors within the 14-3-3ζ interactome. Further characterization of additional proteins within the 14-3-3ζ interactome may help identify novel targets to block obesity-associated expansion of adipose tissues. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

SPF45-related SPLICING FACTOR FOR PHYTOCHROME SIGNALING promotes photomorphogensis by regulating pre-mRNA splicing in arabidopsis

USDA-ARS?s Scientific Manuscript database

Light signals regulate plant growth and development by controlling a plethora of gene expression changes. Post-transcriptional regulation, especially pre-mRNA processing, is a key modulator of gene expression; however, the molecular mechanisms linking pre-mRNA processing and light signaling are not ...

SON and its alternatively spliced isoforms control MLL complex-mediated H3K4me3 and transcription of leukemia-associated genes

PubMed Central

Kim, Jung-Hyun; Baddoo, Melody C.; Park, Eun Young; Stone, Joshua K.; Park, Hyeonsoo; Butler, Thomas W.; Huang, Gang; Yan, Xiaomei; Pauli-Behn, Florencia; Myers, Richard M.; Tan, Ming; Flemington, Erik K.; Lim, Ssang-Taek; Erin Ahn, Eun-Young

2016-01-01

SUMMARY Dysregulation of MLL complex-mediated histone methylation plays a pivotal role in gene expression associated with diseases, but little is known about cellular factors modulating MLL complex activity. Here, we report that SON, previously known as an RNA splicing factor, controls MLL complex-mediated transcriptional initiation. SON binds to DNA near transcription start sites, interacts with menin, and inhibits MLL complex assembly, resulting in decreased H3K4me3 and transcriptional repression. Importantly, alternatively spliced short isoforms of SON are markedly upregulated in acute myeloid leukemia. The short isoforms compete with full-length SON for chromatin occupancy, but lack the menin-binding ability, thereby antagonizing full-length SON function in transcriptional repression while not impairing full-length SON-mediated RNA splicing. Furthermore, overexpression of a short isoform of SON enhances replating potential of hematopoietic progenitors. Our findings define SON as a fine-tuner of the MLL-menin interaction and reveal short SON overexpression as a marker indicating aberrant transcriptional initiation in leukemia. PMID:26990989

Selective expression of a splice variant of decay-accelerating factor in c-erbB-2-positive mammary carcinoma cells showing increased transendothelial invasiveness

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brandt, Burkhard; Mikesch, Jan-Hendrik; Simon, Ronald

2005-04-01

By differential-display-PCR a subclone of the SK-BR-3 cell line with high in vitro transendothelial invasiveness was identified to express increased levels of a new alternative splice variant of decay-accelerating factor (DAF). DAF seems to play an important role in some malignant tumours since on the one hand the expression of complement inhibitors on the surface of tumour cells prevents the accumulation of complement factors and in consequence cell lysis. On the other hand, DAF has been identified as a ligand for the CD97 surface receptor which induces cell migration. Immunofluorescence procedures, Western blot analyses, and cDNA clone sequencing were employedmore » to confirm the expression of DAF restricted to invasive tumour cells. Using a radioactive RNA-in situ hybridisation on freshly frozen tissue microarrays and RT-PCR on native tumour tissue, the expression of alternative spliced DAF mRNA was demonstrated in invasive breast cancer. Due to the fact that it could thereby not be detected in normal mammary tissues, it has to be confirmed in larger studies that the DAF splice variant might be a specific tumour marker for invasive breast cancer.« less

Adaptive thermal control of stem gravitropism through alternative RNA splicing in Arabidopsis

PubMed Central

Ryu, Jae Yong; Kim, Joo-Young; Park, Chung-Mo

2015-01-01

Gravitropism is an important growth movement in response to gravity in virtually all higher plants: the roots showing positive gravitropism and the shoots showing negative gravitropism. The gravitropic orientation of plant organs is also influenced by environmental factors, such as light and temperature. It is known that a zinc finger (ZF)-containing transcription factor SHOOT GRAVITROPISM 5/INDETERMINATE DOMAIN 15 (SGR5/IDD15) mediates the early events of gravitropic responses occurring in inflorescence stems. We have recently found that SGR5 gene undergoes alternative splicing to produce 2 protein variants, the full-size SGR5α transcription factor and the truncated SGR5β form lacking functional ZF motifs. The SGR5β form inhibits SGR5α function possibly by forming nonfunctional heterodimers that are excluded from DNA binding. Notably, SGR5 alternative splicing is accelerated at high temperatures, resulting in a high-level accumulation of SGR5β proteins. Accordingly, transgenic plants overexpressing SGR5β exhibit a reduction in the negative gravitropism of inflorescence stems, as observed in the SGR5-defective mutant. It is proposed that the thermos-responsive alternative splicing of SGR5 gene provides an adaptation strategy by which plants protect the shoots from aerial heat frequently occurring in natural habitats. PMID:26452406

A short conserved motif in ALYREF directs cap- and EJC-dependent assembly of export complexes on spliced mRNAs.

PubMed

Gromadzka, Agnieszka M; Steckelberg, Anna-Lena; Singh, Kusum K; Hofmann, Kay; Gehring, Niels H

2016-03-18

The export of messenger RNAs (mRNAs) is the final of several nuclear posttranscriptional steps of gene expression. The formation of export-competent mRNPs involves the recruitment of export factors that are assumed to facilitate transport of the mature mRNAs. Using in vitro splicing assays, we show that a core set of export factors, including ALYREF, UAP56 and DDX39, readily associate with the spliced RNAs in an EJC (exon junction complex)- and cap-dependent manner. In order to elucidate how ALYREF and other export adaptors mediate mRNA export, we conducted a computational analysis and discovered four short, conserved, linear motifs present in RNA-binding proteins. We show that mutation in one of the new motifs (WxHD) in an unstructured region of ALYREF reduced RNA binding and abolished the interaction with eIF4A3 and CBP80. Additionally, the mutation impaired proper localization to nuclear speckles and export of a spliced reporter mRNA. Our results reveal important details of the orchestrated recruitment of export factors during the formation of export competent mRNPs. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Adaptive thermal control of stem gravitropism through alternative RNA splicing in Arabidopsis.

PubMed

Ryu, Jae Yong; Kim, Joo-Young; Park, Chung-Mo

2015-01-01

Gravitropism is an important growth movement in response to gravity in virtually all higher plants: the roots showing positive gravitropism and the shoots showing negative gravitropism. The gravitropic orientation of plant organs is also influenced by environmental factors, such as light and temperature. It is known that a zinc finger (ZF)-containing transcription factor SHOOT GRAVITROPISM 5/INDETERMINATE DOMAIN 15 (SGR5/IDD15) mediates the early events of gravitropic responses occurring in inflorescence stems. We have recently found that SGR5 gene undergoes alternative splicing to produce 2 protein variants, the full-size SGR5α transcription factor and the truncated SGR5β form lacking functional ZF motifs. The SGR5β form inhibits SGR5α function possibly by forming nonfunctional heterodimers that are excluded from DNA binding. Notably, SGR5 alternative splicing is accelerated at high temperatures, resulting in a high-level accumulation of SGR5β proteins. Accordingly, transgenic plants overexpressing SGR5β exhibit a reduction in the negative gravitropism of inflorescence stems, as observed in the SGR5-defective mutant. It is proposed that the thermos-responsive alternative splicing of SGR5 gene provides an adaptation strategy by which plants protect the shoots from aerial heat frequently occurring in natural habitats.

Identification of human short introns

PubMed Central

Abebrese, Emmanuel L.; Arnold, Zachary R.; Armstrong, Katharine; Burns, Lindsay; Day, R. Thomas; Hsu, Daniel G.; Jarrell, Katherine; Luo, Yi; Mugayo, Daphine

2017-01-01

Canonical pre-mRNA splicing requires snRNPs and associated splicing factors to excise conserved intronic sequences, with a minimum intron length required for efficient splicing. Non-canonical splicing–intron excision without the spliceosome–has been documented; most notably, some tRNAs and the XBP1 mRNA contain short introns that are not removed by the spliceosome. There have been some efforts to identify additional short introns, but little is known about how many short introns are processed from mRNAs. Here, we report an approach to identify RNA short introns from RNA-Seq data, discriminating against small genomic deletions. We identify hundreds of short introns conserved among multiple human cell lines. These short introns are often alternatively spliced and are found in a variety of RNAs–both mRNAs and lncRNAs. Short intron splicing efficiency is increased by secondary structure, and we detect both canonical and non-canonical short introns. In many cases, splicing of these short introns from mRNAs is predicted to alter the reading frame and change protein output. Our findings imply that standard gene prediction models which often assume a lower limit for intron size fail to predict short introns effectively. We conclude that short introns are abundant in the human transcriptome, and short intron splicing represents an added layer to mRNA regulation. PMID:28520720

New discoveries of old SON: a link between RNA splicing and cancer.

PubMed

Hickey, Christopher J; Kim, Jung-Hyun; Ahn, Eun-Young Erin

2014-02-01

The SON protein is a ubiquitously expressed DNA- and RNA-binding protein primarily localized to nuclear speckles. Although several early studies implicated SON in DNA-binding, tumorigenesis and apoptosis, functional significance of this protein had not been recognized until recent studies discovered SON as a novel RNA splicing co-factor. During constitutive RNA splicing, SON ensures efficient intron removal from the transcripts containing suboptimal splice sites. Importantly, SON-mediated splicing is required for proper processing of selective transcripts related to cell cycle, microtubules, centrosome maintenance, and genome stability. Moreover, SON regulates alternative splicing of RNAs from the genes involved in apoptosis and epigenetic modification. In addition to the role in RNA splicing, SON has an ability to suppress transcriptional activation at certain promoter/enhancer DNA sequences. Considering the multiple SON target genes which are directly involved in cell proliferation, genome stability and chromatin modifications, SON is an emerging player in gene regulation during cancer development and progression. Here, we summarize available information from several early studies on SON, and highlight recent discoveries describing molecular mechanisms of SON-mediated gene regulation. We propose that our future effort on better understanding of diverse SON functions would reveal novel targets for cancer therapy. © 2013 Wiley Periodicals, Inc.

VizieR Online Data Catalog: Deep Herschel PACS point spread functions (Bocchio+, 2016)

NASA Astrophysics Data System (ADS)

Bocchio, M.; Bianchi, A.; Abergel, S.

2016-06-01

Herschel PACS dedicated PSF observations are scanmaps centred on various objects taken at 70 (blue channel), 100 (green channel) and 160 (red channel) um. The core of the PSF is best characterised observing faint objects (e.g. the asteroid Vesta), while the wings of the PSF can only be seen in observations of bright objects (e.g. Mars). Using a combination of images of bright and faint objects it is therefore possible to have a good characterisation of the PACS PSFs. (2 data files).

Binding of hnRNP H and U2AF65 to Respective G-codes and a Poly-Uridine Tract Collaborate in the N50-5'ss Selection of the REST N Exon in H69 Cells

PubMed Central

Ortuño-Pineda, Carlos; Galindo-Rosales, José Manuel; Calderón-Salinas, José Victor; Villegas-Sepúlveda, Nicolás; Saucedo-Cárdenas, Odila; De Nova-Ocampo, Mónica; Valdés, Jesús

2012-01-01

The splicing of the N exon in the pre-mRNA coding for the RE1-silencing transcription factor (REST) results in a truncated protein that modifies the expression pattern of some of its target genes. A weak 3'ss, three alternative 5'ss (N4-, N50-, and N62-5'ss) and a variety of putative target sites for splicing regulatory proteins are found around the N exon; two GGGG codes (G2-G3) and a poly-Uridine tract (N-PU) are found in front of the N50-5'ss. In this work we analyzed some of the regulatory factors and elements involved in the preferred selection of the N50-5'ss (N50 activation) in the small cell lung cancer cell line H69. Wild type and mutant N exon/β-globin minigenes recapitulated N50 exon splicing in H69 cells, and showed that the N-PU and the G2-G3 elements are required for N50 exon splicing. Biochemical and knockdown experiments identified these elements as U2AF65 and hnRNP H targets, respectively, and that they are also required for N50 exon activation. Compared to normal MRC5 cells, and in keeping with N50 exon activation, U2AF65, hnRNP H and other splicing factors were highly expressed in H69 cells. CLIP experiments revealed that hnRNP H RNA-binding occurs first and is a prerequisite for U2AF65 RNA binding, and EMSA and CLIP experiments suggest that U2AF65-RNA recognition displaces hnRNP H and helps to recruit other splicing factors (at least U1 70K) to the N50-5'ss. Our results evidenced novel hnRNP H and U2AF65 functions: respectively, U2AF65-recruiting to a 5'ss in humans and the hnRNP H-displacing function from two juxtaposed GGGG codes. PMID:22792276

An alternatively spliced heat shock transcription factor, OsHSFA2dI, functions in the heat stress-induced unfolded protein response in rice.

PubMed

Cheng, Q; Zhou, Y; Liu, Z; Zhang, L; Song, G; Guo, Z; Wang, W; Qu, X; Zhu, Y; Yang, D

2015-03-01

As sessile organisms, plants have evolved a wide range of defence pathways to cope with environmental stress such as heat shock. However, the molecular mechanism of these defence pathways remains unclear in rice. In this study, we found that OsHSFA2d, a heat shock transcriptional factor, encodes two main splice variant proteins, OsHSFA2dI and OsHSFA2dII in rice. Under normal conditions, OsHSFA2dII is the dominant but transcriptionally inactive spliced form. However, when the plant suffers heat stress, OsHSFA2d is alternatively spliced into a transcriptionally active form, OsHSFA2dI, which participates in the heat stress response (HSR). Further study found that this alternative splicing was induced by heat shock rather than photoperiod. We found that OsHSFA2dI is localised to the nucleus, whereas OsHSFA2dII is localised to the nucleus and cytoplasm. Moreover, expression of the unfolded protein response (UNFOLDED PROTEIN RESPONSE) sensors, OsIRE1, OsbZIP39/OsbZIP60 and the UNFOLDED PROTEIN RESPONSE marker OsBiP1, was up-regulated. Interestingly, OsbZIP50 was also alternatively spliced under heat stress, indicating that UNFOLDED PROTEIN RESPONSE signalling pathways were activated by heat stress to re-establish cellular protein homeostasis. We further demonstrated that OsHSFA2dI participated in the unfolded protein response by regulating expression of OsBiP1. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

Global profiling of alternative RNA splicing events provides insights into molecular differences between various types of hepatocellular carcinoma.

PubMed

Tremblay, Marie-Pier; Armero, Victoria E S; Allaire, Andréa; Boudreault, Simon; Martenon-Brodeur, Camille; Durand, Mathieu; Lapointe, Elvy; Thibault, Philippe; Tremblay-Létourneau, Maude; Perreault, Jean-Pierre; Scott, Michelle S; Bisaillon, Martin

2016-08-26

Dysregulations in alternative splicing (AS) patterns have been associated with many human diseases including cancer. In the present study, alterations to the global RNA splicing landscape of cellular genes were investigated in a large-scale screen from 377 liver tissue samples using high-throughput RNA sequencing data. Our study identifies modifications in the AS patterns of transcripts encoded by more than 2500 genes such as tumor suppressor genes, transcription factors, and kinases. These findings provide insights into the molecular differences between various types of hepatocellular carcinoma (HCC). Our analysis allowed the identification of 761 unique transcripts for which AS is misregulated in HBV-associated HCC, while 68 are unique to HCV-associated HCC, 54 to HBV&HCV-associated HCC, and 299 to virus-free HCC. Moreover, we demonstrate that the expression pattern of the RNA splicing factor hnRNPC in HCC tissues significantly correlates with patient survival. We also show that the expression of the HBx protein from HBV leads to modifications in the AS profiles of cellular genes. Finally, using RNA interference and a reverse transcription-PCR screening platform, we examined the implications of cellular proteins involved in the splicing of transcripts involved in apoptosis and demonstrate the potential contribution of these proteins in AS control. This study provides the first comprehensive portrait of global changes in the RNA splicing signatures that occur in hepatocellular carcinoma. Moreover, these data allowed us to identify unique signatures of genes for which AS is misregulated in the different types of HCC.

Control of alternative splicing by forskolin through hnRNP K during neuronal differentiation

PubMed Central

Cao, Wenguang; Razanau, Aleh; Feng, Dairong; Lobo, Vincent G.; Xie, Jiuyong

2012-01-01

The molecular basis of cell signal-regulated alternative splicing at the 3′ splice site remains largely unknown. We isolated a protein kinase A-responsive ribonucleic acid (RNA) element from a 3′ splice site of the synaptosomal-associated protein 25 (Snap25) gene for forskolin-inhibited splicing during neuronal differentiation of rat pheochromocytoma PC12 cells. The element binds specifically to heterogeneous nuclear ribonucleo protein (hnRNP) K in a phosphatase-sensitive way, which directly competes with the U2 auxiliary factor U2AF65, an essential component of early spliceosomes. Transcripts with similarly localized hnRNP K target motifs upstream of alternative exons are enriched in genes often associated with neurological diseases. We show that such motifs upstream of the Runx1 exon 6 also bind hnRNP K, and importantly, hnRNP K is required for forskolin-induced repression of the exon. Interestingly, this exon encodes the peptide domain that determines the switch of the transcriptional repressor/activator activity of Runx1, a change known to be critical in specifying neuron lineages. Consistent with an important role of the target genes in neurons, knocking down hnRNP K severely disrupts forskolin-induced neurite growth. Thus, through hnRNP K, the neuronal differentiation stimulus forskolin targets a critical 3′ splice site component of the splicing machinery to control alternative splicing of crucial genes. This also provides a regulated direct competitor of U2AF65 for cell signal control of 3′ splice site usage. PMID:22684629

Genome-Wide Survey of Cold Stress Regulated Alternative Splicing in Arabidopsis thaliana with Tiling Microarray

PubMed Central

Leviatan, Noam; Alkan, Noam; Leshkowitz, Dena; Fluhr, Robert

2013-01-01

Alternative splicing plays a major role in expanding the potential informational content of eukaryotic genomes. It is an important post-transcriptional regulatory mechanism that can increase protein diversity and affect mRNA stability. Alternative splicing is often regulated in a tissue-specific and stress-responsive manner. Cold stress, which adversely affects plant growth and development, regulates the transcription and splicing of plant splicing factors. This can affect the pre-mRNA processing of many genes. To identify cold regulated alternative splicing we applied Affymetrix Arabidopsis tiling arrays to survey the transcriptome under cold treatment conditions. A novel algorithm was used for detection of statistically relevant changes in intron expression within a transcript between control and cold growth conditions. A reverse transcription polymerase chain reaction (RT-PCR) analysis of a number of randomly selected genes confirmed the changes in splicing patterns under cold stress predicted by tiling array. Our analysis revealed new types of cold responsive genes. While their expression level remains relatively unchanged under cold stress their splicing pattern shows detectable changes in the relative abundance of isoforms. The majority of cold regulated alternative splicing introduced a premature termination codon (PTC) into the transcripts creating potential targets for degradation by the nonsense mediated mRNA decay (NMD) process. A number of these genes were analyzed in NMD-defective mutants by RT-PCR and shown to evade NMD. This may result in new and truncated proteins with altered functions or dominant negative effects. The results indicate that cold affects both quantitative and qualitative aspects of gene expression. PMID:23776682

Spliceman2: a computational web server that predicts defects in pre-mRNA splicing.

PubMed

Cygan, Kamil Jan; Sanford, Clayton Hendrick; Fairbrother, William Guy

2017-09-15

Most pre-mRNA transcripts in eukaryotic cells must undergo splicing to remove introns and join exons, and splicing elements present a large mutational target for disease-causing mutations. Splicing elements are strongly position dependent with respect to the transcript annotations. In 2012, we presented Spliceman, an online tool that used positional dependence to predict how likely distant mutations around annotated splice sites were to disrupt splicing. Here, we present an improved version of the previous tool that will be more useful for predicting the likelihood of splicing mutations. We have added industry-standard input options (i.e. Spliceman now accepts variant call format files), which allow much larger inputs than previously available. The tool also can visualize the locations-within exons and introns-of sequence variants to be analyzed and the predicted effects on splicing of the pre-mRNA transcript. In addition, Spliceman2 integrates with RNAcompete motif libraries to provide a prediction of which trans -acting factors binding sites are disrupted/created and links out to the UCSC genome browser. In summary, the new features in Spliceman2 will allow scientists and physicians to better understand the effects of single nucleotide variations on splicing. Freely available on the web at http://fairbrother.biomed.brown.edu/spliceman2 . Website implemented in PHP framework-Laravel 5, PostgreSQL, Apache, and Perl, with all major browsers supported. william_fairbrother@brown.edu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

SU-G-IeP3-08: Image Reconstruction for Scanning Imaging System Based On Shape-Modulated Point Spreading Function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Ruixing; Yang, LV; Xu, Kele

Purpose: Deconvolution is a widely used tool in the field of image reconstruction algorithm when the linear imaging system has been blurred by the imperfect system transfer function. However, due to the nature of Gaussian-liked distribution for point spread function (PSF), the components with coherent high frequency in the image are hard to restored in most of the previous scanning imaging system, even the relatively accurate PSF is acquired. We propose a novel method for deconvolution of images which are obtained by using shape-modulated PSF. Methods: We use two different types of PSF - Gaussian shape and donut shape -more » to convolute the original image in order to simulate the process of scanning imaging. By employing deconvolution of the two images with corresponding given priors, the image quality of the deblurred images are compared. Then we find the critical size of the donut shape compared with the Gaussian shape which has similar deconvolution results. Through calculation of tightened focusing process using radially polarized beam, such size of donut is achievable under same conditions. Results: The effects of different relative size of donut and Gaussian shapes are investigated. When the full width at half maximum (FWHM) ratio of donut and Gaussian shape is set about 1.83, similar resolution results are obtained through our deconvolution method. Decreasing the size of donut will favor the deconvolution method. A mask with both amplitude and phase modulation is used to create a donut-shaped PSF compared with the non-modulated Gaussian PSF. Donut with size smaller than our critical value is obtained. Conclusion: The utility of donutshaped PSF are proved useful and achievable in the imaging and deconvolution processing, which is expected to have potential practical applications in high resolution imaging for biological samples.« less

A single-source photon source model of a linear accelerator for Monte Carlo dose calculation

PubMed Central

Glatting, Gerhard; Wenz, Frederik; Fleckenstein, Jens

2017-01-01

Purpose To introduce a new method of deriving a virtual source model (VSM) of a linear accelerator photon beam from a phase space file (PSF) for Monte Carlo (MC) dose calculation. Materials and methods A PSF of a 6 MV photon beam was generated by simulating the interactions of primary electrons with the relevant geometries of a Synergy linear accelerator (Elekta AB, Stockholm, Sweden) and recording the particles that reach a plane 16 cm downstream the electron source. Probability distribution functions (PDFs) for particle positions and energies were derived from the analysis of the PSF. These PDFs were implemented in the VSM using inverse transform sampling. To model particle directions, the phase space plane was divided into a regular square grid. Each element of the grid corresponds to an area of 1 mm2 in the phase space plane. The average direction cosines, Pearson correlation coefficient (PCC) between photon energies and their direction cosines, as well as the PCC between the direction cosines were calculated for each grid element. Weighted polynomial surfaces were then fitted to these 2D data. The weights are used to correct for heteroscedasticity across the phase space bins. The directions of the particles created by the VSM were calculated from these fitted functions. The VSM was validated against the PSF by comparing the doses calculated by the two methods for different square field sizes. The comparisons were performed with profile and gamma analyses. Results The doses calculated with the PSF and VSM agree to within 3% /1 mm (>95% pixel pass rate) for the evaluated fields. Conclusion A new method of deriving a virtual photon source model of a linear accelerator from a PSF file for MC dose calculation was developed. Validation results show that the doses calculated with the VSM and the PSF agree to within 3% /1 mm. PMID:28886048

A single-source photon source model of a linear accelerator for Monte Carlo dose calculation.

PubMed

Nwankwo, Obioma; Glatting, Gerhard; Wenz, Frederik; Fleckenstein, Jens

2017-01-01

To introduce a new method of deriving a virtual source model (VSM) of a linear accelerator photon beam from a phase space file (PSF) for Monte Carlo (MC) dose calculation. A PSF of a 6 MV photon beam was generated by simulating the interactions of primary electrons with the relevant geometries of a Synergy linear accelerator (Elekta AB, Stockholm, Sweden) and recording the particles that reach a plane 16 cm downstream the electron source. Probability distribution functions (PDFs) for particle positions and energies were derived from the analysis of the PSF. These PDFs were implemented in the VSM using inverse transform sampling. To model particle directions, the phase space plane was divided into a regular square grid. Each element of the grid corresponds to an area of 1 mm2 in the phase space plane. The average direction cosines, Pearson correlation coefficient (PCC) between photon energies and their direction cosines, as well as the PCC between the direction cosines were calculated for each grid element. Weighted polynomial surfaces were then fitted to these 2D data. The weights are used to correct for heteroscedasticity across the phase space bins. The directions of the particles created by the VSM were calculated from these fitted functions. The VSM was validated against the PSF by comparing the doses calculated by the two methods for different square field sizes. The comparisons were performed with profile and gamma analyses. The doses calculated with the PSF and VSM agree to within 3% /1 mm (>95% pixel pass rate) for the evaluated fields. A new method of deriving a virtual photon source model of a linear accelerator from a PSF file for MC dose calculation was developed. Validation results show that the doses calculated with the VSM and the PSF agree to within 3% /1 mm.

Understanding Polymorphism Formation in Electrospun Fibers of Immiscible Poly(vinylidene fluoride) Blends

DOE Office of Scientific and Technical Information (OSTI.GOV)

G Zhong; L Zhang; R Su

2011-12-31

Effects of electric poling, mechanical stretching, and dipolar interaction on the formation of ferroelectric ({beta} and/or {gamma}) phases in poly(vinylidene fluoride) (PVDF) have been studied in electrospun fibers of PVDF/polyacrylonitrile (PAN) and PVDF/polysulfone (PSF) blends with PVDF as the minor component, using wide-angle X-ray diffraction and Fourier transform infrared techniques. Experimental results of as-electrospun neat PVDF fibers (beaded vs. bead-free) showed that mechanical stretching during electrospinning, rather than electric poling, was effective to induce ferroelectric phases. For as-electrospun PVDF blend fibers with the non-polar PSF matrix, mechanical stretching during electrospinning again was capable of inducing some ferroelectric phases in additionmore » to the major paraelectric ({alpha}) phase. However, after removing the mechanical stretching in a confined melt-recrystallization process, only the paraelectric phase was obtained. For as-electrospun PVDF blend fibers with the polar (or ferroelectric) PAN matrix, strong intermolecular interactions between polar PAN and PVDF played an important role in the ferroelectric phase formation in addition to the mechanical stretching effect during electrospinning. Even after the removal of mechanical stretching through the confined melt-recrystallization process, a significant amount of ferroelectric phases persisted. Comparing the ferroelectric phase formation between PVDF/PSF and PVDF/PAN blend fibers, we concluded that the local electric field-dipole interactions were the determining factor for the nucleation and growth of polar PVDF phases.« less

Preparation and characterization of polysulfone/zeolite mixed matrix membranes for removal of low-concentration ammonia from aquaculture wastewater.

PubMed

Moradihamedani, Pourya; Abdullah, Abdul Halim

2018-01-01

Removal of low-concentration ammonia (1-10 ppm) from aquaculture wastewater was investigated via polysulfone (PSf)/zeolite mixed matrix membrane. PSf/zeolite mixed matrix membranes with different weight ratios (90/10, 80/20, 70/30 and 60/40 wt.%) were prepared and characterized. Results indicate that PSf/zeolite (80/20) was the most efficient membrane for removal of low-concentration ammonia. The ammonia elimination by PSf/zeolite (80/20) from aqueous solution for 10, 7, 5, 3 and 1 ppm of ammonia was 100%, 99%, 98.8%, 96% and 95% respectively. The recorded results revealed that pure water flux declined in higher loading of zeolite in the membrane matrix due to surface pore blockage caused by zeolite particles. On the other hand, ammonia elimination from water was decreased in higher contents of zeolite because of formation of cavities and macrovoids in the membrane substructure.

Perturbed cooperative-state feedback strategy for model predictive networked control of interconnected systems.

PubMed

Tran, Tri; Ha, Q P

2018-01-01

A perturbed cooperative-state feedback (PSF) strategy is presented for the control of interconnected systems in this paper. The subsystems of an interconnected system can exchange data via the communication network that has multiple connection topologies. The PSF strategy can resolve both issues, the sensor data losses and the communication network breaks, thanks to the two components of the control including a cooperative-state feedback and a perturbation variable, e.g., u i =K ij x j +w i . The PSF is implemented in a decentralized model predictive control scheme with a stability constraint and a non-monotonic storage function (ΔV(x(k))≥0), derived from the dissipative systems theory. Numerical simulation for the automatic generation control problem in power systems is studied to illustrate the effectiveness of the presented PSF strategy. Copyright © 2017 ISA. Published by Elsevier Ltd. All rights reserved.

Evidence that "brain-specific" FOX-1, FOX-2, and nPTB alternatively spliced isoforms are produced in the lens.

PubMed

Bitel, Claudine L; Nathan, Rachel; Wong, Patrick; Kuppasani, Sunil; Matsushita, Masafumi; Kanazawa, Hrioshi; Frederikse, Peter H

2011-04-01

Alternative RNA splicing is essential in development and more rapid physiological processes that include disease mechanisms. Studies over the last 20 years demonstrated that RNA binding protein families, which mediate the alternative splicing of a large percentage of genes in mammals, contain isoforms with mutually exclusive expression in non-neural and neural progenitor cells vs. post-mitotic neurons, and regulate the comprehensive reprogramming of alternative splicing during neurogenesis. Polypyrimidine tract binding (PTB) proteins and Fox-1 proteins also undergo mutually exclusive alternative splicing in neural and non-neural cells that regulates their tissue-specific expression and splicing activities. Over the past 50 years, striking morphological similarities noted between lens fiber cells and neurons suggested that cell biology processes and gene expression profiles may be shared as well. Here, we examined mouse and rat lenses to determine if alternative splicing of neuronal nPTB and Fox-1/Fox-2 isoforms also occurs in lenses. Immunoblot, immunofluorescence, and RT-PCR were used to examine expression and alternative splicing of transcripts in lens and brain. We demonstrated that exon 10 is predominantly included in nPTB transcripts consistent with nPTB protein in lenses, and that alternatively spliced Fox-1/-2 lens transcripts contain exons that have been considered neuron-specific. We identified a 3' alternative Fox-1 exon in lenses that encodes a nuclear localization signal consistent with its protein distribution detected in fiber cells. Neuronal alternative splicing of kinesin KIF1Bβ2 has been associated with PTB/nPTB and Fox-2, and we found that two 'neuron-specific' exons are also included in lenses. The present study provides evidence that alternative neuronal nPTB and Fox-1/Fox-2 isoforms are also produced in lenses. These findings raise questions regarding the extent these factors contribute to a similar reprogramming of alternative splicing during lens differentiation, and the degree that alternative gene transcripts produced during neurogenesis are also expressed in the lens.

Alternative RNA splicing and gastric cancer.

PubMed

Li, Ying; Yuan, Yuan

2017-07-01

Alternative splicing (AS) linked to diseases, especially to tumors. Recently, more and more studies focused on the relationship between AS and gastric cancer (GC). This review surveyed the hot topic from four aspects: First, the common types of AS in cancer, including exon skipping, intron retention, mutually exclusive exon, alternative 5 ' or 3' splice site, alternative first or last exon and alternative 3' untranslated regions. Second, basic mechanisms of AS and its relationship with cancer. RNA splicing in eukaryotes follows the GT-AG rule by both cis-elements and trans-acting factors regulatory. Through RNA splicing, different proteins with different forms and functions can be produced and may be associated with carcinogenesis. Third, AS types of GC-related genes and their splicing variants. In this paper, we listed 10 common genes with AS and illustrated its possible molecular mechanisms owing to genetic variation (mutation and /or polymorphism). Fourth, the splicing variants of GC-associated genes and gastric carcinogenesis, invasion and metastasis. Many studies have found that the different splicing variants of the same gene are differentially expressed in GC and its precancerous diseases, suggesting AS has important implications in GC development. Taking together, this review highlighted the role of AS and splicing variants in the process of GC. We hope that this is not only beneficial to advances in the study field of GC, but also can provide valuable information to other similar tumor research.Although we already know some gene splicing and splicing variants play an important role in the development of GC, but many phenomena and mechanisms are still unknown. For example, how the tumor microenvironment and signal transduction pathway effect the forming and function of AS? Unfortunately, this review did not cover the contents because the current study is limited. It is no doubt that clarifying the phenomena and mechanisms of these unknown may help to reveal the relationship of AS with complex tumor genetic variation and the occurrence and development of tumors. Copyright © 2016 Elsevier B.V. All rights reserved.

Abnormal splicing switch of DMD's penultimate exon compromises muscle fibre maintenance in myotonic dystrophy.

PubMed

Rau, Frédérique; Lainé, Jeanne; Ramanoudjame, Laetitita; Ferry, Arnaud; Arandel, Ludovic; Delalande, Olivier; Jollet, Arnaud; Dingli, Florent; Lee, Kuang-Yung; Peccate, Cécile; Lorain, Stéphanie; Kabashi, Edor; Athanasopoulos, Takis; Koo, Taeyoung; Loew, Damarys; Swanson, Maurice S; Le Rumeur, Elisabeth; Dickson, George; Allamand, Valérie; Marie, Joëlle; Furling, Denis

2015-05-28

Myotonic Dystrophy type 1 (DM1) is a dominant neuromuscular disease caused by nuclear-retained RNAs containing expanded CUG repeats. These toxic RNAs alter the activities of RNA splicing factors resulting in alternative splicing misregulation and muscular dysfunction. Here we show that the abnormal splicing of DMD exon 78 found in dystrophic muscles of DM1 patients is due to the functional loss of MBNL1 and leads to the re-expression of an embryonic dystrophin in place of the adult isoform. Forced expression of embryonic dystrophin in zebrafish using an exon-skipping approach severely impairs the mobility and muscle architecture. Moreover, reproducing Dmd exon 78 missplicing switch in mice induces muscle fibre remodelling and ultrastructural abnormalities including ringed fibres, sarcoplasmic masses or Z-band disorganization, which are characteristic features of dystrophic DM1 skeletal muscles. Thus, we propose that splicing misregulation of DMD exon 78 compromises muscle fibre maintenance and contributes to the progressive dystrophic process in DM1.

Abnormal splicing switch of DMD's penultimate exon compromises muscle fibre maintenance in myotonic dystrophy

PubMed Central

Rau, Frédérique; Lainé, Jeanne; Ramanoudjame, Laetitita; Ferry, Arnaud; Arandel, Ludovic; Delalande, Olivier; Jollet, Arnaud; Dingli, Florent; Lee, Kuang-Yung; Peccate, Cécile; Lorain, Stéphanie; Kabashi, Edor; Athanasopoulos, Takis; Koo, Taeyoung; Loew, Damarys; Swanson, Maurice S.; Le Rumeur, Elisabeth; Dickson, George; Allamand, Valérie; Marie, Joëlle; Furling, Denis

2015-01-01

Myotonic Dystrophy type 1 (DM1) is a dominant neuromuscular disease caused by nuclear-retained RNAs containing expanded CUG repeats. These toxic RNAs alter the activities of RNA splicing factors resulting in alternative splicing misregulation and muscular dysfunction. Here we show that the abnormal splicing of DMD exon 78 found in dystrophic muscles of DM1 patients is due to the functional loss of MBNL1 and leads to the re-expression of an embryonic dystrophin in place of the adult isoform. Forced expression of embryonic dystrophin in zebrafish using an exon-skipping approach severely impairs the mobility and muscle architecture. Moreover, reproducing Dmd exon 78 missplicing switch in mice induces muscle fibre remodelling and ultrastructural abnormalities including ringed fibres, sarcoplasmic masses or Z-band disorganization, which are characteristic features of dystrophic DM1 skeletal muscles. Thus, we propose that splicing misregulation of DMD exon 78 compromises muscle fibre maintenance and contributes to the progressive dystrophic process in DM1. PMID:26018658

When SUMO met splicing.

PubMed

Pozzi, Berta; Mammi, Pablo; Bragado, Laureano; Giono, Luciana E; Srebrow, Anabella

2018-05-09

Spliceosomal proteins have been revealed as SUMO conjugation targets. Moreover, we have reported that many of these are in a SUMO-conjugated form when bound to a pre-mRNA substrate during a splicing reaction. We demonstrated that SUMOylation of Prp3 (PRPF3), a component of the U4/U6 di-snRNP, is required for U4/U6•U5 tri-snRNP formation and/or recruitment to active spliceosomes. Expanding upon our previous results, we have shown that the splicing factor SRSF1 stimulates SUMO conjugation to several spliceosomal proteins. Given the relevance of the splicing process, as well as the complex and dynamic nature of its governing machinery, the spliceosome, the molecular mechanisms that modulate its function represent an attractive topic of research. We posit that SUMO conjugation could represent a way of modulating spliceosome assembly and thus, splicing efficiency. How cycles of SUMOylation/de-SUMOylation of spliceosomal proteins become integrated throughout the highly choreographed spliceosomal cycle awaits further investigation.

Neural network simulation of the atmospheric point spread function for the adjacency effect research

NASA Astrophysics Data System (ADS)

Ma, Xiaoshan; Wang, Haidong; Li, Ligang; Yang, Zhen; Meng, Xin

2016-10-01

Adjacency effect could be regarded as the convolution of the atmospheric point spread function (PSF) and the surface leaving radiance. Monte Carlo is a common method to simulate the atmospheric PSF. But it can't obtain analytic expression and the meaningful results can be only acquired by statistical analysis of millions of data. A backward Monte Carlo algorithm was employed to simulate photon emitting and propagating in the atmosphere under different conditions. The PSF was determined by recording the photon-receiving numbers in fixed bin at different position. A multilayer feed-forward neural network with a single hidden layer was designed to learn the relationship between the PSF's and the input condition parameters. The neural network used the back-propagation learning rule for training. Its input parameters involved atmosphere condition, spectrum range, observing geometry. The outputs of the network were photon-receiving numbers in the corresponding bin. Because the output units were too many to be allowed by neural network, the large network was divided into a collection of smaller ones. These small networks could be ran simultaneously on many workstations and/or PCs to speed up the training. It is important to note that the simulated PSF's by Monte Carlo technique in non-nadir viewing angles are more complicated than that in nadir conditions which brings difficulties in the design of the neural network. The results obtained show that the neural network approach could be very useful to compute the atmospheric PSF based on the simulated data generated by Monte Carlo method.

Non-stationary blind deconvolution of medical ultrasound scans

NASA Astrophysics Data System (ADS)

Michailovich, Oleg V.

2017-03-01

In linear approximation, the formation of a radio-frequency (RF) ultrasound image can be described based on a standard convolution model in which the image is obtained as a result of convolution of the point spread function (PSF) of the ultrasound scanner in use with a tissue reflectivity function (TRF). Due to the band-limited nature of the PSF, the RF images can only be acquired at a finite spatial resolution, which is often insufficient for proper representation of the diagnostic information contained in the TRF. One particular way to alleviate this problem is by means of image deconvolution, which is usually performed in a "blind" mode, when both PSF and TRF are estimated at the same time. Despite its proven effectiveness, blind deconvolution (BD) still suffers from a number of drawbacks, chief among which stems from its dependence on a stationary convolution model, which is incapable of accounting for the spatial variability of the PSF. As a result, virtually all existing BD algorithms are applied to localized segments of RF images. In this work, we introduce a novel method for non-stationary BD, which is capable of recovering the TRF concurrently with the spatially variable PSF. Particularly, our approach is based on semigroup theory which allows one to describe the effect of such a PSF in terms of the action of a properly defined linear semigroup. The approach leads to a tractable optimization problem, which can be solved using standard numerical methods. The effectiveness of the proposed solution is supported by experiments with in vivo ultrasound data.

Conservation of CD44 exon v3 functional elements in mammals

PubMed Central

Vela, Elena; Hilari, Josep M; Delclaux, María; Fernández-Bellon, Hugo; Isamat, Marcos

2008-01-01

Background The human CD44 gene contains 10 variable exons (v1 to v10) that can be alternatively spliced to generate hundreds of different CD44 protein isoforms. Human CD44 variable exon v3 inclusion in the final mRNA depends on a multisite bipartite splicing enhancer located within the exon itself, which we have recently described, and provides the protein domain responsible for growth factor binding to CD44. Findings We have analyzed the sequence of CD44v3 in 95 mammalian species to report high conservation levels for both its splicing regulatory elements (the 3' splice site and the exonic splicing enhancer), and the functional glycosaminglycan binding site coded by v3. We also report the functional expression of CD44v3 isoforms in peripheral blood cells of different mammalian taxa with both consensus and variant v3 sequences. Conclusion CD44v3 mammalian sequences maintain all functional splicing regulatory elements as well as the GAG binding site with the same relative positions and sequence identity previously described during alternative splicing of human CD44. The sequence within the GAG attachment site, which in turn contains the Y motif of the exonic splicing enhancer, is more conserved relative to the rest of exon. Amplification of CD44v3 sequence from mammalian species but not from birds, fish or reptiles, may lead to classify CD44v3 as an exclusive mammalian gene trait. PMID:18710510

X-linked CHARGE-like Abruzzo-Erickson syndrome and classic cleft palate with ankyloglossia result from TBX22 splicing mutations.

PubMed

Pauws, E; Peskett, E; Boissin, C; Hoshino, A; Mengrelis, K; Carta, E; Abruzzo, M A; Lees, M; Moore, G E; Erickson, R P; Stanier, P

2013-04-01

X-linked cleft palate (CPX) is caused by mutations in the gene encoding the TBX22 transcription factor and is known to exhibit phenotypic variability, usually involving either a complete, partial or submucous cleft palate, with or without ankyloglossia. This study hypothesized a possible involvement of TBX22 in a family with X-linked, CHARGE-like Abruzzo-Erickson syndrome, of unknown etiology. The phenotype extends to additional features including sensorineural deafness and coloboma, which are suggested by the Tbx22 developmental expression pattern but not previously associated in CPX patients. A novel TBX22 splice acceptor mutation (c.593-5T>A) was identified that tracked with the phenotype in this family. A novel splice donor variant (c.767+5G>A) and a known canonical splice donor mutation (c.767+1G>A) affecting the same exon were identified in patients with classic CPX phenotypes and were comparatively analyzed using both in silico and in vitro splicing studies. All three variants were predicted to abolish normal mRNA splicing and an in vitro assay indicated that use of alternative splice sites was a likely outcome. Collectively, the data showed the functional effect of several novel intronic splice site variants but most importantly confirms that TBX22 is the gene underlying Abruzzo-Erickson syndrome, expanding the phenotypic spectrum of TBX22 mutations. © 2012 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.

Misregulation of Alternative Splicing in a Mouse Model of Rett Syndrome

PubMed Central

Li, Ronghui; Dong, Qiping; Yuan, Xinni; Zeng, Xin; Gao, Yu; Li, Hongda; Keles, Sunduz; Wang, Zefeng; Chang, Qiang

2016-01-01

Mutations in the human MECP2 gene cause Rett syndrome (RTT), a severe neurodevelopmental disorder that predominantly affects girls. Despite decades of work, the molecular function of MeCP2 is not fully understood. Here we report a systematic identification of MeCP2-interacting proteins in the mouse brain. In addition to transcription regulators, we found that MeCP2 physically interacts with several modulators of RNA splicing, including LEDGF and DHX9. These interactions are disrupted by RTT causing mutations, suggesting that they may play a role in RTT pathogenesis. Consistent with the idea, deep RNA sequencing revealed misregulation of hundreds of splicing events in the cortex of Mecp2 knockout mice. To reveal the functional consequence of altered RNA splicing due to the loss of MeCP2, we focused on the regulation of the splicing of the flip/flop exon of Gria2 and other AMPAR genes. We found a significant splicing shift in the flip/flop exon toward the flop inclusion, leading to a faster decay in the AMPAR gated current and altered synaptic transmission. In summary, our study identified direct physical interaction between MeCP2 and splicing factors, a novel MeCP2 target gene, and established functional connection between a specific RNA splicing change and synaptic phenotypes in RTT mice. These results not only help our understanding of the molecular function of MeCP2, but also reveal potential drug targets for future therapies. PMID:27352031

Spliced XBP1 promotes macrophage survival and autophagy by interacting with Beclin-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tian, Ping-Ge; Jiang, Zhi-Xin; Li, Jian-Hua

Macrophage autophagy plays an important role in the development of atherosclerosis, but the precise mechanism mediating this process is unclear. The potential role of the X-box binding protein 1 (XBP1), a crucial transduction factor that is involved in endoplasmic reticulum stress and the unfolded protein response, in bone marrow-derived macrophage autophagy is unknown. This study mainly explores the roles of XBP1 mRNA splicing in bone marrow-derived macrophage autophagy. The present study shows that the transient overexpression of spliced XBP1 via adenovirus-mediated gene transfer induces autophagy and promotes proliferation in bone marrow-derived macrophages via the down-regulation of Beclin-1, but that themore » sustained overexpression of spliced XBP1 leads to apoptosis. When XBP1 is down-regulated in bone marrow-derived macrophages using siRNA, rapamycin-induced autophagosome formation is ablated. Furthermore, we have detected the overexpression of XBP1 in areas of atherosclerotic plaques in the arteries of ApoE−/− mice. These results demonstrate that XBP1 mRNA splicing plays an important role in maintaining the function of bone marrow-derived macrophages and provide new insight into the study and treatment of atherosclerosis. - Highlights: • XBP1 was up-regulated in atherosclerotic plaques of ApoE−/− mice. • Transient spliced XBP1 overexpression induced macrophages autophagy via Beclin-1. • Sustained spliced XBP1 overexpression triggered macrophages apoptosis. • Spliced XBP1 plays a key role in maintaining the macrophages survival.« less

Polypyrimidine Tract Binding Protein Homologs from Arabidopsis Are Key Regulators of Alternative Splicing with Implications in Fundamental Developmental Processes[W

PubMed Central

Rühl, Christina; Stauffer, Eva; Kahles, André; Wagner, Gabriele; Drechsel, Gabriele; Rätsch, Gunnar; Wachter, Andreas

2012-01-01

Alternative splicing (AS) generates transcript variants by variable exon/intron definition and massively expands transcriptome diversity. Changes in AS patterns have been found to be linked to manifold biological processes, yet fundamental aspects, such as the regulation of AS and its functional implications, largely remain to be addressed. In this work, widespread AS regulation by Arabidopsis thaliana Polypyrimidine tract binding protein homologs (PTBs) was revealed. In total, 452 AS events derived from 307 distinct genes were found to be responsive to the levels of the splicing factors PTB1 and PTB2, which predominantly triggered splicing of regulated introns, inclusion of cassette exons, and usage of upstream 5′ splice sites. By contrast, no major AS regulatory function of the distantly related PTB3 was found. Dependent on their position within the mRNA, PTB-regulated events can both modify the untranslated regions and give rise to alternative protein products. We find that PTB-mediated AS events are connected to diverse biological processes, and the functional implications of selected instances were further elucidated. Specifically, PTB misexpression changes AS of PHYTOCHROME INTERACTING FACTOR6, coinciding with altered rates of abscisic acid–dependent seed germination. Furthermore, AS patterns as well as the expression of key flowering regulators were massively changed in a PTB1/2 level-dependent manner. PMID:23192226

Isoforms of receptors of fibroblast growth factors.

PubMed

Gong, Siew-Ging

2014-12-01

The breadth and scope of Fibroblast Growth Factor signaling is immense, with documentation of its role in almost every organism and system studied so far. FGF ligands signal through a family of four distinct tyrosine kinase receptors, the FGF receptors (FGFRs). One contribution to the diversity of function and signaling of FGFs and their receptors arises from the numerous alternative splicing variants that have been documented in the FGFR literature. The present review discusses the types and roles of alternatively spliced variants of the FGFR family members and the significant impact of alternative splicing on the physiological functions of five broad classes of FGFR isoforms. Some characterized known regulatory mechanisms of alternative splicing and future directions in studies of FGFR alternative splicing are also discussed. Presence, absence, and/or the combination of specific exons within each FGFR protein impart upon each individual isoform its unique function and expression pattern during normal function and in diseased states (e.g., in cancers and birth defects). A better understanding of the diversity of FGF signaling in different developmental contexts and diseased states can be achieved through increased knowledge of the presence of specific FGFR isoforms and their impact on downstream signaling and functions. Modern high-throughput techniques afford an opportunity to explore the distribution and function of isoforms of FGFR during development and in diseases. © 2014 Wiley Periodicals, Inc.

Variants of the Xenopus laevis ribosomal transcription factor xUBF are developmentally regulated by differential splicing.

PubMed

Guimond, A; Moss, T

1992-07-11

XUBF is a Xenopus ribosomal transcription factor of the HMG-box family which contains five tandemly disposed homologies to the HMG1 & 2 DNA binding domains. XUBF has been isolated as a protein doublet and two cDNAs encoding the two molecular weight variants have been characterised. The major two forms of xUBF identified differ by the presence or absence of a 22 amino acid segment lying between HMG-boxes 3 and 4. Here we show that the mRNAs for these two forms of xUBF are regulated during development and differentiation over a range of nearly 20 fold. By isolating two of the xUBF genes, it was possible to show that both encoded the variable 22 amino acid segment in exon 12. Oocyte splicing assays and the sequencing of PCR-generated cDNA fragments, demonstrated that the transcripts from one of these genes were differentially spliced in a developmentally regulated manner. Transcripts from the second gene were found to be predominantly or exclusively spliced to produce the lower molecular weight form of xUBF. Expression of a high molecular weight form from yet a third gene was also detected. Although the intron-exon structures of the Xenopus and mouse UBF genes were found to be essentially identical, the differential splicing of exon 8 found in mammals, was not detected in Xenopus.

Perispeckles are major assembly sites for the exon junction core complex

PubMed Central

Daguenet, Elisabeth; Baguet, Aurélie; Degot, Sébastien; Schmidt, Ute; Alpy, Fabien; Wendling, Corinne; Spiegelhalter, Coralie; Kessler, Pascal; Rio, Marie-Christine; Le Hir, Hervé; Bertrand, Edouard; Tomasetto, Catherine

2012-01-01

The exon junction complex (EJC) is loaded onto mRNAs as a consequence of splicing and regulates multiple posttranscriptional events. MLN51, Magoh, Y14, and eIF4A3 form a highly stable EJC core, but where this tetrameric complex is assembled in the cell remains unclear. Here we show that EJC factors are enriched in domains that we term perispeckles and are visible as doughnuts around nuclear speckles. Fluorescence resonance energy transfer analyses and EJC assembly mutants show that perispeckles do not store free subunits, but instead are enriched for assembled cores. At the ultrastructural level, perispeckles are distinct from interchromatin granule clusters that may function as storage sites for splicing factors and intermingle with perichromatin fibrils, where nascent RNAs and active RNA Pol II are present. These results support a model in which perispeckles are major assembly sites for the tetrameric EJC core. This subnuclear territory thus represents an intermediate region important for mRNA maturation, between transcription sites and splicing factor reservoirs and assembly sites. PMID:22419818

Reading the tea leaves: Dead transposon copies reveal novel host and transposon biology.

PubMed

McLaughlin, Richard N

2018-03-01

Transposable elements comprise a huge portion of most animal genomes. Unlike many pathogens, these elements leave a mark of their impact via their insertion into host genomes. With proper teasing, these sequences can relay information about the evolutionary history of transposons and their hosts. In a new publication, Larson and colleagues describe a previously unappreciated density of long interspersed element-1 (LINE-1) sequences that have been spliced (LINE-1 and other reverse transcribing elements are necessarily intronless). They provide data to suggest that the retention of these potentially deleterious splice sites in LINE-1 results from the sites' overlap with an important transcription factor binding site. These spliced LINE-1s (i.e., spliced integrated retrotransposed elements [SpiREs]) lose their ability to replicate, suggesting they are evolutionary dead ends. However, the lethality of this splicing could be an efficient means of blocking continued replication of LINE-1. In this way, the record of inactive LINE-1 sequences in the human genome revealed a new, though infrequent, event in the LINE-1 replication cycle and motivates future studies to test whether splicing might be another weapon in the anti-LINE-1 arsenal of host genomes.

Short linear motif acquisition, exon formation and alternative splicing determine a pathway to diversity for NCoR-family co-repressors

PubMed Central

Short, Stephen; Peterkin, Tessa; Guille, Matthew; Patient, Roger; Sharpe, Colin

2015-01-01

Vertebrate NCoR-family co-repressors play central roles in the timing of embryo and stem cell differentiation by repressing the activity of a range of transcription factors. They interact with nuclear receptors using short linear motifs (SLiMs) termed co-repressor for nuclear receptor (CoRNR) boxes. Here, we identify the pathway leading to increasing co-repressor diversity across the deuterostomes. The final complement of CoRNR boxes arose in an ancestral cephalochordate, and was encoded in one large exon; the urochordates and vertebrates then split this region between 10 and 12 exons. In Xenopus, alternative splicing is prevalent in NCoR2, but absent in NCoR1. We show for one NCoR1 exon that alternative splicing can be recovered by a single point mutation, suggesting NCoR1 lost the capacity for alternative splicing. Analyses in Xenopus and zebrafish identify that cellular context, rather than gene sequence, predominantly determines species differences in alternative splicing. We identify a pathway to diversity for the NCoR family beginning with the addition of a SLiM, followed by gene duplication, the generation of alternatively spliced isoforms and their differential deployment. PMID:26289800

Identification and functional analysis of two alternatively spliced transcripts of ABSCISIC ACID INSENSITIVE3 (ABI3) in linseed flax (Linum usitatissimum L.).

PubMed

Wang, Yanyan; Zhang, Tianbao; Song, Xiaxia; Zhang, Jianping; Dang, Zhanhai; Pei, Xinwu; Long, Yan

2018-01-01

Alternative splicing is a popular phenomenon in different types of plants. It can produce alternative spliced transcripts that encode proteins with altered functions. Previous studies have shown that one transcription factor, ABSCISIC ACID INSENSITIVE3 (ABI3), which encodes an important component in abscisic acid (ABA) signaling, is subjected to alternative splicing in both mono- and dicotyledons. In the current study, we identified two homologs of ABI3 in the genome of linseed flax. We screened two alternatively spliced flax LuABI3 transcripts, LuABI3-2 and LuABI3-3, and one normal flax LuABI3 transcript, LuABI3-1. Sequence analysis revealed that one of the alternatively spliced transcripts, LuABI3-3, retained a 6 bp intron. RNA accumulation analysis showed that all three transcripts were expressed during seed development, while subcellular localization and transgene experiments showed that LuABI3-3 had no biological function. The two normal transcripts, LuABI3-1 and LuABI3-2, are the important functional isoforms in flax and play significant roles in the ABA regulatory pathway during seed development, germination, and maturation.

Skipping of Exons by Premature Termination of Transcription and Alternative Splicing within Intron-5 of the Sheep SCF Gene: A Novel Splice Variant

PubMed Central

Saravanaperumal, Siva Arumugam; Pediconi, Dario; Renieri, Carlo; La Terza, Antonietta

2012-01-01

Stem cell factor (SCF) is a growth factor, essential for haemopoiesis, mast cell development and melanogenesis. In the hematopoietic microenvironment (HM), SCF is produced either as a membrane-bound (−) or soluble (+) forms. Skin expression of SCF stimulates melanocyte migration, proliferation, differentiation, and survival. We report for the first time, a novel mRNA splice variant of SCF from the skin of white merino sheep via cloning and sequencing. Reverse transcriptase (RT)-PCR and molecular prediction revealed two different cDNA products of SCF. Full-length cDNA libraries were enriched by the method of rapid amplification of cDNA ends (RACE-PCR). Nucleotide sequencing and molecular prediction revealed that the primary 1519 base pair (bp) cDNA encodes a precursor protein of 274 amino acids (aa), commonly known as ‘soluble’ isoform. In contrast, the shorter (835 and/or 725 bp) cDNA was found to be a ‘novel’ mRNA splice variant. It contains an open reading frame (ORF) corresponding to a truncated protein of 181 aa (vs 245 aa) with an unique C-terminus lacking the primary proteolytic segment (28 aa) right after the D175G site which is necessary to produce ‘soluble’ form of SCF. This alternative splice (AS) variant was explained by the complete nucleotide sequencing of splice junction covering exon 5-intron (5)-exon 6 (948 bp) with a premature termination codon (PTC) whereby exons 6 to 9/10 are skipped (Cassette Exon, CE 6–9/10). We also demonstrated that the Northern blot analysis at transcript level is mediated via an intron-5 splicing event. Our data refine the structure of SCF gene; clarify the presence (+) and/or absence (−) of primary proteolytic-cleavage site specific SCF splice variants. This work provides a basis for understanding the functional role and regulation of SCF in hair follicle melanogenesis in sheep beyond what was known in mice, humans and other mammals. PMID:22719917

Systematic Profiling of Poly(A)+ Transcripts Modulated by Core 3’ End Processing and Splicing Factors Reveals Regulatory Rules of Alternative Cleavage and Polyadenylation

PubMed Central

Li, Wencheng; You, Bei; Hoque, Mainul; Zheng, Dinghai; Luo, Wenting; Ji, Zhe; Park, Ji Yeon; Gunderson, Samuel I.; Kalsotra, Auinash; Manley, James L.; Tian, Bin

2015-01-01

Alternative cleavage and polyadenylation (APA) results in mRNA isoforms containing different 3’ untranslated regions (3’UTRs) and/or coding sequences. How core cleavage/polyadenylation (C/P) factors regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3’UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A) sites (pAs), CFI-25/68, PABPN1 and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI-25/68 or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly inhibiting C/P events in introns near the 5’ end of gene and U2 suppressing those in introns with features for efficient splicing. Furthermore, PABPN1 inhibits expression of transcripts with pAs near the transcription start site (TSS), a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results support an APA code where an APA event in a given cellular context is regulated by a number of parameters, including relative location to the TSS, splicing context, distance between competing pAs, surrounding cis elements and concentrations of core C/P factors. PMID:25906188

SmD1 Modulates the miRNA Pathway Independently of Its Pre-mRNA Splicing Function.

PubMed

Xiong, Xiao-Peng; Vogler, Georg; Kurthkoti, Krishna; Samsonova, Anastasia; Zhou, Rui

2015-08-01

microRNAs (miRNAs) are a class of endogenous regulatory RNAs that play a key role in myriad biological processes. Upon transcription, primary miRNA transcripts are sequentially processed by Drosha and Dicer ribonucleases into ~22-24 nt miRNAs. Subsequently, miRNAs are incorporated into the RNA-induced silencing complexes (RISCs) that contain Argonaute (AGO) family proteins and guide RISC to target RNAs via complementary base pairing, leading to post-transcriptional gene silencing by a combination of translation inhibition and mRNA destabilization. Select pre-mRNA splicing factors have been implicated in small RNA-mediated gene silencing pathways in fission yeast, worms, flies and mammals, but the underlying molecular mechanisms are not well understood. Here, we show that SmD1, a core component of the Drosophila small nuclear ribonucleoprotein particle (snRNP) implicated in splicing, is required for miRNA biogenesis and function. SmD1 interacts with both the microprocessor component Pasha and pri-miRNAs, and is indispensable for optimal miRNA biogenesis. Depletion of SmD1 impairs the assembly and function of the miRISC without significantly affecting the expression of major canonical miRNA pathway components. Moreover, SmD1 physically and functionally associates with components of the miRISC, including AGO1 and GW182. Notably, miRNA defects resulting from SmD1 silencing can be uncoupled from defects in pre-mRNA splicing, and the miRNA and splicing machineries are physically and functionally distinct entities. Finally, photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) analysis identifies numerous SmD1-binding events across the transcriptome and reveals direct SmD1-miRNA interactions. Our study suggests that SmD1 plays a direct role in miRNA-mediated gene silencing independently of its pre-mRNA splicing activity and indicates that the dual roles of splicing factors in post-transcriptional gene regulation may be evolutionarily widespread.

Disturbed alternative splicing of FIR (PUF60) directed cyclin E overexpression in esophageal cancers.

PubMed

Ogura, Yukiko; Hoshino, Tyuji; Tanaka, Nobuko; Ailiken, Guzhanuer; Kobayashi, Sohei; Kitamura, Kouichi; Rahmutulla, Bahityar; Kano, Masayuki; Murakami, Kentarou; Akutsu, Yasunori; Nomura, Fumio; Itoga, Sakae; Matsubara, Hisahiro; Matsushita, Kazuyuki

2018-05-01

Overexpression of alternative splicing of far upstream element binding protein 1 (FUBP1) interacting repressor (FIR; poly(U) binding splicing factor 60 [PUF60]) and cyclin E were detected in esophageal squamous cell carcinomas (ESCC). Accordingly, the expression of FBW7 was examined by which cyclin E is degraded as a substrate via the proteasome system. Expectedly, FBW7 expression was decreased significantly in ESCC. Conversely, c-myc gene transcriptional repressor FIR (alias PUF60; U2AF-related protein) and its alternative splicing variant form (FIRΔexon2) were overexpressed in ESCC. Further, anticancer drugs (cis-diaminedichloroplatinum/cisplatin [CDDP] or 5-fluorouracil [5-FU]) and knockdown of FIR by small interfering RNA (siRNA) increased cyclin E while knockdown of FIRΔexon2 by siRNA decreased cyclin E expression in ESCC cell lines (TE1, TE2, and T.Tn) or cervical SCC cells (HeLa cells). Especially, knockdown of SAP155 (SF3b1), a splicing factor required for proper alternative splicing of FIR pre-mRNA, decreased cyclin E. Therefore, disturbed alternative splicing of FIR generated FIR/FIRΔexon2 with cyclin E overexpression in esophageal cancers, indicating that SAP155 siRNA potentially rescued FBW7 function by reducing expression of FIR and/or FIRΔexon2. Remarkably, Three-dimensional structure analysis revealed the hypothetical inhibitory mechanism of FBW7 function by FIR/FIRΔexon2, a novel mechanism of cyclin E overexpression by FIR/FIRΔexon2-FBW7 interaction was discussed. Clinically, elevated FIR expression potentially is an indicator of the number of lymph metastases and anti-FIR/FIRΔexon2 antibodies in sera as cancer diagnosis, indicating chemical inhibitors of FIR/FIRΔexon2-FBW7 interaction could be potential candidate drugs for cancer therapy. In conclusion, elevated cyclin E expression was, in part, induced owing to potential FIR/FIRΔexon2-FBW7 interaction in ESCC.

VizieR Online Data Catalog: Optical/NIR photometry of OGLE-2012-SN-006 (Pastorello+, 2015)

NASA Astrophysics Data System (ADS)

Pastorello, A.; Wyrzykowski, L.; Valenti, S.; Prieto, J. L.; Kozlowski, S.; Udalski, A.; Elias-Rosa, N.; Morales-Garoffolo, A.; Anderson, J. P.; Benetti, S.; Bersten, M.; Botticella, M. T.; Cappellaro, E.; Fasano, G.; Fraser, M.; Gal-Yam, A.; Gillone, M.; Graham, M. L.; Greiner, J.; Hachinger, S.; Howell, D. A.; Inserra, C.; Parrent, J.; Rau, A.; Schulze, S.; Smartt, S. J.; Smith, K. W.; Turatto, M.; Yaron, O.; Young, D. R.; Kubiak, M.; Szymanski, M. K.; Pietrzynski, G.; Soszynski, I.; Ulaczyk, K.; Poleski, R.; Pietrukowicz, P.; Skowron, J.; Mroz, P.

2017-11-01

Photometric measurements in the optical and NIR bands were obtained through the PSF-fitting technique. A template PSF was built using stars in the SN field. With this PSF model along with a low-order polynomial surface, we finally performed a fit to the SN and the underlying background. OGLE-IV photometry was obtained using the difference imaging analysis, which is a template subtraction method adapted to the OGLE data and detailed in Wyrzykowski et al. 2014, J/AcA/64/197 (see also Wozniak 2000, J/AcA/50/421). (2 data files).

Shack-Hartmann-based objective straylight assessment of the human eye in an increased scattering angle range

NASA Astrophysics Data System (ADS)

Schramm, Stefan; Schikowski, Patrick; Lerm, Elena; Kaeding, André; Haueisen, Jens; Baumgarten, Daniel

2016-07-01

Objective measurement of straylight in the human eye with a Shack-Hartmann (SH) wavefront aberrometer is limited in imaging angle. We propose a measurement principle and a point spread function (PSF) reconstruction algorithm to overcome this limitation. In our optical setup, a variable stop replaces the stop conventionally used to suppress reflections and scatter in SH aberrometers. We record images with 21 diameters of the stop. From each SH image, the average intensity of the pupil is computed and normalized. The intensities represent integral values of the PSF. We reconstruct the PSF, which is the derivative of the intensities with respect to the visual angle. A modified Stiles Holladay approximation is fitted to the reconstructed PSF, resulting in a straylight parameter. A proof-of-principle study was carried out on eight healthy young volunteers. Scatter filters were positioned in front of the volunteers' eyes to simulate straylight. The straylight parameter was compared to the C-Quant measurements and the filter values. The PSF parameter shows strong correlation with the density of the filters and a linear relation to the C-Quant straylight parameter. Our measurement and reconstruction techniques allow for objective straylight analysis of visual angles up to 4 deg.

Determination of the Point-Spread Function for the FERMI Large Area Telescope from On-Orbit Data and Limits on Pair Halos of Active Galactic Nuclei

DOE PAGES

Ackermann, M.; Ajello, M.; Allafort, A.; ...

2013-02-15

We present the Large Area Telescope (LAT) on the Fermi Gamma-ray Space Telescope is a pair-conversion telescope designed to detect photons with energies from ≈20 MeV to >300 GeV. The pre-launch response functions of the LAT were determined through extensive Monte Carlo simulations and beam tests. The point-spread function (PSF) characterizing the angular distribution of reconstructed photons as a function of energy and geometry in the detector is determined here from two years of on-orbit data by examining the distributions of γ rays from pulsars and active galactic nuclei (AGNs). Above 3 GeV, the PSF is found to be broadermore » than the pre-launch PSF. We checked for dependence of the PSF on the class of γ-ray source and observation epoch and found none. We also investigated several possible spatial models for pair-halo emission around BL Lac AGNs. Finally, we found no evidence for a component with spatial extension larger than the PSF and set upper limits on the amplitude of halo emission in stacked images of low- and high-redshift BL Lac AGNs and the TeV blazars 1ES0229+200 and 1ES0347–121.« less

Negatively charged polysulfone membranes with hydrophilicity and antifouling properties based on in situ cross-linked polymerization.

PubMed

Zhu, Lijing; Song, Haiming; Zhang, Dawei; Wang, Gang; Zeng, Zhixiang; Xue, Qunji

2017-07-15

Polysulfone (PSf) membrane has been widely used in water separation and purification, although, membrane fouling is still a serious problem limiting its potential. We aim to improve the antifouling of PSf membranes via a very simple and efficient method. In this work, antifouling PSf membranes were fabricated via in situ cross-linked polymerization coupled with non-solvent induced phase separation. In brief, acrylic acid (AA) and vinyltriethoxysilane (VTEOS) were copolymerized in PSf solution, then directly casted into membranes without purification. With the increase of monomers concentration, the morphology of the as-cast membranes changed from a finger-like morphology to a fully sponge-like structure due to the increased viscosity and decreased precipitation rate of the polymer solutions. Meanwhile, the hydrophilicity and electronegativity of modified membranes were highly improved leading to inhibited protein adsorption and improved antifouling property. Furthermore, in order to further find out the different roles player by AA and VTESO, the modified membrane without VTEOS was prepared and characterized. The results indicated that AA is more effective in the membrane hydrophilicity improvement, VTEOS is more crucial to improve membrane stability. This work provides valuable guidance for fabricating PSf membranes with hydrophilicity and antifouling property via in situ cross-linked polymerization. Copyright © 2017 Elsevier Inc. All rights reserved.

Increased losses of organic carbon and destabilising of tropical peatlands following deforestation, drainage and burning. (Invited)

NASA Astrophysics Data System (ADS)

Moore, S.; Gauci, V.; Evans, C.; Page, S. E.

2013-12-01

Tropical peatlands contain one of the largest pools of terrestrial organic carbon, amounting to about 89,000 teragrams. Approximately 65% of this carbon store is in Indonesia, where extensive anthropogenic degradation in the form of deforestation, drainage and associated fire is converting it into a globally significant source of atmospheric carbon dioxide. Unlike boreal and temperate forests and higher-latitude wetlands, however, the loss of fluvial organic carbon from tropical peats has yet to be fully quantified. Here, we present the first data from intact and degraded peat swamp forest (PSF) catchments in Central Kalimantan, Borneo, that indicate a doubling of fluvial organic carbon losses from tropical peatlands following deforestation and drainage. Through carbon-14 dating of dissolved organic carbon (DO14C), we find that leaching of DOC from intact PSF is derived mainly from recent primary production. In contrast, DOC from disturbed PSF consists mostly of much older carbon from deep within the peat column. When we include this fluvial carbon loss, which is often ignored in peatland carbon budgets, we find that it increases the estimate of total carbon lost from the disturbed peatlands in our study by 22%. We further estimate that since 1990, peatland disturbance has resulted in a 32% increase in fluvial organic carbon flux from Southeast Asia - an increase that equates to more than half of the entire annual fluvial organic carbon flux from all European peatlands. Finally, we monitored fluvial organic carbon fluxes following large-scale peatland fires in 2009/10 within the study sub-catchments and found fluvial carbon fluxes to be 30-70% larger in the fire-affected catchments when compared to fluxes during the same interval in the previous year (pre-fire). This is in marked contrast to the intact catchment (control/no fire) where there were no differences observed in fluxes 'pre to post fire years'. Our sub-catchment findings were also found to be representative at a larger river basin scale and we estimate the fluvial carbon flux from the Sebangau River basin (5,200 km2) to the Java Sea to be 0.58 Tg year-1. This is a 25% increase on the flux calculated for the River Sebangau the preceding year (pre-fire; 0.46 Tg). These new data are the first to demonstrate a large and sustained pulse of fluvial carbon following large scale human-induced fires in carbon rich tropical PSF. (L) Undisturbed PSF, (R) Disturbed PSF Borneo study sites and land-cover class properties Area = area of each catchment (PSF1 = intact, PSF2 & PSF3 = disturbed). Rainfall = total annual. Total annual discharge = standardized by area. TOC concentrations & fluxes = mean × standard error of site means.

Experimental evaluation and basis function optimization of the spatially variant image-space PSF on the Ingenuity PET/MR scanner

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kotasidis, Fotis A., E-mail: Fotis.Kotasidis@unige.ch; Zaidi, Habib; Geneva Neuroscience Centre, Geneva University, CH-1205 Geneva

2014-06-15

Purpose: The Ingenuity time-of-flight (TF) PET/MR is a recently developed hybrid scanner combining the molecular imaging capabilities of PET with the excellent soft tissue contrast of MRI. It is becoming common practice to characterize the system's point spread function (PSF) and understand its variation under spatial transformations to guide clinical studies and potentially use it within resolution recovery image reconstruction algorithms. Furthermore, due to the system's utilization of overlapping and spherical symmetric Kaiser-Bessel basis functions during image reconstruction, its image space PSF and reconstructed spatial resolution could be affected by the selection of the basis function parameters. Hence, a detailedmore » investigation into the multidimensional basis function parameter space is needed to evaluate the impact of these parameters on spatial resolution. Methods: Using an array of 12 × 7 printed point sources, along with a custom made phantom, and with the MR magnet on, the system's spatially variant image-based PSF was characterized in detail. Moreover, basis function parameters were systematically varied during reconstruction (list-mode TF OSEM) to evaluate their impact on the reconstructed resolution and the image space PSF. Following the spatial resolution optimization, phantom, and clinical studies were subsequently reconstructed using representative basis function parameters. Results: Based on the analysis and under standard basis function parameters, the axial and tangential components of the PSF were found to be almost invariant under spatial transformations (∼4 mm) while the radial component varied modestly from 4 to 6.7 mm. Using a systematic investigation into the basis function parameter space, the spatial resolution was found to degrade for basis functions with a large radius and small shape parameter. However, it was found that optimizing the spatial resolution in the reconstructed PET images, while having a good basis function superposition and keeping the image representation error to a minimum, is feasible, with the parameter combination range depending upon the scanner's intrinsic resolution characteristics. Conclusions: Using the printed point source array as a MR compatible methodology for experimentally measuring the scanner's PSF, the system's spatially variant resolution properties were successfully evaluated in image space. Overall the PET subsystem exhibits excellent resolution characteristics mainly due to the fact that the raw data are not under-sampled/rebinned, enabling the spatial resolution to be dictated by the scanner's intrinsic resolution and the image reconstruction parameters. Due to the impact of these parameters on the resolution properties of the reconstructed images, the image space PSF varies both under spatial transformations and due to basis function parameter selection. Nonetheless, for a range of basis function parameters, the image space PSF remains unaffected, with the range depending on the scanner's intrinsic resolution properties.« less

RNA splicing and splicing regulator changes in prostate cancer pathology.

PubMed

Munkley, Jennifer; Livermore, Karen; Rajan, Prabhakar; Elliott, David J

2017-09-01

Changes in mRNA splice patterns have been associated with key pathological mechanisms in prostate cancer progression. The androgen receptor (abbreviated AR) transcription factor is a major driver of prostate cancer pathology and activated by androgen steroid hormones. Selection of alternative promoters by the activated AR can critically alter gene function by switching mRNA isoform production, including creating a pro-oncogenic isoform of the normally tumour suppressor gene TSC2. A number of androgen-regulated genes generate alternatively spliced mRNA isoforms, including a prostate-specific splice isoform of ST6GALNAC1 mRNA. ST6GALNAC1 encodes a sialyltransferase that catalyses the synthesis of the cancer-associated sTn antigen important for cell mobility. Genetic rearrangements occurring early in prostate cancer development place ERG oncogene expression under the control of the androgen-regulated TMPRSS2 promoter to hijack cell behaviour. This TMPRSS2-ERG fusion gene shows different patterns of alternative splicing in invasive versus localised prostate cancer. Alternative AR mRNA isoforms play a key role in the generation of prostate cancer drug resistance, by providing a mechanism through which prostate cancer cells can grow in limited serum androgen concentrations. A number of splicing regulator proteins change expression patterns in prostate cancer and may help drive key stages of disease progression. Up-regulation of SRRM4 establishes neuronal splicing patterns in neuroendocrine prostate cancer. The splicing regulators Sam68 and Tra2β increase expression in prostate cancer. The SR protein kinase SRPK1 that modulates the activity of SR proteins is up-regulated in prostate cancer and has already given encouraging results as a potential therapeutic target in mouse models.

Intragenic motifs regulate the transcriptional complexity of Pkhd1/PKHD1

PubMed Central

Boddu, Ravindra; Yang, Chaozhe; O’Connor, Amber K.; Hendrickson, Robert Curtis; Boone, Braden; Cui, Xiangqin; Garcia-Gonzalez, Miguel; Igarashi, Peter; Onuchic, Luiz F.; Germino, Gregory G.

2014-01-01

Autosomal recessive polycystic kidney disease (ARPKD) results from mutations in the human PKHD1 gene. Both this gene, and its mouse ortholog, Pkhd1, are primarily expressed in renal and biliary ductal structures. The mouse protein product, fibrocystin/polyductin complex (FPC), is a 445-kDa protein encoded by a 67-exon transcript that spans >500 kb of genomic DNA. In the current study, we observed multiple alternatively spliced Pkhd1 transcripts that varied in size and exon composition in embryonic mouse kidney, liver, and placenta samples, as well as among adult mouse pancreas, brain, heart, lung, testes, liver, and kidney. Using reverse transcription PCR and RNASeq, we identified 22 novel Pkhd1 kidney transcripts with unique exon junctions. Various mechanisms of alternative splicing were observed, including exon skipping, use of alternate acceptor/donor splice sites, and inclusion of novel exons. Bioinformatic analyses identified, and exon-trapping minigene experiments validated, consensus binding sites for serine/arginine-rich proteins that modulate alternative splicing. Using site-directed mutagenesis, we examined the functional importance of selected splice enhancers. In addition, we demonstrated that many of the novel transcripts were polysome bound, thus likely translated. Finally, we determined that the human PKHD1 R760H missense variant alters a splice enhancer motif that disrupts exon splicing in vitro and is predicted to truncate the protein. Taken together, these data provide evidence of the complex transcriptional regulation of Pkhd1/PKHD1 and identified motifs that regulate its splicing. Our studies indicate that Pkhd1/PKHD1 transcription is modulated, in part by intragenic factors, suggesting that aberrant PKHD1 splicing represents an unappreciated pathogenic mechanism in ARPKD. PMID:24984783

Reflections on the history of pre-mRNA processing and highlights of current knowledge: A unified picture

PubMed Central

Darnell, James E.

2013-01-01

Several strong conclusions emerge concerning pre-mRNA processing from both old and newer experiments. The RNAPII complex is involved with pre-mRNA processing through binding of processing proteins to the CTD (carboxyl terminal domain) of the largest RNAPII subunit. These interactions are necessary for efficient processing, but whether factor binding to the CTD and delivery to splicing sites is obligatory or facilitatory is unsettled. Capping, addition of an m7Gppp residue (cap) to the initial transcribed residue of a pre-mRNA, occurs within seconds. Splicing of pre-mRNA by spliceosomes at particular sites is most likely committed during transcription by the binding of initiating processing factors and ∼50% of the time is completed in mammalian cells before completion of the primary transcript. This fact has led to an outpouring in the literature about “cotranscriptional splicing.” However splicing requires several minutes for completion and can take longer. The RNAPII complex moves through very long introns and also through regions dense with alternating exons and introns at an average rate of ∼3 kb per min and is, therefore, not likely detained at each splice site for more than a few seconds, if at all. Cleavage of the primary transcript at the 3′ end and polyadenylation occurs within 30 sec or less at recognized polyA sites, and the majority of newly polyadenylated pre-mRNA molecules are much larger than the average mRNA. Finally, it seems quite likely that the nascent RNA most often remains associated with the chromosomal locus being transcribed until processing is complete, possibly acquiring factors related to the transport of the new mRNA to the cytoplasm. PMID:23440351

Genome-based identification of spliceosomal proteins in the silk moth Bombyx mori.

PubMed

Somarelli, Jason A; Mesa, Annia; Fuller, Myron E; Torres, Jacqueline O; Rodriguez, Carol E; Ferrer, Christina M; Herrera, Rene J

2010-12-01

Pre-messenger RNA splicing is a highly conserved eukaryotic cellular function that takes place by way of a large, RNA-protein assembly known as the spliceosome. In the mammalian system, nearly 300 proteins associate with uridine-rich small nuclear (sn)RNAs to form this complex. Some of these splicing factors are ubiquitously present in the spliceosome, whereas others are involved only in the processing of specific transcripts. Several proteomics analyses have delineated the proteins of the spliceosome in several species. In this study, we mine multiple sequence data sets of the silk moth Bombyx mori in an attempt to identify the entire set of known spliceosomal proteins. Five data sets were utilized, including the 3X, 6X, and Build 2.0 genomic contigs as well as the expressed sequence tag and protein libraries. While homologs for 88% of vertebrate splicing factors were delineated in the Bombyx mori genome, there appear to be several spliceosomal polypeptides absent in Bombyx mori and seven additional insect species. This apparent increase in spliceosomal complexity in vertebrates may reflect the tissue-specific and developmental stage-specific alternative pre-mRNA splicing requirements in vertebrates. Phylogenetic analyses of 15 eukaryotic taxa using the core splicing factors suggest that the essential functional units of the pre-mRNA processing machinery have remained highly conserved from yeast to humans. The Sm and LSm proteins are the most conserved, whereas proteins of the U1 small nuclear ribonucleoprotein particle are the most divergent. These data highlight both the differential conservation and relative phylogenetic signals of the essential spliceosomal components throughout evolution. © 2010 Wiley Periodicals, Inc.

Evidence that C9ORF72 Dipeptide Repeat Proteins Associate with U2 snRNP to Cause Mis-splicing in ALS/FTD Patients.

PubMed

Yin, Shanye; Lopez-Gonzalez, Rodrigo; Kunz, Ryan C; Gangopadhyay, Jaya; Borufka, Carl; Gygi, Steven P; Gao, Fen-Biao; Reed, Robin

2017-06-13

Hexanucleotide repeat expansion in the C9ORF72 gene results in production of dipeptide repeat (DPR) proteins that may disrupt pre-mRNA splicing in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients. At present, the mechanisms underlying this mis-splicing are not understood. Here, we show that addition of proline-arginine (PR) and glycine-arginine (GR) toxic DPR peptides to nuclear extracts blocks spliceosome assembly and splicing, but not other types of RNA processing. Proteomic and biochemical analyses identified the U2 small nuclear ribonucleoprotein particle (snRNP) as a major interactor of PR and GR peptides. In addition, U2 snRNP, but not other splicing factors, mislocalizes from the nucleus to the cytoplasm both in C9ORF72 patient induced pluripotent stem cell (iPSC)-derived motor neurons and in HeLa cells treated with the toxic peptides. Bioinformatic studies support a specific role for U2-snRNP-dependent mis-splicing in C9ORF72 patient brains. Together, our data indicate that DPR-mediated dysfunction of U2 snRNP could account for as much as ∼44% of the mis-spliced cassette exons in C9ORF72 patient brains. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Interplay between Alternative Splicing and Alternative Polyadenylation Defines the Expression Outcome of the Plant Unique OXIDATIVE TOLERANT-6 Gene.

PubMed

Li, Qingshun Q; Liu, Zhaoyang; Lu, Wenjia; Liu, Man

2017-05-17

Pre-mRNA alternative splicing and alternative polyadenylation have been implicated to play important roles during eukaryotic gene expression. However, much remains unknown regarding the regulatory mechanisms and the interactions of these two processes in plants. Here we focus on an Arabidopsis gene OXT6 (Oxidative Tolerant-6) that has been demonstrated to encode two proteins through alternative splicing and alternative polyadenylation. Specifically, alternative polyadenylation at Intron-2 of OXT6 produces a transcript coding for AtCPSF30, an Arabidopsis ortholog of 30 kDa subunit of the Cleavage and Polyadenylation Specificity Factor. On the other hand, alternative splicing of Intron-2 generates a longer transcript encoding a protein named AtC30Y, a polypeptide including most part of AtCPSF30 and a YT521B domain. To investigate the expression outcome of OXT6 in plants, a set of mutations were constructed to alter the splicing and polyadenylation patterns of OXT6. Analysis of transgenic plants bearing these mutations by quantitative RT-PCR revealed a competition relationship between these two processes. Moreover, when both splice sites and poly(A) signals were mutated, polyadenylation became the preferred mode of OXT6 processing. These results demonstrate the interplay between alternative splicing and alternative polyadenylation, and it is their concerted actions that define a gene's expression outcome.

Nuclear poly(A)-binding protein aggregates misplace a pre-mRNA outside of SC35 speckle causing its abnormal splicing

PubMed Central

Klein, Pierre; Oloko, Martine; Roth, Fanny; Montel, Valérie; Malerba, Alberto; Jarmin, Susan; Gidaro, Teresa; Popplewell, Linda; Perie, Sophie; Lacau St Guily, Jean; de la Grange, Pierre; Antoniou, Michael N.; Dickson, George; Butler-Browne, Gillian; Bastide, Bruno; Mouly, Vincent; Trollet, Capucine

2016-01-01

A short abnormal polyalanine expansion in the polyadenylate-binding protein nuclear-1 (PABPN1) protein causes oculopharyngeal muscular dystrophy (OPMD). Mutated PABPN1 proteins accumulate as insoluble intranuclear aggregates in muscles of OPMD patients. While the roles of PABPN1 in nuclear polyadenylation and regulation of alternative poly(A) site choice have been established, the molecular mechanisms which trigger pathological defects in OPMD and the role of aggregates remain to be determined. Using exon array, for the first time we have identified several splicing defects in OPMD. In particular, we have demonstrated a defect in the splicing regulation of the muscle-specific Troponin T3 (TNNT3) mutually exclusive exons 16 and 17 in OPMD samples compared to controls. This splicing defect is directly linked to the SC35 (SRSF2) splicing factor and to the presence of nuclear aggregates. As reported here, PABPN1 aggregates are able to trap TNNT3 pre-mRNA, driving it outside nuclear speckles, leading to an altered SC35-mediated splicing. This results in a decreased calcium sensitivity of muscle fibers, which could in turn plays a role in muscle pathology. We thus report a novel mechanism of alternative splicing deregulation that may play a role in various other diseases with nuclear inclusions or foci containing an RNA binding protein. PMID:27507886

Tissue-specific expression and regulation of the alternatively-spliced forms of lysyl hydroxylase 2 (LH2) in human kidney cells and skin fibroblasts.

PubMed

Walker, Linda C; Overstreet, Mayra A; Yeowell, Heather N

2005-01-01

Lysyl hydroxylases 1, 2, and 3 catalyse the hydroxylation of specific lysines in collagen. A small percentage of these hydroxylysine residues are precursors for the cross-link formation essential for the tensile strength of collagen. Lysyl hydroxylase 2 (LH2) exists as two alternatively-spliced forms; the long transcript (the major ubiquitously-expressed form) includes a 63 bp exon (13A) that is spliced out in the short form (expressed, together with the long form, in human kidney, spleen, liver, and placenta). This study shows that this alternative splicing event can be regulated by both cell density and cycloheximide (CHX). Although only the long form of LH2 is detected in untreated confluent human skin fibroblasts, after 24 h treatment with CHX the short LH2 transcript is also expressed. In kidney cells, in which both LH2 transcripts are equally expressed, the long LH2 transcript is significantly decreased after 24 h CHX treatment, whereas expression of the short transcript is slightly increased. This suggests that, in kidney cells, the splicing mechanism for the inclusion of exon 13A in LH2 requires a newly-synthesized protein factor that is suppressed by CHX, whereas, in skin fibroblasts in which levels of LH2 (long) are unaffected, CHX appears to suppress a factor that inhibits exclusion of exon 13A, thereby promoting expression of LH2 (short). As these alternate transcripts of LH2 may have specificity for hydroxylation of lysines in either telopeptide or helical collagen domains, their relative expression determines the type of cross-links formed, thereby affecting collagen strength. Therefore, any perturbation of the regulation of LH2 splicing could influence the stability of the extracellular matrix and contribute to specific connective tissue disorders.

Long genes and genes with multiple splice variants are enriched in pathways linked to cancer and other multigenic diseases.

PubMed

Sahakyan, Aleksandr B; Balasubramanian, Shankar

2016-03-12

The role of random mutations and genetic errors in defining the etiology of cancer and other multigenic diseases has recently received much attention. With the view that complex genes should be particularly vulnerable to such events, here we explore the link between the simple properties of the human genes, such as transcript length, number of splice variants, exon/intron composition, and their involvement in the pathways linked to cancer and other multigenic diseases. We reveal a substantial enrichment of cancer pathways with long genes and genes that have multiple splice variants. Although the latter two factors are interdependent, we show that the overall gene length and splicing complexity increase in cancer pathways in a partially decoupled manner. Our systematic survey for the pathways enriched with top lengthy genes and with genes that have multiple splice variants reveal, along with cancer pathways, the pathways involved in various neuronal processes, cardiomyopathies and type II diabetes. We outline a correlation between the gene length and the number of somatic mutations. Our work is a step forward in the assessment of the role of simple gene characteristics in cancer and a wider range of multigenic diseases. We demonstrate a significant accumulation of long genes and genes with multiple splice variants in pathways of multigenic diseases that have already been associated with de novo mutations. Unlike the cancer pathways, we note that the pathways of neuronal processes, cardiomyopathies and type II diabetes contain genes long enough for topoisomerase-dependent gene expression to also be a potential contributing factor in the emergence of pathologies, should topoisomerases become impaired.

Splicing factor SR34b mutation reduces cadmium tolerance in Arabidopsis by regulating iron-regulated transporter 1 gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Wentao; Du, Bojing; Liu, Di

Highlights: • Arabidopsis splicing factor SR34b gene is cadmium-inducible. • SR34b T-DNA insertion mutant is sensitive to cadmium due to high cadmium uptake. • SR34b is a regulator of cadmium transporter IRT1 at the posttranscription level. • These results highlight the roles of splicing factors in cadmium tolerance of plant. - Abstract: Serine/arginine-rich (SR) proteins are important splicing factors. However, the biological functions of plant SR proteins remain unclear especially in abiotic stresses. Cadmium (Cd) is a non-essential element that negatively affects plant growth and development. In this study, we provided clear evidence for SR gene involved in Cd tolerancemore » in planta. Systemic expression analysis of 17 Arabidopsis SR genes revealed that SR34b is the only SR gene upregulated by Cd, suggesting its potential roles in Arabidopsis Cd tolerance. Consistent with this, a SR34b T-DNA insertion mutant (sr34b) was moderately sensitive to Cd, which had higher Cd{sup 2+} uptake rate and accumulated Cd in greater amounts than wild-type. This was due to the altered expression of iron-regulated transporter 1 (IRT1) gene in sr34b mutant. Under normal growth conditions, IRT1 mRNAs highly accumulated in sr34b mutant, which was a result of increased stability of IRT1 mRNA. Under Cd stress, however, sr34b mutant plants had a splicing defect in IRT1 gene, thus reducing the IRT1 mRNA accumulation. Despite of this, sr34b mutant plants still constitutively expressed IRT1 proteins under Cd stress, thereby resulting in Cd stress-sensitive phenotype. We therefore propose the essential roles of SR34b in posttranscriptional regulation of IRT1 expression and identify it as a regulator of Arabidopsis Cd tolerance.« less

Vimentin, a Novel NF-κB Regulator, Is Required for Meningitic Escherichia coli K1-Induced Pathogen Invasion and PMN Transmigration across the Blood-Brain Barrier.

PubMed

Huang, Sheng-He; Chi, Feng; Peng, Liang; Bo, Tao; Zhang, Bao; Liu, Li-Qun; Wu, Xuedong; Mor-Vaknin, Nirit; Markovitz, David M; Cao, Hong; Zhou, Yan-Hong

NF-κB activation, pathogen invasion, polymorphonuclear leukocytes (PMN) transmigration (PMNT) across the blood-brain barrier (BBB) are the pathogenic triad hallmark features of bacterial meningitis, but the mechanisms underlying these events remain largely unknown. Vimentin, which is a novel NF-κB regulator, is the primary receptor for the major Escherichia coli K1 virulence factor IbeA that contributes to the pathogenesis of neonatal bacterial sepsis and meningitis (NSM). We have previously shown that IbeA-induced NF-κB signaling through its primary receptor vimentin as well as its co-receptor PTB-associated splicing factor (PSF) is required for pathogen penetration and leukocyte transmigration across the BBB. This is the first in vivo study to demonstrate how vimentin and related factors contributed to the pathogenic triad of bacterial meningitis. The role of vimentin in IbeA+ E. coli K1-induced NF-κB activation, pathogen invasion, leukocyte transmigration across the BBB has now been demonstrated by using vimentin knockout (KO) mice. In the in vivo studies presented here, IbeA-induced NF-κB activation, E. coli K1 invasion and polymorphonuclear neutrophil (PMN) transmigration across the BBB were significantly reduced in Vim-/- mice. Decreased neuronal injury in the hippocampal dentate gyrus was observed in Vim-/- mice with meningitis. The major inflammatory regulator α7 nAChR and several signaling molecules contributing to NF-κB activation (p65 and p-CamKII) were significantly reduced in the brain tissues of the Vim-/- mice with E. coli meningitis. Furthermore, Vim KO resulted in significant reduction in neuronal injury and in α7 nAChR-mediated calcium signaling. Vimentin, a novel NF-κB regulator, plays a detrimental role in the host defense against meningitic infection by modulating the NF-κB signaling pathway to increase pathogen invasion, PMN recruitment, BBB permeability and neuronal inflammation. Our findings provide the first evidence for Vim-dependent mechanisms underlying the pathogenic triad of bacterial meningitis.

Vimentin, a Novel NF-κB Regulator, Is Required for Meningitic Escherichia coli K1-Induced Pathogen Invasion and PMN Transmigration across the Blood-Brain Barrier

PubMed Central

Zhang, Bao; Liu, Li-Qun; Wu, Xuedong; Mor-Vaknin, Nirit; Markovitz, David M.; Cao, Hong; Zhou, Yan-Hong

2016-01-01

Background NF-κB activation, pathogen invasion, polymorphonuclear leukocytes (PMN) transmigration (PMNT) across the blood-brain barrier (BBB) are the pathogenic triad hallmark features of bacterial meningitis, but the mechanisms underlying these events remain largely unknown. Vimentin, which is a novel NF-κB regulator, is the primary receptor for the major Escherichia coli K1 virulence factor IbeA that contributes to the pathogenesis of neonatal bacterial sepsis and meningitis (NSM). We have previously shown that IbeA-induced NF-κB signaling through its primary receptor vimentin as well as its co-receptor PTB-associated splicing factor (PSF) is required for pathogen penetration and leukocyte transmigration across the BBB. This is the first in vivo study to demonstrate how vimentin and related factors contributed to the pathogenic triad of bacterial meningitis. Methodology/Principal Findings The role of vimentin in IbeA+ E. coli K1-induced NF-κB activation, pathogen invasion, leukocyte transmigration across the BBB has now been demonstrated by using vimentin knockout (KO) mice. In the in vivo studies presented here, IbeA-induced NF-κB activation, E. coli K1 invasion and polymorphonuclear neutrophil (PMN) transmigration across the BBB were significantly reduced in Vim-/- mice. Decreased neuronal injury in the hippocampal dentate gyrus was observed in Vim-/- mice with meningitis. The major inflammatory regulator α7 nAChR and several signaling molecules contributing to NF-κB activation (p65 and p-CamKII) were significantly reduced in the brain tissues of the Vim-/- mice with E. coli meningitis. Furthermore, Vim KO resulted in significant reduction in neuronal injury and in α7 nAChR-mediated calcium signaling. Conclusion/Significance Vimentin, a novel NF-κB regulator, plays a detrimental role in the host defense against meningitic infection by modulating the NF-κB signaling pathway to increase pathogen invasion, PMN recruitment, BBB permeability and neuronal inflammation. Our findings provide the first evidence for Vim-dependent mechanisms underlying the pathogenic triad of bacterial meningitis. PMID:27657497

MeV gamma-ray observation with a well-defined point spread function based on electron tracking

NASA Astrophysics Data System (ADS)

Takada, A.; Tanimori, T.; Kubo, H.; Mizumoto, T.; Mizumura, Y.; Komura, S.; Kishimoto, T.; Takemura, T.; Yoshikawa, K.; Nakamasu, Y.; Matsuoka, Y.; Oda, M.; Miyamoto, S.; Sonoda, S.; Tomono, D.; Miuchi, K.; Kurosawa, S.; Sawano, T.

2016-07-01

The field of MeV gamma-ray astronomy has not opened up until recently owing to imaging difficulties. Compton telescopes and coded-aperture imaging cameras are used as conventional MeV gamma-ray telescopes; however their observations are obstructed by huge background, leading to uncertainty of the point spread function (PSF). Conventional MeV gamma-ray telescopes imaging utilize optimizing algorithms such as the ML-EM method, making it difficult to define the correct PSF, which is the uncertainty of a gamma-ray image on the celestial sphere. Recently, we have defined and evaluated the PSF of an electron-tracking Compton camera (ETCC) and a conventional Compton telescope, and thereby obtained an important result: The PSF strongly depends on the precision of the recoil direction of electron (scatter plane deviation, SPD) and is not equal to the angular resolution measure (ARM). Now, we are constructing a 30 cm-cubic ETCC for a second balloon experiment, Sub-MeV gamma ray Imaging Loaded-on-balloon Experiment: SMILE-II. The current ETCC has an effective area of 1 cm2 at 300 keV, a PSF of 10° at FWHM for 662 keV, and a large field of view of 3 sr. We will upgrade this ETCC to have an effective area of several cm2 and a PSF of 5° using a CF4-based gas. Using the upgraded ETCC, our observation plan for SMILE-II is to map of the electron-positron annihilation line and the 1.8 MeV line from 26Al. In this paper, we will report on the current performance of the ETCC and on our observation plan.

High precision wavefront control in point spread function engineering for single emitter localization

NASA Astrophysics Data System (ADS)

Siemons, M.; Hulleman, C. N.; Thorsen, R. Ø.; Smith, C. S.; Stallinga, S.

2018-04-01

Point Spread Function (PSF) engineering is used in single emitter localization to measure the emitter position in 3D and possibly other parameters such as the emission color or dipole orientation as well. Advanced PSF models such as spline fits to experimental PSFs or the vectorial PSF model can be used in the corresponding localization algorithms in order to model the intricate spot shape and deformations correctly. The complexity of the optical architecture and fit model makes PSF engineering approaches particularly sensitive to optical aberrations. Here, we present a calibration and alignment protocol for fluorescence microscopes equipped with a spatial light modulator (SLM) with the goal of establishing a wavefront error well below the diffraction limit for optimum application of complex engineered PSFs. We achieve high-precision wavefront control, to a level below 20 m$\\lambda$ wavefront aberration over a 30 minute time window after the calibration procedure, using a separate light path for calibrating the pixel-to-pixel variations of the SLM, and alignment of the SLM with respect to the optical axis and Fourier plane within 3 $\\mu$m ($x/y$) and 100 $\\mu$m ($z$) error. Aberrations are retrieved from a fit of the vectorial PSF model to a bead $z$-stack and compensated with a residual wavefront error comparable to the error of the SLM calibration step. This well-calibrated and corrected setup makes it possible to create complex `3D+$\\lambda$' PSFs that fit very well to the vectorial PSF model. Proof-of-principle bead experiments show precisions below 10~nm in $x$, $y$, and $\\lambda$, and below 20~nm in $z$ over an axial range of 1 $\\mu$m with 2000 signal photons and 12 background photons.

Pancreas Oxygen Persufflation Increases ATP Levels as Shown by Nuclear Magnetic Resonance

PubMed Central

Scott, W.E.; Weegman, B.P.; Ferrer-Fabrega, J.; Stein, S.A.; Anazawa, T.; Kirchner, V.A.; Rizzari, M.D.; Stone, J.; Matsumoto, S.; Hammer, B.E.; Balamurugan, A.N.; Kidder, L.S.; Suszynski, T.M.; Avgoustiniatos, E.S.; Stone, S.G.; Tempelman, L.A.; Sutherland, D.E.R.; Hering, B.J.; Papas, K.K.

2010-01-01

Background Islet transplantation is a promising treatment for type 1 diabetes. Due to a shortage of suitable human pancreata, high cost, and the large dose of islets presently required for long-term diabetes reversal; it is important to maximize viable islet yield. Traditional methods of pancreas preservation have been identified as suboptimal due to insufficient oxygenation. Enhanced oxygen delivery is a key area of improvement. In this paper, we explored improved oxygen delivery by persufflation (PSF), ie, vascular gas perfusion. Methods Human pancreata were obtained from brain-dead donors. Porcine pancreata were procured by en bloc viscerectomy from heparinized donation after cardiac death donors and were either preserved by either two-layer method (TLM) or PSF. Following procurement, organs were transported to a 1.5-T magnetic resonance (MR) system for 31P nuclear magnetic resonance spectroscopy to investigate their bioenergetic status by measuring the ratio of adenosine triphosphate to inorganic phosphate (ATP:Pi) and for assessing PSF homogeneity by MRI. Results Human and porcine pancreata can be effectively preserved by PSF. MRI showed that pancreatic tissue was homogeneously filled with gas. TLM can effectively raise ATP:Pi levels in rat pancreata but not in larger porcine pancreata. ATP:Pi levels were almost undetectable in porcine organs preserved with TLM. When human or porcine organs were preserved by PSF, ATP:Pi was elevated to levels similar to those observed in rat pancreata. Conclusion The methods developed for human and porcine pancreas PSF homogeneously deliver oxygen throughout the organ. This elevates ATP levels during preservation and may improve islet isolation outcomes while enabling the use of marginal donors, thus expanding the usable donor pool. PMID:20692395

Wavelength-Dependent PSFs and their Impact on Weak Lensing Measurements

NASA Astrophysics Data System (ADS)

Carlsten, S. G.; Strauss, Michael A.; Lupton, Robert H.; Meyers, Joshua E.; Miyazaki, Satoshi

2018-06-01

We measure and model the wavelength dependence of the point spread function (PSF) in the Hyper Suprime-Cam Subaru Strategic Program survey. We find that PSF chromaticity is present in that redder stars appear smaller than bluer stars in the g, r, and i-bands at the 1-2 per cent level and in the z and y-bands at the 0.1-0.2 per cent level. From the color dependence of the PSF, we fit a model between the monochromatic PSF size based on weighted second moments, R, and wavelength of the form R(λ)∝λ-b. We find values of b between 0.2 and 0.5, depending on the epoch and filter. This is consistent with the expectations of a turbulent atmosphere with an outer scale length of ˜10 - 100 m, indicating that the atmosphere is dominating the chromaticity. In the best seeing data, we find that the optical system and detector also contribute some wavelength dependence. Meyers & Burchat (2015b) showed that b must be measured to an accuracy of ˜0.02 not to dominate the systematic error budget of the Large Synoptic Survey Telescope (LSST) weak lensing (WL) survey. Using simple image simulations, we find that b can be inferred with this accuracy in the r and i-bands for all positions in the LSST focal plane, assuming a stellar density of 1 star arcmin-2 and that the optical component of the PSF can be accurately modeled. Therefore, it is possible to correct for most, if not all, of the bias that the wavelength-dependent PSF will introduce into an LSST-like WL survey.

Modeling and Correcting the Time-Dependent ACS PSF

NASA Technical Reports Server (NTRS)

Rhodes, Jason; Massey, Richard; Albert, Justin; Taylor, James E.; Koekemoer, Anton M.; Leauthaud, Alexie

2006-01-01

The ability to accurately measure the shapes of faint objects in images taken with the Advanced Camera for Surveys (ACS) on the Hubble Space Telescope (HST) depends upon detailed knowledge of the Point Spread Function (PSF). We show that thermal fluctuations cause the PSF of the ACS Wide Field Camera (WFC) to vary over time. We describe a modified version of the TinyTim PSF modeling software to create artificial grids of stars across the ACS field of view at a range of telescope focus values. These models closely resemble the stars in real ACS images. Using 10 bright stars in a real image, we have been able to measure HST s apparent focus at the time of the exposure. TinyTim can then be used to model the PSF at any position on the ACS field of view. This obviates the need for images of dense stellar fields at different focus values, or interpolation between the few observed stars. We show that residual differences between our TinyTim models and real data are likely due to the effects of Charge Transfer Efficiency (CTE) degradation. Furthermore, we discuss stochastic noise that is added to the shape of point sources when distortion is removed, and we present MultiDrizzle parameters that are optimal for weak lensing science. Specifically, we find that reducing the MultiDrizzle output pixel scale and choosing a Gaussian kernel significantly stabilizes the resulting PSF after image combination, while still eliminating cosmic rays/bad pixels, and correcting the large geometric distortion in the ACS. We discuss future plans, which include more detailed study of the effects of CTE degradation on object shapes and releasing our TinyTim models to the astronomical community.

Redirecting RNA splicing by SMAD3 turns TGF-β into a tumor promoter.

PubMed

Tripathi, Veenu; Zhang, Ying E

2017-01-01

Transforming growth factor β (TGF-β) is a well-known growth inhibitor of normal epithelial cells, but it is also secreted by solid tumors to promote cancer progression. Our recent discovery of SMAD3-PCBP1 complex with direct RNA-binding properties has shed light on how this conversion is implemented by controlling pre-mRNA splicing patterns.

Alternative RNA splicing of the MEAF6 gene facilitates neuroendocrine prostate cancer progression.

PubMed

Lee, Ahn R; Li, Yinan; Xie, Ning; Gleave, Martin E; Cox, Michael E; Collins, Colin C; Dong, Xuesen

2017-04-25

Although potent androgen receptor pathway inhibitors (ARPI) improve overall survival of metastatic prostate cancer patients, treatment-induced neuroendocrine prostate cancer (t-NEPC) as a consequence of the selection pressures of ARPI is becoming a more common clinical issue. Improved understanding of the molecular biology of t-NEPC is essential for the development of new effective management approaches for t-NEPC. In this study, we identify a splice variant of the MYST/Esa1-associated factor 6 (MEAF6) gene, MEAF6-1, that is highly expressed in both t-NEPC tumor biopsies and neuroendocrine cell lines of prostate and lung cancers. We show that MEAF6-1 splicing is stimulated by neuronal RNA splicing factor SRRM4. Rather than inducing neuroendocrine trans-differentiation of cells in prostate adenocarcinoma, MEAF6-1 upregulation stimulates cell proliferation, anchorage-independent cell growth, invasion and xenograft tumor growth. Gene microarray identifies that these MEAF6-1 actions are in part mediated by the ID1 and ID3 genes. These findings suggest that the MEAF6-1 variant does not induce neuroendocrine differentiation of prostate cancer cells, but rather facilitates t-NEPC progression by increasing the proliferation rate of cells that have acquired neuroendocrine phenotypes.

Characterization of CaV1.2 exon 33 heterozygous knockout mice and negative correlation between Rbfox1 and CaV1.2 exon 33 expressions in human heart failure.

PubMed

Wang, Juejin; Li, Guang; Yu, Dejie; Wong, Yuk Peng; Yong, Tan Fong; Liang, Mui Cheng; Liao, Ping; Foo, Roger; Hoppe, Uta C; Soong, Tuck Wah

2018-01-01

Recently, we reported that homozygous deletion of alternative exon 33 of Ca V 1.2 calcium channel in the mouse resulted in ventricular arrhythmias arising from increased Ca V 1.2 Δ33 I CaL current density in the cardiomyocytes. We wondered whether heterozygous deletion of exon 33 might produce cardiac phenotype in a dose-dependent manner, and whether the expression levels of RNA splicing factors known to regulate alternative splicing of exon 33 might change in human heart failure. Unexpectedly, we found that exon 33 +/- cardiomyocytes showed similar Ca V 1.2 channel properties as wild-type cardiomyocyte, even though Ca V 1.2 Δ33 channels exhibit a gain-in-function. In human hearts, we found that the mRNA level of splicing factor Rbfox1, but not Rbfox2, was downregulated in dilated cardiomyopathy, and CACNA1C mRNA level was dramatically decreased in the both of dilated and ischemic cardiomyopathy. These data imply Rbfox1 may be involved in the development of cardiomyopathies via regulating the alternative splicing of Ca V 1.2 exon 33. (149 words).

Application of the Hotelling and ideal observers to detection and localization of exoplanets.

PubMed

Caucci, Luca; Barrett, Harrison H; Devaney, Nicholas; Rodríguez, Jeffrey J

2007-12-01

The ideal linear discriminant or Hotelling observer is widely used for detection tasks and image-quality assessment in medical imaging, but it has had little application in other imaging fields. We apply it to detection of planets outside of our solar system with long-exposure images obtained from ground-based or space-based telescopes. The statistical limitations in this problem include Poisson noise arising mainly from the host star, electronic noise in the image detector, randomness or uncertainty in the point-spread function (PSF) of the telescope, and possibly a random background. PSF randomness is reduced but not eliminated by the use of adaptive optics. We concentrate here on the effects of Poisson and electronic noise, but we also show how to extend the calculation to include a random PSF. For the case where the PSF is known exactly, we compare the Hotelling observer to other observers commonly used for planet detection; comparison is based on receiver operating characteristic (ROC) and localization ROC (LROC) curves.

Application of the Hotelling and ideal observers to detection and localization of exoplanets

PubMed Central

Caucci, Luca; Barrett, Harrison H.; Devaney, Nicholas; Rodríguez, Jeffrey J.

2008-01-01

The ideal linear discriminant or Hotelling observer is widely used for detection tasks and image-quality assessment in medical imaging, but it has had little application in other imaging fields. We apply it to detection of planets outside of our solar system with long-exposure images obtained from ground-based or space-based telescopes. The statistical limitations in this problem include Poisson noise arising mainly from the host star, electronic noise in the image detector, randomness or uncertainty in the point-spread function (PSF) of the telescope, and possibly a random background. PSF randomness is reduced but not eliminated by the use of adaptive optics. We concentrate here on the effects of Poisson and electronic noise, but we also show how to extend the calculation to include a random PSF. For the case where the PSF is known exactly, we compare the Hotelling observer to other observers commonly used for planet detection; comparison is based on receiver operating characteristic (ROC) and localization ROC (LROC) curves. PMID:18059905

Weak Lensing from Space I: Instrumentation and Survey Strategy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rhodes, Jason; Refregier, Alexandre; Massey, Richard

A wide field space-based imaging telescope is necessary to fully exploit the technique of observing dark matter via weak gravitational lensing. This first paper in a three part series outlines the survey strategies and relevant instrumental parameters for such a mission. As a concrete example of hardware design, we consider the proposed Supernova/Acceleration Probe (SNAP). Using SNAP engineering models, we quantify the major contributions to this telescope's Point Spread Function (PSF). These PSF contributions are relevant to any similar wide field space telescope. We further show that the PSF of SNAP or a similar telescope will be smaller than currentmore » ground-based PSFs, and more isotropic and stable over time than the PSF of the Hubble Space Telescope. We outline survey strategies for two different regimes - a ''wide'' 300 square degree survey and a ''deep'' 15 square degree survey that will accomplish various weak lensing goals including statistical studies and dark matter mapping.« less

Effects of structures of bidentate Schiff base type bonded-ligands derived from benzaldehyde on the photoluminescence performance of polymer-rare earth complexes.

PubMed

Gao, Baojiao; Zhang, Liqin; Zhang, Dandan

2018-02-07

Two kinds of bidentate Schiff base ligands derived from benzaldehyde, benzaldehyde/m-aminophenol (BAMA) type and benzaldehyde/glutamic acid (BAGL) type ligands, were synchronously synthesized and bonded on the backbone of polysulfone (PSF) through molecular design and by polymer reactions, and two functional polymers, PSF-BAMA and PSF-BAGL, were obtained. Then two series of novel luminescent Schiff base-type polymer-rare earth complexes were prepared via coordination reactions. In this work, the effects of the structures of the bonded ligands on the photoluminescence performance of the complexes were investigated in detail, and for the different photophysical properties of the prepared complexes, relevant theoretical explanations were given. The experimental results show that the bonded ligand BAMA can strongly sensitize the fluorescence emission of Eu(iii) ions, and the binary complex PSF-(BAMA) 3 -Eu(iii) emits strong red fluorescence under UV light. The reason for this lies in the fact that a larger conjugate π-bond system is contained in the structure of BAMA, and so the triplet state of BAMA can be matched with the resonant energy level of the Eu(iii) ion. While the bonded ligand BAGL can effectively sensitize the fluorescence emission of Tb(iii) ions, the binary complex PSF-(BAGL) 3 -Tb(iii) exhibits very strong green fluorescence under UV light. The reason is that a smaller conjugate π-bond system is contained in the structure of BAGL and there is a good energy level matching between the triplet state of BAGL and the resonant energy level of the Tb(iii) ion. The fluorescence intensities of the two ternary complexes, PSF-(BAMA) 3 -Eu(iii)-(Phen) 1 (phenanthroline, Phen) and PSF-(BAGL) 3 -Tb(iii)-(Phen) 1 , are much stronger than that of the corresponding binary complex because Phen as the second ligand has two effects, the effect of synergistic coordination with the first ligand and the effect of replacing the coordinated water around the central ion, and it has been confirmed by fluorescence spectroscopy and thermogravimetric analysis.

Impact of Time-of-Flight on PET Tumor Detection

PubMed Central

Kadrmas, Dan J.; Casey, Michael E.; Conti, Maurizio; Jakoby, Bjoern W.; Lois, Cristina; Townsend, David W.

2009-01-01

Time-of-flight (TOF) PET uses very fast detectors to improve localization of events along coincidence lines-of-response. This information is then utilized to improve the tomographic reconstruction. This work evaluates the effect of TOF upon an observer's performance for detecting and localizing focal warm lesions in noisy PET images. Methods An advanced anthropomorphic lesion-detection phantom was scanned 12 times over 3 days on a prototype TOF PET/CT scanner (Siemens Medical Solutions). The phantom was devised to mimic whole-body oncologic 18F-FDG PET imaging, and a number of spheric lesions (diameters 6–16 mm) were distributed throughout the phantom. The data were reconstructed with the baseline line-of-response ordered-subsets expectation-maximization algorithm, with the baseline algorithm plus point spread function model (PSF), baseline plus TOF, and with both PSF+TOF. The lesion-detection performance of each reconstruction was compared and ranked using localization receiver operating characteristics (LROC) analysis with both human and numeric observers. The phantom results were then subjectively compared to 2 illustrative patient scans reconstructed with PSF and with PSF+TOF. Results Inclusion of TOF information provides a significant improvement in the area under the LROC curve compared to the baseline algorithm without TOF data (P = 0.002), providing a degree of improvement similar to that obtained with the PSF model. Use of both PSF+TOF together provided a cumulative benefit in lesion-detection performance, significantly outperforming either PSF or TOF alone (P < 0.002). Example patient images reflected the same image characteristics that gave rise to improved performance in the phantom data. Conclusion Time-of-flight PET provides a significant improvement in observer performance for detecting focal warm lesions in a noisy background. These improvements in image quality can be expected to improve performance for the clinical tasks of detecting lesions and staging disease. Further study in a large clinical population is warranted to assess the benefit of TOF for various patient sizes and count levels, and to demonstrate effective performance in the clinical environment. PMID:19617317

Image processing tools dedicated to quantification in 3D fluorescence microscopy

NASA Astrophysics Data System (ADS)

Dieterlen, A.; De Meyer, A.; Colicchio, B.; Le Calvez, S.; Haeberlé, O.; Jacquey, S.

2006-05-01

3-D optical fluorescent microscopy now becomes an efficient tool for the volume investigation of living biological samples. Developments in instrumentation have permitted to beat off the conventional Abbe limit. In any case the recorded image can be described by the convolution equation between the original object and the Point Spread Function (PSF) of the acquisition system. Due to the finite resolution of the instrument, the original object is recorded with distortions and blurring, and contaminated by noise. This induces that relevant biological information cannot be extracted directly from raw data stacks. If the goal is 3-D quantitative analysis, then to assess optimal performance of the instrument and to ensure the data acquisition reproducibility, the system characterization is mandatory. The PSF represents the properties of the image acquisition system; we have proposed the use of statistical tools and Zernike moments to describe a 3-D PSF system and to quantify the variation of the PSF. This first step toward standardization is helpful to define an acquisition protocol optimizing exploitation of the microscope depending on the studied biological sample. Before the extraction of geometrical information and/or intensities quantification, the data restoration is mandatory. Reduction of out-of-focus light is carried out computationally by deconvolution process. But other phenomena occur during acquisition, like fluorescence photo degradation named "bleaching", inducing an alteration of information needed for restoration. Therefore, we have developed a protocol to pre-process data before the application of deconvolution algorithms. A large number of deconvolution methods have been described and are now available in commercial package. One major difficulty to use this software is the introduction by the user of the "best" regularization parameters. We have pointed out that automating the choice of the regularization level; also greatly improves the reliability of the measurements although it facilitates the use. Furthermore, to increase the quality and the repeatability of quantitative measurements a pre-filtering of images improves the stability of deconvolution process. In the same way, the PSF prefiltering stabilizes the deconvolution process. We have shown that Zemike polynomials can be used to reconstruct experimental PSF, preserving system characteristics and removing the noise contained in the PSF.

Sexy splicing: regulatory interplays governing sex determination from Drosophila to mammals.

PubMed

Lalli, Enzo; Ohe, Kenji; Latorre, Elisa; Bianchi, Marco E; Sassone-Corsi, Paolo

2003-02-01

A remarkable array of strategies is used to produce sexual differentiation in different species. Complex gene hierarchies govern sex determination pathways, as exemplified by the classic D. melanogaster paradigm, where an interplay of transcriptional, splicing and translational mechanisms operate. Molecular studies support the hypothesis that genetic sex determination pathways evolved in reverse order, from downstream to upstream genes, in the cascade. The recent identification of a role for the key regulatory factors SRY and WT1(+KTS) in pre-mRNA splicing indicates that important steps in the mammalian sex determination process are likely to operate at the post-transcriptional level.

In vivo transcriptional profile analysis reveals RNA splicing and chromatin remodeling as prominent processes for adult neurogenesis.

PubMed

Lim, Daniel A; Suárez-Fariñas, Mayte; Naef, Felix; Hacker, Coleen R; Menn, Benedicte; Takebayashi, Hirohide; Magnasco, Marcelo; Patil, Nila; Alvarez-Buylla, Arturo

2006-01-01

Neural stem cells and neurogenesis persist in the adult mammalian brain subventricular zone (SVZ). Cells born in the rodent SVZ migrate to the olfactory bulb (Ob) where they differentiate into interneurons. To determine the gene expression and functional profile of SVZ neurogenesis, we performed three complementary sets of transcriptional analysis experiments using Affymetrix GeneChips: (1) comparison of adult mouse SVZ and Ob gene expression profiles with those of the striatum, cerebral cortex, and hippocampus; (2) profiling of SVZ stem cells and ependyma isolated by fluorescent-activated cell sorting (FACS); and (3) analysis of gene expression changes during in vivo SVZ regeneration after anti-mitotic treatment. Gene Ontology (GO) analysis of data from these three separate approaches showed that in adult SVZ neurogenesis, RNA splicing and chromatin remodeling are biological processes as statistically significant as cell proliferation, transcription, and neurogenesis. In non-neurogenic brain regions, RNA splicing and chromatin remodeling were not prominent processes. Fourteen mRNA splicing factors including Sf3b1, Sfrs2, Lsm4, and Khdrbs1/Sam68 were detected along with 9 chromatin remodeling genes including Mll, Bmi1, Smarcad1, Baf53a, and Hat1. We validated the transcriptional profile data with Northern blot analysis and in situ hybridization. The data greatly expand the catalogue of cell cycle components, transcription factors, and migration genes for adult SVZ neurogenesis and reveal RNA splicing and chromatin remodeling as prominent biological processes for these germinal cells.

Antisense-based RNA therapy of factor V deficiency: in vitro and ex vivo rescue of a F5 deep-intronic splicing mutation.

PubMed

Nuzzo, Francesca; Radu, Claudia; Baralle, Marco; Spiezia, Luca; Hackeng, Tilman M; Simioni, Paolo; Castoldi, Elisabetta

2013-11-28

Antisense molecules are emerging as a powerful tool to correct splicing defects. Recently, we identified a homozygous deep-intronic mutation (F5 c.1296+268A>G) activating a cryptic donor splice site in a patient with severe coagulation factor V (FV) deficiency and life-threatening bleeding episodes. Here, we assessed the ability of 2 mutation-specific antisense molecules (a morpholino oligonucleotide [MO] and an engineered U7 small nuclear RNA [snRNA]) to correct this splicing defect. COS-1 and HepG2 cells transfected with a F5 minigene construct containing the patient's mutation expressed aberrant messenger RNA (mRNA) in excess of normal mRNA. Treatment with mutation-specific antisense MO (1-5 µM) or a construct expressing antisense U7snRNA (0.25-2 µg) dose-dependently increased the relative amount of correctly spliced mRNA by 1 to 2 orders of magnitude, whereas control MO and U7snRNA were ineffective. Patient-derived megakaryocytes obtained by differentiation of circulating progenitor cells did not express FV, but became positive for FV at immunofluorescence staining after administration of antisense MO or U7snRNA. However, treatment adversely affected cell viability, mainly because of the transfection reagents used to deliver the antisense molecules. Our data provide in vitro and ex vivo proof of principle for the efficacy of RNA therapy in severe FV deficiency, but additional cytotoxicity studies are warranted.

SRSF3 represses the expression of PDCD4 protein by coordinated regulation of alternative splicing, export and translation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Seung Kuk; Jeong, Sunjoo, E-mail: sjsj@dankook.ac.kr

2016-02-05

Gene expression is regulated at multiple steps, such as transcription, splicing, export, degradation and translation. Considering diverse roles of SR proteins, we determined whether the tumor-related splicing factor SRSF3 regulates the expression of the tumor-suppressor protein, PDCD4, at multiple steps. As we have reported previously, knockdown of SRSF3 increased the PDCD4 protein level in SW480 colon cancer cells. More interestingly, here we showed that the alternative splicing and the nuclear export of minor isoforms of pdcd4 mRNA were repressed by SRSF3, but the translation step was unaffected. In contrast, only the translation step of the major isoform of pdcd4 mRNAmore » was repressed by SRSF3. Therefore, overexpression of SRSF3 might be relevant to the repression of all isoforms of PDCD4 protein levels in most types of cancer cell. We propose that SRSF3 could act as a coordinator of the expression of PDCD4 protein via two mechanisms on two alternatively spliced mRNA isoforms.« less

Impact of Selected Parameters on the Fatigue Strength of Splices on Multiply Textile Conveyor Belts

NASA Astrophysics Data System (ADS)

Bajda, Mirosław; Błażej, Ryszard; Hardygóra, Monika

2016-10-01

Splices are the weakest points in the conveyor belt loop. The strength of these joints, and thus their design as well as the method and quality of splicing, determine the strength of the whole conveyor belt loop. A special zone in a splice exists, where the stresses in the adjacent plies or cables differ considerably from each other. This results in differences in the elongation of these elements and in additional shearing stresses in the rubber layer. The strength of the joints depends on several factors, among others on the parameters of the joined belt, on the connecting layer and the technology of joining, as well as on the materials used to make the joint. The strength of the joint constitutes a criterion for the selection of a belt suitable for the operating conditions, and therefore methods of testing such joints are of great importance. This paper presents the method of testing fatigue strength of splices made on multi-ply textile conveyor belts and the results of these studies.

Blind deconvolution of 2-D and 3-D fluorescent micrographs

NASA Astrophysics Data System (ADS)

Krishnamurthi, Vijaykumar; Liu, Yi-Hwa; Holmes, Timothy J.; Roysam, Badrinath; Turner, James N.

1992-06-01

This paper presents recent results of our reconstructions of 3-D data from Drosophila chromosomes as well as our simulations with a refined version of the algorithm used in the former. It is well known that the calibration of the point spread function (PSF) of a fluorescence microscope is a tedious process and involves esoteric techniques in most cases. This problem is further compounded in the case of confocal microscopy where the measured intensities are usually low. A number of techniques have been developed to solve this problem, all of which are methods in blind deconvolution. These are so called because the measured PSF is not required in the deconvolution of degraded images from any optical system. Our own efforts in this area involved the maximum likelihood (ML) method, the numerical solution to which is obtained by the expectation maximization (EM) algorithm. Based on the reasonable early results obtained during our simulations with 2-D phantoms, we carried out experiments with real 3-D data. We found that the blind deconvolution method using the ML approach gave reasonable reconstructions. Next we tried to perform the reconstructions using some 2-D data, but we found that the results were not encouraging. We surmised that the poor reconstructions were primarily due to the large values of dark current in the input data. This, coupled with the fact that we are likely to have similar data with considerable dark current from a confocal microscope prompted us to look into ways of constraining the solution of the PSF. We observed that in the 2-D case, the reconstructed PSF has a tendency to retain values larger than those of the theoretical PSF in regions away from the center (outside of those we considered to be its region of support). This observation motivated us to apply an upper bound constraint on the PSF in these regions. Furthermore, we constrain the solution of the PSF to be a bandlimited function, as in the case in the true situation. We have derived two separate approaches for implementing the constraint. One approach involves the mathematical rigors of Lagrange multipliers. This approach is discussed in another paper. The second approach involves an adaptation of the Gershberg Saxton algorithm, which ensures bandlimitedness and non-negativity of the PSF. Although the latter approach is mathematically less rigorous than the former, we currently favor it because it has a simpler implementation on a computer and has smaller memory requirements. The next section describes briefly the theory and derivation of these constraint equations using Lagrange multipliers.

Characterization of Two Late-Stage Enzymes Involved in Fosfomycin Biosynthesis in Pseudomonads.

PubMed

Olivares, Philip; Ulrich, Emily C; Chekan, Jonathan R; van der Donk, Wilfred A; Nair, Satish K

2017-02-17

The broad-spectrum phosphonate antibiotic fosfomycin is currently in use for clinical treatment of infections caused by both Gram-positive and Gram-negative uropathogens. The antibiotic is biosynthesized by various streptomycetes, as well as by pseudomonads. Notably, the biosynthetic strategies used by the two genera share only two steps: the first step in which primary metabolite phosphoenolpyruvate (PEP) is converted to phosphonopyruvate (PnPy) and the terminal step in which 2-hydroxypropylphosphonate (2-HPP) is converted to fosfomycin. Otherwise, distinct enzymatic paths are employed. Here, we biochemically confirm the last two steps in the fosfomycin biosynthetic pathway of Pseudomonas syringae PB-5123, showing that Psf3 performs the reduction of 2-oxopropylphosphonate (2-OPP) to (S)-2-HPP, followed by the Psf4-catalyzed epoxidation of (S)-2-HPP to fosfomycin. Psf4 can also accept (R)-2-HPP as a substrate but instead performs an oxidation to make 2-OPP. We show that the combined activities of Psf3 and Psf4 can be used to convert racemic 2-HPP to fosfomycin in an enantioconvergent process. X-ray structures of each enzyme with bound substrates provide insights into the stereospecificity of each conversion. These studies shed light on the reaction mechanisms of the two terminal enzymes in a distinct pathway employed by pseudomonads for the production of a potent antimicrobial agent.

Effect of incorporating graphene oxide and surface imprinting on polysulfone membranes on flux, hydrophilicity and rejection of salt and polycyclic aromatic hydrocarbons from water

NASA Astrophysics Data System (ADS)

Kibechu, Rose Waithiegeni; Ndinteh, Derek Tantoh; Msagati, Titus Alfred Makudali; Mamba, Bhekie Briliance; Sampath, S.

2017-08-01

We report a significant enhancement of hydrophillity of polysulfone (Psf) membranes after modification with graphene oxide (GO) as a filler followed by surface imprinting on the surface of GO/Psf composite imprinted membranes (CIMs). The surface imprinting on the GO-Psf membrane was employed in order to enhance its selectivity towards polycyclic aromatic hydrocarbons (PAHs) in water. The CIMs were prepared through a process of phase inversion of a mixture of graphene oxide and polysulfone (Psf) in N-methylpyrrolidone (NMP). Fourier-transform spectroscopy (FT-IR) of the imprinted showed new peaks at 935 cm-1 and 1638 cm-1 indicating success in surface imprinting on the GO-Psf membrane. The CIM also showed improvement in flux from 8.56 LM-2 h-1 of unmodified polysulfone membrane to 15.3 LM-2 h-1 in the CIM, salt rejection increased from 57.2 ± 4.2% of polysulfone membrane to 76 ± 4.5%. The results obtained from the contact angle measurements showed a decrease with increase in GO content from 72 ± 2.7% of neat polysulfone membrane to 62.3 ± 2.1% of CIM indicating an improvement in surface hydrophilicity. The results from this study shows that, it is possible to improve the hydrophilicity of the membranes without affecting the performance of the membranes.

The effect of time-of-flight and point spread function modeling on 82Rb myocardial perfusion imaging of obese patients.

PubMed

Dasari, Paul K R; Jones, Judson P; Casey, Michael E; Liang, Yuanyuan; Dilsizian, Vasken; Smith, Mark F

2018-06-15

The effect of time-of-flight (TOF) and point spread function (PSF) modeling in image reconstruction has not been well studied for cardiac PET. This study assesses their separate and combined influence on 82 Rb myocardial perfusion imaging in obese patients. Thirty-six obese patients underwent rest-stress 82 Rb cardiac PET. Images were reconstructed with and without TOF and PSF modeling. Perfusion was quantitatively compared using the AHA 17-segment model for patients grouped by BMI, cross-sectional body area in the scanner field of view, gender, and left ventricular myocardial volume. Summed rest scores (SRS), summed stress scores (SSS), and summed difference scores (SDS) were compared. TOF improved polar map visual uniformity and increased septal wall perfusion by up to 10%. This increase was greater for larger patients, more evident for patients grouped by cross-sectional area than by BMI, and more prominent for females. PSF modeling increased perfusion by about 1.5% in all cardiac segments. TOF modeling generally decreased SRS and SSS with significant decreases between 2.4 and 3.0 (P < .05), which could affect risk stratification; SDS remained about the same. With PSF modeling, SRS, SSS, and SDS were largely unchanged. TOF and PSF modeling affect regional and global perfusion, SRS, and SSS. Clinicians should consider these effects and gender-dependent differences when interpreting 82 Rb perfusion studies.

A detailed transcript-level probe annotation reveals alternative splicing based microarray platform differences

PubMed Central

Lee, Joseph C; Stiles, David; Lu, Jun; Cam, Margaret C

2007-01-01

Background Microarrays are a popular tool used in experiments to measure gene expression levels. Improving the reproducibility of microarray results produced by different chips from various manufacturers is important to create comparable and combinable experimental results. Alternative splicing has been cited as a possible cause of differences in expression measurements across platforms, though no study to this point has been conducted to show its influence in cross-platform differences. Results Using probe sequence data, a new microarray probe/transcript annotation was created based on the AceView Aug05 release that allowed for the categorization of genes based on their expression measurements' susceptibility to alternative splicing differences across microarray platforms. Examining gene expression data from multiple platforms in light of the new categorization, genes unsusceptible to alternative splicing differences showed higher signal agreement than those genes most susceptible to alternative splicing differences. The analysis gave rise to a different probe-level visualization method that can highlight probe differences according to transcript specificity. Conclusion The results highlight the need for detailed probe annotation at the transcriptome level. The presence of alternative splicing within a given sample can affect gene expression measurements and is a contributing factor to overall technical differences across platforms. PMID:17708771

Global analysis of epigenetic regulation of gene expression in response to drought stress in Sorghum.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reddy, Anireddy; Ben-Hur, Asa

Abiotic stresses including drought are major limiting factors of crop yields and cause significant crop losses. Acquisition of stress tolerance to abiotic stresses requires coordinated regulation of a multitude of biochemical and physiological changes, and most of these changes depend on alterations in gene expression. The goal of this work is to perform global analysis of differential regulation of gene expression and alternative splicing, and their relationship with chromatin landscape in drought sensitive and tolerant cultivars. our Iso-Seq study revealed transcriptome-wide full-length isoforms at an unprecedented scale with over 11000 novel splice isoforms. Additionally, we uncovered alternative polyadenylation sites ofmore » ~11000 expressed genes and many novel genes. Overall, Iso-Seq results greatly enhanced sorghum gene annotations that are not only useful in analyzing all our RNA-seq, ChIP-seq and ATAC-seq data but also serve as a great resource to the plant biology community. Our studies identified differentially expressed genes and splicing events that are correlated with the drought-resistant phenotype. An association between alternative splicing and chromatin accessibility was also revealed. Several computational tools developed here (TAPIS and iDiffIR) have been made freely available to the research community in analyzing alternative splicing and differential alternative splicing.« less

Computational Identification of Tissue-Specific Splicing Regulatory Elements in Human Genes from RNA-Seq Data.

PubMed

Badr, Eman; ElHefnawi, Mahmoud; Heath, Lenwood S

2016-01-01

Alternative splicing is a vital process for regulating gene expression and promoting proteomic diversity. It plays a key role in tissue-specific expressed genes. This specificity is mainly regulated by splicing factors that bind to specific sequences called splicing regulatory elements (SREs). Here, we report a genome-wide analysis to study alternative splicing on multiple tissues, including brain, heart, liver, and muscle. We propose a pipeline to identify differential exons across tissues and hence tissue-specific SREs. In our pipeline, we utilize the DEXSeq package along with our previously reported algorithms. Utilizing the publicly available RNA-Seq data set from the Human BodyMap project, we identified 28,100 differentially used exons across the four tissues. We identified tissue-specific exonic splicing enhancers that overlap with various previously published experimental and computational databases. A complicated exonic enhancer regulatory network was revealed, where multiple exonic enhancers were found across multiple tissues while some were found only in specific tissues. Putative combinatorial exonic enhancers and silencers were discovered as well, which may be responsible for exon inclusion or exclusion across tissues. Some of the exonic enhancers are found to be co-occurring with multiple exonic silencers and vice versa, which demonstrates a complicated relationship between tissue-specific exonic enhancers and silencers.

The bromodomain protein BRD4 regulates splicing during heat shock

PubMed Central

Hussong, Michelle; Kaehler, Christian; Kerick, Martin; Grimm, Christina; Franz, Alexandra; Timmermann, Bernd; Welzel, Franziska; Isensee, Jörg; Hucho, Tim; Krobitsch, Sylvia; Schweiger, Michal R.

2017-01-01

The cellular response to heat stress is an ancient and evolutionarily highly conserved defence mechanism characterised by the transcriptional up-regulation of cyto-protective genes and a partial inhibition of splicing. These features closely resemble the proteotoxic stress response during tumor development. The bromodomain protein BRD4 has been identified as an integral member of the oxidative stress as well as of the inflammatory response, mainly due to its role in the transcriptional regulation process. In addition, there are also several lines of evidence implicating BRD4 in the splicing process. Using RNA-sequencing we found a significant increase in splicing inhibition, in particular intron retentions (IR), following heat treatment in BRD4-depleted cells. This leads to a decrease of mRNA abundancy of the affected transcripts, most likely due to premature termination codons. Subsequent experiments revealed that BRD4 interacts with the heat shock factor 1 (HSF1) such that under heat stress BRD4 is recruited to nuclear stress bodies and non-coding SatIII RNA transcripts are up-regulated. These findings implicate BRD4 as an important regulator of splicing during heat stress. Our data which links BRD4 to the stress induced splicing process may provide novel mechanisms of BRD4 inhibitors in regard to anti-cancer therapies. PMID:27536004

Nuclear Organization and Myt1 Interaction in Transcriptional Control of Neural Cell Differentiation

DTIC Science & Technology

2002-01-01

secreted from the notochord and floor plate [4]. Oligodendrocytes also respond to cell contact- dependent interactions from the notch-signaling pathway...appendix A 1 mature oligodendrocytes sending out multiple processes to begin myelinating axons primarily during the postnatal period of...snRNA transcription [32]. 7 Gene regulation also occurs post-transcriptionally in processes such as RNA splicing. Many splicing factors are

Fanconi anemia FANCD2 and FANCI proteins regulate the nuclear dynamics of splicing factors.

PubMed

Moriel-Carretero, María; Ovejero, Sara; Gérus-Durand, Marie; Vryzas, Dimos; Constantinou, Angelos

2017-12-04

Proteins disabled in the cancer-prone disorder Fanconi anemia (FA) ensure the maintenance of chromosomal stability during DNA replication. FA proteins regulate replication dynamics, coordinate replication-coupled repair of interstrand DNA cross-links, and mitigate conflicts between replication and transcription. Here we show that FANCI and FANCD2 associate with splicing factor 3B1 (SF3B1), a key spliceosomal protein of the U2 small nuclear ribonucleoprotein (U2 snRNP). FANCI is in close proximity to SF3B1 in the nucleoplasm of interphase and mitotic cells. Furthermore, we find that DNA replication stress induces the release of SF3B1 from nuclear speckles in a manner that depends on FANCI and on the activity of the checkpoint kinase ATR. In chromatin, both FANCD2 and FANCI associate with SF3B1, prevent accumulation of postcatalytic intron lariats, and contribute to the timely eviction of splicing factors. We propose that FANCD2 and FANCI contribute to the organization of functional domains in chromatin, ensuring the coordination of DNA replication and cotranscriptional processes. © 2017 Moriel-Carretero et al.

Functional analyses of interacting factors involved in both pre-mRNA splicing and cell cycle progression in Saccharomyces cerevisiae.

PubMed Central

Russell, C S; Ben-Yehuda, S; Dix, I; Kupiec, M; Beggs, J D

2000-01-01

Through a genetic screen to search for factors that interact with Prp17/Cdc40p, a protein involved in both cell cycle progression and pre-mRNA splicing, we identify three novel factors, which we call Syf1p, Syf2p, and Syf3 (SYnthetic lethal with cdc Forty). Here we present evidence that all three proteins are spliceosome associated, that they associate weakly or transiently with U6 and U5 snRNAs, and that Syf1p and Syf3p (also known as Clf1p) are required for pre-mRNA splicing. In addition we show that depletion of Syf1p or Syf3p results in cell cycle arrest at the G2/M transition. Thus, like Prp17/Cdc40p, Syf1p and Syf3p are involved in two distinct cellular processes. We discuss the likelihood that Syf1p, Syf2p, and Syf3p are components of a protein complex that assembles into spliceosomes and also regulates cell cycle progression. PMID:11105756

Functional analyses of interacting factors involved in both pre-mRNA splicing and cell cycle progression in Saccharomyces cerevisiae.

PubMed

Russell, C S; Ben-Yehuda, S; Dix, I; Kupiec, M; Beggs, J D

2000-11-01

Through a genetic screen to search for factors that interact with Prp17/Cdc40p, a protein involved in both cell cycle progression and pre-mRNA splicing, we identify three novel factors, which we call Syf1p, Syf2p, and Syf3 (SYnthetic lethal with cdc Forty). Here we present evidence that all three proteins are spliceosome associated, that they associate weakly or transiently with U6 and U5 snRNAs, and that Syf1p and Syf3p (also known as Clf1p) are required for pre-mRNA splicing. In addition we show that depletion of Syf1p or Syf3p results in cell cycle arrest at the G2/M transition. Thus, like Prp17/Cdc40p, Syf1p and Syf3p are involved in two distinct cellular processes. We discuss the likelihood that Syf1p, Syf2p, and Syf3p are components of a protein complex that assembles into spliceosomes and also regulates cell cycle progression.

Novel splice site mutation in the growth hormone receptor gene in Turkish patients with Laron-type dwarfism.

PubMed

Arman, Ahmet; Ozon, Alev; Isguven, Pinar S; Coker, Ajda; Peker, Ismail; Yordam, Nursen

2008-01-01

Growth hormone (GH) is involved in growth, and fat and carbohydrate metabolism. Interaction of GH with the GH receptor (GHR) is necessary for systemic and local production of insulin-like growth factor-I (IGF-I) which mediates GH actions. Mutations in the GHR cause severe postnatal growth failure; the disorder is an autosomal recessive genetic disease resulting in GH insensitivity, called Laron syndrome. It is characterized by dwarfism with elevated serum GH and low levels of IGF-I. We analyzed the GHR gene for mutations and polymorphisms in eight patients with Laron-type dwarfism from six families. We found three missense mutations (S40L, V125A, I526L), one nonsense mutation (W157X), and one splice site mutation in the extracellular domain of GHR. Furthermore, G168G and exon 3 deletion polymorphisms were detected in patients with Laron syndrome. The splice site mutation, which is a novel mutation, was located at the donor splice site of exon 2/ intron 2 within GHR. Although this mutation changed the highly conserved donor splice site consensus sequence GT to GGT by insertion of a G residue, the intron splicing between exon 2 and exon 3 was detected in the patient. These results imply that the splicing occurs arthe GT site in intron 2, leaving the extra inserted G residue at the end of exon 2, thus changing the open reading frame of GHR resulting in a premature termination codon in exon 3.

4β-Hydroxywithanolide E Modulates Alternative Splicing of Apoptotic Genes in Human Hepatocellular Carcinoma Huh-7 Cells.

PubMed

Lee, Chien-Chin; Chang, Wen-Hsin; Chang, Ya-Sian; Liu, Ting-Yuan; Chen, Yu-Chia; Wu, Yang-Chang; Chang, Jan-Gowth

2017-08-04

Alternative splicing is a mechanism for increasing protein diversity from a limited number of genes. Studies have demonstrated that aberrant regulation in the alternative splicing of apoptotic gene transcripts may contribute to the development of cancer. In this study, we isolated 4β-Hydroxywithanolide E (4bHWE) from the traditional herb Physalis peruviana and investigated its biological effect in cancer cells. The results demonstrated that 4bHWE modulates the alternative splicing of various apoptotic genes, including HIPK3, SMAC/DIABLO, and SURVIVIN. We also discovered that the levels of SRSF1 phospho-isoform were decreased and the levels of H3K36me3 were increased in 4bHWE treatment. Knockdown experiments revealed that the splicing site selection of SMAC/DIABLO could be mediated by changes in the level of H3K36me3 in 4bHWE-treated cells. Furthermore, we extended our study to apoptosis-associated molecules, and detected increased levels of poly ADP-ribose polymerase cleavage and the active form of CASPASE-3 in 4bHWE-induced apoptosis. In vivo experiments indicated that the treatment of tumor-bearing mice with 4bHWE resulted in a marked decrease in tumor size. This study is the first to demonstrate that 4bHWE affects alternative splicing by modulating splicing factors and histone modifications, and provides a novel view of the antitumor mechanism of 4bHWE.

A contracted DNA repeat in LHX3 intron 5 is associated with aberrant splicing and pituitary dwarfism in German shepherd dogs.

PubMed

Voorbij, Annemarie M W Y; van Steenbeek, Frank G; Vos-Loohuis, Manon; Martens, Ellen E C P; Hanson-Nilsson, Jeanette M; van Oost, Bernard A; Kooistra, Hans S; Leegwater, Peter A

2011-01-01

Dwarfism in German shepherd dogs is due to combined pituitary hormone deficiency of unknown genetic cause. We localized the recessively inherited defect by a genome wide approach to a region on chromosome 9 with a lod score of 9.8. The region contains LHX3, which codes for a transcription factor essential for pituitary development. Dwarfs have a deletion of one of six 7 bp repeats in intron 5 of LHX3, reducing the intron size to 68 bp. One dwarf was compound heterozygous for the deletion and an insertion of an asparagine residue in the DNA-binding homeodomain of LHX3, suggesting involvement of the gene in the disorder. An exon trapping assay indicated that the shortened intron is not spliced efficiently, probably because it is too small. We applied bisulfite conversion of cytosine to uracil in RNA followed by RT-PCR to analyze the splicing products. The aberrantly spliced RNA molecules resulted from either skipping of exon 5 or retention of intron 5. The same splicing defects were observed in cDNA derived from the pituitary of dwarfs. A survey of similarly mutated introns suggests that there is a minimal distance requirement between the splice donor and branch site of 50 nucleotides. In conclusion, a contraction of a DNA repeat in intron 5 of canine LHX3 leads to deficient splicing and is associated with pituitary dwarfism.

A Contracted DNA Repeat in LHX3 Intron 5 Is Associated with Aberrant Splicing and Pituitary Dwarfism in German Shepherd Dogs

PubMed Central

Voorbij, Annemarie M. W. Y.; van Steenbeek, Frank G.; Vos-Loohuis, Manon; Martens, Ellen E. C. P.; Hanson-Nilsson, Jeanette M.; van Oost, Bernard A.; Kooistra, Hans S.; Leegwater, Peter A.

2011-01-01

Dwarfism in German shepherd dogs is due to combined pituitary hormone deficiency of unknown genetic cause. We localized the recessively inherited defect by a genome wide approach to a region on chromosome 9 with a lod score of 9.8. The region contains LHX3, which codes for a transcription factor essential for pituitary development. Dwarfs have a deletion of one of six 7 bp repeats in intron 5 of LHX3, reducing the intron size to 68 bp. One dwarf was compound heterozygous for the deletion and an insertion of an asparagine residue in the DNA-binding homeodomain of LHX3, suggesting involvement of the gene in the disorder. An exon trapping assay indicated that the shortened intron is not spliced efficiently, probably because it is too small. We applied bisulfite conversion of cytosine to uracil in RNA followed by RT-PCR to analyze the splicing products. The aberrantly spliced RNA molecules resulted from either skipping of exon 5 or retention of intron 5. The same splicing defects were observed in cDNA derived from the pituitary of dwarfs. A survey of similarly mutated introns suggests that there is a minimal distance requirement between the splice donor and branch site of 50 nucleotides. In conclusion, a contraction of a DNA repeat in intron 5 of canine LHX3 leads to deficient splicing and is associated with pituitary dwarfism. PMID:22132174

Detecting Near-Earth Objects Using Cross-Correlation with a Point Spread Function

DTIC Science & Technology

2009-03-01

greater than .001 seconds [Goodman, 2000]. Cross-Correlation Cross-Correlation measures the strength and direction of the linear relationship between...real(ifft2(fftshift(otf_long)))); %normalize point spread funtion 55 if (Corner == 1) psf_source = makeshift(psf*source_img(ccd_x/2,ccd_y/2

Posterior fusion only for thoracic adolescent idiopathic scoliosis of more than 80 degrees: pedicle screws versus hybrid instrumentation.

PubMed

Di Silvestre, Mario; Bakaloudis, Georgios; Lolli, Francesco; Vommaro, Francesco; Martikos, Konstantinos; Parisini, Patrizio

2008-10-01

The treatment of thoracic adolescent idiopathic scoliosis (AIS) of more than 80 degrees traditionally consisted of a combined procedure, an anterior release performed through an open thoracotomy followed by a posterior fusion. Recently, some studies have reassessed the role of posterior fusion only as treatment for severe thoracic AIS; the correction rate of the thoracic curves was comparable to most series of combined anterior and posterior surgery, with shorter surgery time and without the negative effect on pulmonary function of anterior transthoracic exposure. Compared with other studies published so far on the use of posterior fusion alone for severe thoracic AIS, the present study examines a larger group of patients (52 cases) reviewed at a longer follow-up (average 6.7 years, range 4.5-8.5 years). The aim of the study was to evaluate the clinical and radiographic outcome of surgical treatment for severe thoracic (>80 degrees) AIS treated with posterior spinal fusion alone, and compare comprehensively the results of posterior fusion with a hybrid construct (proximal hooks and distal pedicle screws) versus a pedicle screw instrumentation. All patients (n = 52) with main thoracic AIS curves greater than 80 degrees (Lenke type 1, 2, 3, and 4), surgically treated between 1996 and 2000 at one institution, by posterior spinal fusion either with hybrid instrumentation (PSF-H group; n = 27 patients), or with pedicle screw-only construct (PSF-S group; n = 25 patients) were reviewed. There were no differences between the two groups in terms of age, Risser's sign, Cobb preoperative main thoracic (MT) curve magnitude (PSF-H: 92 degrees vs. PSF-S: 88 degrees), or flexibility on bending films (PSF-H: 27% vs. PSF-S: 25%). Statistical analysis was performed using the t test (paired and unpaired), Wilcoxon test for non-parametric paired analysis, and the Mann-Whitney test for non-parametric unpaired analysis. At the last follow-up, the PSF-S group, when compared to the PSF-H group had a final MT correction rate of 52.4 versus 44.52% (P = 0.001), with a loss of -1.9 degrees versus -11.3 degrees (P = 0.0005), a TL/L correction of 50 versus 43% (ns), a greater correction of the lowest instrumented vertebra translation (-1.00 vs. -0.54 cm; P = 0.04), and tilt (-19 degrees vs. -10 degrees; P = 0.005) on the coronal plane. There were no statistically significant differences in sagittal and global coronal alignment between the two groups (C7-S1 offset: PSF-H = 0.5 cm vs. PSF-S = 0 cm). In the hybrid series (27 patients) surgery-related complications necessitated three revision surgeries, whereas in the screw group (25 patients) one revision surgery was performed. No neurological complications or deep wound infection occurred in this series. In conclusion, posterior spinal fusion for severe thoracic AIS with pedicle screws only, when compared to hybrid construct, allowed a greater coronal correction of both main thoracic and secondary lumbar curves, less loss of the postoperative correction achieved, and fewer revision surgeries. Posterior-only fusion with pedicle screws enabled a good and stable correction of severe scoliosis. However, severe curves may be amenable to hybrid instrumentation that produced analogous results to the screws-only constructs concerning patient satisfaction; at the latest follow-up, SRS-30 and SF-36 scores did not show any statistical differences between the two groups.

A hybrid method for synthetic aperture ladar phase-error compensation

NASA Astrophysics Data System (ADS)

Hua, Zhili; Li, Hongping; Gu, Yongjian

2009-07-01

As a high resolution imaging sensor, synthetic aperture ladar data contain phase-error whose source include uncompensated platform motion and atmospheric turbulence distortion errors. Two previously devised methods, rank one phase-error estimation algorithm and iterative blind deconvolution are reexamined, of which a hybrid method that can recover both the images and PSF's without any a priori information on the PSF is built to speed up the convergence rate by the consideration in the choice of initialization. To be integrated into spotlight mode SAL imaging model respectively, three methods all can effectively reduce the phase-error distortion. For each approach, signal to noise ratio, root mean square error and CPU time are computed, from which we can see the convergence rate of the hybrid method can be improved because a more efficient initialization set of blind deconvolution. Moreover, by making a further discussion of the hybrid method, the weight distribution of ROPE and IBD is found to be an important factor that affects the final result of the whole compensation process.

Point spread function engineering for iris recognition system design.

PubMed

Ashok, Amit; Neifeld, Mark A

2010-04-01

Undersampling in the detector array degrades the performance of iris-recognition imaging systems. We find that an undersampling of 8 x 8 reduces the iris-recognition performance by nearly a factor of 4 (on CASIA iris database), as measured by the false rejection ratio (FRR) metric. We employ optical point spread function (PSF) engineering via a Zernike phase mask in conjunction with multiple subpixel shifted image measurements (frames) to mitigate the effect of undersampling. A task-specific optimization framework is used to engineer the optical PSF and optimize the postprocessing parameters to minimize the FRR. The optimized Zernike phase enhanced lens (ZPEL) imager design with one frame yields an improvement of nearly 33% relative to a thin observation module by bounded optics (TOMBO) imager with one frame. With four frames the optimized ZPEL imager achieves a FRR equal to that of the conventional imager without undersampling. Further, the ZPEL imager design using 16 frames yields a FRR that is actually 15% lower than that obtained with the conventional imager without undersampling.

CIR, a corepressor of CBF1, binds to PAP-1 and effects alternative splicing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maita, Hiroshi; CREST, Japan Science and Technology Corporation, Saitama 332-0012; Kitaura, Hirotake

2005-02-15

We have reported that PAP-1, a product of a causative gene for autosomal retinitis pigmentosa, plays a role in splicing. In this study, CIR, a protein originally identified as a CBF1-interacting protein and reported to act as a transcriptional corepressor, was identified as a PAP-1 binding protein and its function as a splicing factor was investigated. In addition to a basic lysine and acidic serine-rich (BA) domain and a zinc knuckle-like motif, CIR has an arginine/serine dipeptide repeat (RS) domain in its C terminal region. The RS domain has been reported to be present in the superfamily of SR proteins,more » which are involved in splicing reactions. We generated CIR mutants with deletions of each BA and RS domain and studied their subcellular localizations and interactions with PAP-1 and other SR proteins, including SC35, SF2/ASF, and U2AF{sup 35}. CIR was found to interact with U2AF{sup 35} through the BA domain, with SC35 and SF2/ASF through the RS domain, and with PAP-1 outside the BA domain in vivo and in vitro. CIR was found to be colocalized with SC35 and PAP-1 in nuclear speckles. Then the effect of CIR on splicing was investigated using the E1a minigene as a reporter in HeLa cells. Ectopic expression of CIR with the E1a minigene changed the ratio of spliced isoforms of E1a that were produced by alternative selection of 5'-splice sites. These results indicate that CIR is a member of the family of SR-related proteins and that CIR plays a role in splicing regulation.« less

Predicting Gene Structure Changes Resulting from Genetic Variants via Exon Definition Features.

PubMed

Majoros, William H; Holt, Carson; Campbell, Michael S; Ware, Doreen; Yandell, Mark; Reddy, Timothy E

2018-04-25

Genetic variation that disrupts gene function by altering gene splicing between individuals can substantially influence traits and disease. In those cases, accurately predicting the effects of genetic variation on splicing can be highly valuable for investigating the mechanisms underlying those traits and diseases. While methods have been developed to generate high quality computational predictions of gene structures in reference genomes, the same methods perform poorly when used to predict the potentially deleterious effects of genetic changes that alter gene splicing between individuals. Underlying that discrepancy in predictive ability are the common assumptions by reference gene finding algorithms that genes are conserved, well-formed, and produce functional proteins. We describe a probabilistic approach for predicting recent changes to gene structure that may or may not conserve function. The model is applicable to both coding and noncoding genes, and can be trained on existing gene annotations without requiring curated examples of aberrant splicing. We apply this model to the problem of predicting altered splicing patterns in the genomes of individual humans, and we demonstrate that performing gene-structure prediction without relying on conserved coding features is feasible. The model predicts an unexpected abundance of variants that create de novo splice sites, an observation supported by both simulations and empirical data from RNA-seq experiments. While these de novo splice variants are commonly misinterpreted by other tools as coding or noncoding variants of little or no effect, we find that in some cases they can have large effects on splicing activity and protein products, and we propose that they may commonly act as cryptic factors in disease. The software is available from geneprediction.org/SGRF. bmajoros@duke.edu. Supplementary information is available at Bioinformatics online.

SU-F-T-66: Characteristics of Electron Beams From Varian Trubeam

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dimofte, A; Kennedy, C; Zhu, T

2016-06-15

Purpose: The purpose of this study was to compare the electron beam data between Truebeam and 2300ix Varian accelerators for percent depth dose for broad beam and small circular cutouts, cone factors, head scatter factor as a function of cone size and SSD, phantom scatter factor, blocking factor, distance factor and virtual source position. Methods: Measurements were performed for Truebeam and 2300ix Varian accelerators. The main energies used were: 6, 9, 12, 16 and 20 MeV. PDD was measured at SSD = 100 cm for open beam and small circular cutouts (r = 0.5, 1.0, 1.5, 2.0, 3.0, 4.0 andmore » 6.6cm) for different energies. Measurements to determine the head scatter factor (H) were done as a function of radius for six representative energies and five cone sizes (6, 10, 15, 20 and 25cm2). The phantom scatter factor (PSF) is defined as the ratio of blocking factor in water at reference depth and head scatter factor in air. PSF was measured as a function of radius and electron energy. Distance factor was measured for all energies and cones for three SSD’s (100, 110 and 120cm). Results: The percent depth dose (PDD) was measured for small cutouts of radius r = 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0 and 5.6cm. Blocking factor (BF) was measured for Truebeam and 2300ix accelerators, for different circular cutouts and energies for a 10×10 cone. Cone factors were compared between the two accelerators for different energies and applicator sizes. Conclusion: Cone factors measured for the two accelerator types differ by up to 5% for the largest applicator size. Blocking factors differs by up to 3%, with the largest variation for the smallest field size (0.5cm). Distance factor for different SSD’s differ by up to 4.5%.« less

Nuclear degradation of Wilms tumor 1-associating protein and survivin splice variant switching underlie IGF-1-mediated survival.

PubMed

Small, Theodore W; Pickering, J Geoffrey

2009-09-11

WTAP (Wilms tumor 1-associating protein) is a recently identified nuclear protein that is essential for mouse embryo development. The Drosophila homolog of WTAP, Fl(2)d, regulates pre-mRNA splicing; however, the role of WTAP in mammalian cells is uncertain. To elucidate a context for WTAP action, we screened growth and survival factors for their effects on WTAP expression in vascular smooth muscle cells (SMCs), a cell type previously found to express WTAP dynamically. This revealed that insulin-like growth factor-1 (IGF-1) uniquely reduced WTAP abundance. This decline in WTAP proved to be necessary for IGF-1 to confer its antiapoptotic properties, which were blocked by transducing the WTAP gene into SMCs. WTAP down-regulation by IGF-1 was mediated by an IGF-1 receptor-phosphatidylinositol 3-kinase-Akt signaling axis that directed WTAP degradation via a nuclear 26 S proteasome. Moreover, by promoting the degradation of WTAP, IGF-1 shifted the pre-mRNA splicing program for the survival factor, survivin, to reduce expression of survivin-2B, which is proapoptotic, and increase expression of survivin, which is antiapoptotic. Knockdown of survivin-2B rescued the ability of IGF-1 to promote survival when WTAP was overexpressed. These data uncover a novel regulatory cascade for human SMC survival based on adjusting the nuclear abundance of WTAP to define the splice variant balance among survivin isoforms.

Alteration of the SETBP1 gene and splicing pathway genes SF3B1, U2AF1, and SRSF2 in childhood acute myeloid leukemia.

PubMed

Choi, Hyun-Woo; Kim, Hye-Ran; Baek, Hee-Jo; Kook, Hoon; Cho, Duck; Shin, Jong-Hee; Suh, Soon-Pal; Ryang, Dong-Wook; Shin, Myung-Geun

2015-01-01

Recurrent somatic SET-binding protein 1 (SETBP1) and splicing pathway gene mutations have recently been found in atypical chronic myeloid leukemia and other hematologic malignancies. These mutations have been comprehensively analyzed in adult AML, but not in childhood AML. We investigated possible alteration of the SETBP1, splicing factor 3B subunit 1 (SF3B1), U2 small nuclear RNA auxiliary factor 1 (U2AF1), and serine/arginine-rich splicing factor 2 (SRSF2) genes in childhood AML. Cytogenetic and molecular analyses were performed to reveal chromosomal and genetic alterations. Sequence alterations in the SETBP1, SF3B1, U2AF1, and SRSF2 genes were examined by using direct sequencing in a cohort of 53 childhood AML patients. Childhood AML patients did not harbor any recurrent SETBP1 gene mutations, although our study did identify a synonymous mutation in one patient. None of the previously reported aberrations in the mutational hotspot of SF3B1, U2AF1, and SRSF2 were identified in any of the 53 patients. Alterations of the SETBP1 gene or SF3B1, U2AF1, and SRSF2 genes are not common genetic events in childhood AML, implying that the mutations are unlikely to exert a driver effect in myeloid leukemogenesis during childhood.

Axial field shaping under high-numerical-aperture focusing

NASA Astrophysics Data System (ADS)

Jabbour, Toufic G.; Kuebler, Stephen M.

2007-03-01

Kant reported [J. Mod. Optics47, 905 (2000)] a formulation for solving the inverse problem of vector diffraction, which accurately models high-NA focusing. Here, Kant's formulation is adapted to the method of generalized projections to obtain an algorithm for designing diffractive optical elements (DOEs) that reshape the axial point-spread function (PSF). The algorithm is applied to design a binary phase-only DOE that superresolves the axial PSF with controlled increase in axial sidelobes. An 11-zone DOE is identified that axially narrows the PSF central lobe by 29% while maintaining the sidelobe intensity at or below 52% of the peak intensity. This DOE could improve the resolution achievable in several applications without significantly complicating the optical system.

Efficient generalized cross-validation with applications to parametric image restoration and resolution enhancement.

PubMed

Nguyen, N; Milanfar, P; Golub, G

2001-01-01

In many image restoration/resolution enhancement applications, the blurring process, i.e., point spread function (PSF) of the imaging system, is not known or is known only to within a set of parameters. We estimate these PSF parameters for this ill-posed class of inverse problem from raw data, along with the regularization parameters required to stabilize the solution, using the generalized cross-validation method (GCV). We propose efficient approximation techniques based on the Lanczos algorithm and Gauss quadrature theory, reducing the computational complexity of the GCV. Data-driven PSF and regularization parameter estimation experiments with synthetic and real image sequences are presented to demonstrate the effectiveness and robustness of our method.

State Education Finance and Governance Profile: Texas

ERIC Educational Resources Information Center

Boswell, Courtney

2010-01-01

This article presents the state education finance and governance profile of Texas. In 1854, the Permanent School Fund (PSF) was established by a legislative appropriation of $2 million. Today, the PSF has an approximate balance of $25 billion and continues to receive revenue from state taxes on land and minerals. The State Board of Education…

Transport of polar and non-polar volatile compounds in polystyrene foam and oriented strand board

NASA Astrophysics Data System (ADS)

Yuan, Huali; Little, John C.; Hodgson, Alfred T.

Transport of hexanal and styrene in polystyrene foam (PSF) and oriented strand board (OSB) was characterized. A microbalance was used to measure sorption/desorption kinetics and equilibrium data. While styrene transport in PSF can be described by Fickian diffusion with a symmetrical and reversible sorption/desorption process, hexanal transport in both PSF and OSB exhibited significant hysteresis, with desorption being much slower than sorption. A porous media diffusion model that assumes instantaneous local equilibrium governed by a nonlinear Freundlich isotherm was found to explain the hysteresis in hexanal transport. A new nonlinear sorption and porous diffusion emissions model was, therefore, developed and partially validated using independent chamber data. The results were also compared to the more conventional linear Fickian-diffusion emissions model. While the linear emissions model predicts styrene emissions from PSF with reasonable accuracy, it substantially underestimates the rate of hexanal emissions from OSB. Although further research and more rigorous validation is needed, the new nonlinear emissions model holds promise for predicting emissions of polar VOCs such as hexanal from porous building materials.

In Vivo Evaluation of the Biocompatibility of Surface Modified Hemodialysis Polysulfone Hollow Fibers in Rat

PubMed Central

Sabale, Siddharth S.; Kadam, Dattatray P.; Sarkate, Laxman B.; Bellare, Jayesh R.

2011-01-01

Polysulfone (Psf) hollow fiber membranes (HFMs) have been widely used in blood purification but their biocompatibility remains a concern. To enhance their biocompatibility, Psf/TPGS (d-α-tocopheryl polyethylene glycol 1000 succinate) composite HFMs and 2-methacryloyloxyethyl phosphorylcholine (MPC) coated Psf HFMs have been prepared. They have been evaluated for in vivo biocompatibility and graft acceptance and compared with sham and commercial membranes by intra-peritoneal implantation in rats at day 7 and 21. Normal body weights, tissue formation and angiogenesis indicate acceptance of implants by the animals. Hematological observations show presence of post-surgical stress which subsides over time. Serum biochemistry results reveal normal organ function and elevated liver ALP levels at day 21. Histological studies exhibit fibroblast recruitment cells, angiogenesis and collagen deposition at the implant surface indicating new tissue formation. Immuno-histochemistry studies show non-activation of MHC molecules signifying biocompatibilty. Additionally, Psf/TPGS exhibit most favorable tissue response as compared with other HFMs making them the material of choice for HFM preparation for hemodialysis applications. PMID:22046236

Increasing circular synthetic aperture sonar resolution via adapted wave atoms deconvolution.

PubMed

Pailhas, Yan; Petillot, Yvan; Mulgrew, Bernard

2017-04-01

Circular Synthetic Aperture Sonar (CSAS) processing computes coherently Synthetic Aperture Sonar (SAS) data acquired along a circular trajectory. This approach has a number of advantages, in particular it maximises the aperture length of a SAS system, producing very high resolution sonar images. CSAS image reconstruction using back-projection algorithms, however, introduces a dissymmetry in the impulse response, as the imaged point moves away from the centre of the acquisition circle. This paper proposes a sampling scheme for the CSAS image reconstruction which allows every point, within the full field of view of the system, to be considered as the centre of a virtual CSAS acquisition scheme. As a direct consequence of using the proposed resampling scheme, the point spread function (PSF) is uniform for the full CSAS image. Closed form solutions for the CSAS PSF are derived analytically, both in the image and the Fourier domain. The thorough knowledge of the PSF leads naturally to the proposed adapted atom waves basis for CSAS image decomposition. The atom wave deconvolution is successfully applied to simulated data, increasing the image resolution by reducing the PSF energy leakage.

Correlation between optical properties surface morphology of porous silicon electrodeposited by Fe3+ ion

NASA Astrophysics Data System (ADS)

Mabrouk, Asma; Lorrain, N.; Haji, M. L.; Oueslati, Meherzi

2015-01-01

In this paper, we analyze the photoluminescence spectra (PL) of porous silicon (PS) layer which is elaborated by electrochemical etching and passivated by Fe3+ ions (PSF) via current density, electro-deposition and temperature measurements. We observe unusual surface morphology of PSF surface and anomalous emission behavior. The PSF surface shows regular distribution of cracks, leaving isolated regions or ;platelets; of nearly uniform thickness. These cracks become more pronounced for high current densities. The temperature dependence of the PL peak energy (EPL) presents anomalous behaviors, i.e., the PL peak energy shows a successive red/blue/redshift (S-shaped behavior) with increasing temperature that we attribute to the existence of strong potential fluctuations induced by the electrochemical etching of PS layers. A competition process between localized and delocalized excitons is used to discuss these PL properties. In this case, the potential confinement plays a key role on the enhancement of PL intensity in PSF. To explain the temperature dependence of the PL intensity, we have proposed a recombination model based on the tunneling and dissociation of excitons.

A spatially-variant deconvolution method based on total variation for optical coherence tomography images

NASA Astrophysics Data System (ADS)

Almasganj, Mohammad; Adabi, Saba; Fatemizadeh, Emad; Xu, Qiuyun; Sadeghi, Hamid; Daveluy, Steven; Nasiriavanaki, Mohammadreza

2017-03-01

Optical Coherence Tomography (OCT) has a great potential to elicit clinically useful information from tissues due to its high axial and transversal resolution. In practice, an OCT setup cannot reach to its theoretical resolution due to imperfections of its components, which make its images blurry. The blurriness is different alongside regions of image; thus, they cannot be modeled by a unique point spread function (PSF). In this paper, we investigate the use of solid phantoms to estimate the PSF of each sub-region of imaging system. We then utilize Lucy-Richardson, Hybr and total variation (TV) based iterative deconvolution methods for mitigating occurred spatially variant blurriness. It is shown that the TV based method will suppress the so-called speckle noise in OCT images better than the two other approaches. The performance of proposed algorithm is tested on various samples, including several skin tissues besides the test image blurred with synthetic PSF-map, demonstrating qualitatively and quantitatively the advantage of TV based deconvolution method using spatially-variant PSF for enhancing image quality.

PSF estimation for defocus blurred image based on quantum back-propagation neural network

NASA Astrophysics Data System (ADS)

Gao, Kun; Zhang, Yan; Shao, Xiao-guang; Liu, Ying-hui; Ni, Guoqiang

2010-11-01

Images obtained by an aberration-free system are defocused blur due to motion in depth and/or zooming. The precondition of restoring the degraded image is to estimate point spread function (PSF) of the imaging system as precisely as possible. But it is difficult to identify the analytic model of PSF precisely due to the complexity of the degradation process. Inspired by the similarity between the quantum process and imaging process in the probability and statistics fields, one reformed multilayer quantum neural network (QNN) is proposed to estimate PSF of the defocus blurred image. Different from the conventional artificial neural network (ANN), an improved quantum neuron model is used in the hidden layer instead, which introduces a 2-bit controlled NOT quantum gate to control output and adopts 2 texture and edge features as the input vectors. The supervised back-propagation learning rule is adopted to train network based on training sets from the historical images. Test results show that this method owns excellent features of high precision and strong generalization ability.

Optimal apodization design for medical ultrasound using constrained least squares part I: theory.

PubMed

Guenther, Drake A; Walker, William F

2007-02-01

Aperture weighting functions are critical design parameters in the development of ultrasound systems because beam characteristics affect the contrast and point resolution of the final output image. In previous work by our group, we developed a metric that quantifies a broadband imaging system's contrast resolution performance. We now use this metric to formulate a novel general ultrasound beamformer design method. In our algorithm, we use constrained least squares (CLS) techniques and a linear algebra formulation to describe the system point spread function (PSF) as a function of the aperture weightings. In one approach, we minimize the energy of the PSF outside a certain boundary and impose a linear constraint on the aperture weights. In a second approach, we minimize the energy of the PSF outside a certain boundary while imposing a quadratic constraint on the energy of the PSF inside the boundary. We present detailed analysis for an arbitrary ultrasound imaging system and discuss several possible applications of the CLS techniques, such as designing aperture weightings to maximize contrast resolution and improve the system depth of field.

Shear Recovery Accuracy in Weak-Lensing Analysis with the Elliptical Gauss-Laguerre Method

NASA Astrophysics Data System (ADS)

Nakajima, Reiko; Bernstein, Gary

2007-04-01

We implement the elliptical Gauss-Laguerre (EGL) galaxy-shape measurement method proposed by Bernstein & Jarvis and quantify the shear recovery accuracy in weak-lensing analysis. This method uses a deconvolution fitting scheme to remove the effects of the point-spread function (PSF). The test simulates >107 noisy galaxy images convolved with anisotropic PSFs and attempts to recover an input shear. The tests are designed to be immune to statistical (random) distributions of shapes, selection biases, and crowding, in order to test more rigorously the effects of detection significance (signal-to-noise ratio [S/N]), PSF, and galaxy resolution. The systematic error in shear recovery is divided into two classes, calibration (multiplicative) and additive, with the latter arising from PSF anisotropy. At S/N > 50, the deconvolution method measures the galaxy shape and input shear to ~1% multiplicative accuracy and suppresses >99% of the PSF anisotropy. These systematic errors increase to ~4% for the worst conditions, with poorly resolved galaxies at S/N simeq 20. The EGL weak-lensing analysis has the best demonstrated accuracy to date, sufficient for the next generation of weak-lensing surveys.

Deciphering the Developmental Dynamics of the Mouse Liver Transcriptome

PubMed Central

Gunewardena, Sumedha S.; Yoo, Byunggil; Peng, Lai; Lu, Hong; Zhong, Xiaobo; Klaassen, Curtis D.; Cui, Julia Yue

2015-01-01

During development, liver undergoes a rapid transition from a hematopoietic organ to a major organ for drug metabolism and nutrient homeostasis. However, little is known on a transcriptome level of the genes and RNA-splicing variants that are differentially regulated with age, and which up-stream regulators orchestrate age-specific biological functions in liver. We used RNA-Seq to interrogate the developmental dynamics of the liver transcriptome in mice at 12 ages from late embryonic stage (2-days before birth) to maturity (60-days after birth). Among 21,889 unique NCBI RefSeq-annotated genes, 9,641 were significantly expressed in at least one age, 7,289 were differently regulated with age, and 859 had multiple (> = 2) RNA splicing-variants. Factor analysis showed that the dynamics of hepatic genes fall into six distinct groups based on their temporal expression. The average expression of cytokines, ion channels, kinases, phosphatases, transcription regulators and translation regulators decreased with age, whereas the average expression of peptidases, enzymes and transmembrane receptors increased with age. The average expression of growth factors peak between Day-3 and Day-10, and decrease thereafter. We identified critical biological functions, upstream regulators, and putative transcription modules that seem to govern age-specific gene expression. We also observed differential ontogenic expression of known splicing variants of certain genes, and 1,455 novel splicing isoform candidates. In conclusion, the hepatic ontogeny of the transcriptome ontogeny has unveiled critical networks and up-stream regulators that orchestrate age-specific biological functions in liver, and suggest that age contributes to the complexity of the alternative splicing landscape of the hepatic transcriptome. PMID:26496202

The Exon Junction Complex and Srp54 Contribute to Hedgehog Signaling via ci RNA Splicing in Drosophila melanogaster.

PubMed

Garcia-Garcia, Elisa; Little, Jamie C; Kalderon, Daniel

2017-08-01

Hedgehog (Hh) regulates the Cubitus interruptus (Ci) transcription factor in Drosophila melanogaster by activating full-length Ci-155 and blocking processing to the Ci-75 repressor. However, the interplay between the regulation of Ci-155 levels and activity, as well as processing-independent mechanisms that affect Ci-155 levels, have not been explored extensively. Here, we identified Mago Nashi (Mago) and Y14 core Exon Junction Complex (EJC) proteins, as well as the Srp54 splicing factor, as modifiers of Hh pathway activity under sensitized conditions. Mago inhibition reduced Hh pathway activity by altering the splicing pattern of ci to reduce Ci-155 levels. Srp54 inhibition also affected pathway activity by reducing ci RNA levels but additionally altered Ci-155 levels and activity independently of ci splicing. Further tests using ci transgenes and ci mutations confirmed evidence from studying the effects of Mago and Srp54 that relatively small changes in the level of Ci-155 primary translation product alter Hh pathway activity under a variety of sensitized conditions. We additionally used ci transgenes lacking intron sequences or the presumed translation initiation codon for an alternatively spliced ci RNA to provide further evidence that Mago acts principally by modulating the levels of the major ci RNA encoding Ci-155, and to show that ci introns are necessary to support the production of sufficient Ci-155 for robust Hh signaling and may also be important mediators of regulatory inputs. Copyright © 2017 by the Genetics Society of America.