Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C promoter.

Lentiviral vectors almost universally use heterologous internal promoters to express transgenes. One of the most commonly used promoter fragments is a 1.2-kb sequence from the human ubiquitin C (UBC) gene, encompassing the promoter, some enhancers, first exon, first intron and a small part of the second exon of UBC. Because splicing can occur after transcription of the vector genome during vector production, we investigated whether the intron within the UBC promoter fragment is faithfully transmitted to target cells. Genetic analysis revealed that more than 80% of proviral forms lack the intron of the UBC promoter. The human elongation factor 1 alpha (EEF1A1) promoter fragment intron was not lost during lentiviral packaging, and this difference between the UBC and EEF1A1 promoter introns was conferred by promoter exonic sequences. UBC promoter intron loss caused a 4-fold reduction in transgene expression. Movement of the expression cassette to the opposite strand prevented intron loss and restored full expression. This increase in expression was mostly due to non-classical enhancer activity within the intron, and movement of putative intronic enhancer sequences to multiple promoter-proximal sites actually repressed expression. Reversal of the UBC promoter also prevented intron loss and restored full expression in bidirectional lentiviral vectors

The Origin of Double-peaked Narrow Lines in Active Galactic Nuclei. IV. Association with Galaxy Mergers

Double-peaked narrow emission lines in active galactic nucleus (AGN) spectra can be produced by AGN outflows, rotation, or dual AGNs, which are AGN pairs in ongoing galaxy mergers. Consequently, double-peaked narrow AGN emission lines are useful tracers of the coevolution of galaxies and their supermassive black holes, as driven by AGN feedback and AGN fueling. We investigate this concept further with follow-up optical longslit observations of a sample of 95 Sloan Digital Sky Survey (SDSS) galaxies that have double-peaked narrow AGN emission lines. Based on a kinematic analysis of the longslit spectra, we confirm previous work that finds that the majority of double-peaked narrow AGN emission lines are associated with outflows. We also find that eight of the galaxies have companion galaxies with line-of-sight velocity separations < 500 km/s and physical separations <30 kpc. Since we find evidence of AGNs in both galaxies, all eight of these systems are compelling dual AGN candidates. Galaxies with double-peaked narrow AGN emission lines occur in such galaxy mergers at least twice as often as typical active galaxies. Finally, we conclude that at least 3% of SDSS galaxies with double-peaked narrow AGN emission lines are found in galaxy mergers where both galaxies are resolved in SDSS imaging.Comment: 14 pages, 2 figures, ApJ in pres

Reliable and accurate diagnostics from highly multiplexed sequencing assays

Scalable, inexpensive, and secure testing for SARS-CoV-2 infection is crucial for control of the novel coronavirus pandemic. Recently developed highly multiplexed sequencing assays (HMSAs) that rely on high-throughput sequencing can, in principle, meet these demands, and present promising alternatives to currently used RT-qPCR-based tests. However, reliable analysis, interpretation, and clinical use of HMSAs requires overcoming several computational, statistical and engineering challenges. Using recently acquired experimental data, we present and validate a computational workflow based on kallisto and bustools, that utilizes robust statistical methods and fast, memory efficient algorithms, to quickly, accurately and reliably process high-throughput sequencing data. We show that our workflow is effective at processing data from all recently proposed SARS-CoV-2 sequencing based diagnostic tests, and is generally applicable to any diagnostic HMSA

Reactivating Fetal Hemoglobin Expression in Human Adult Erythroblasts Through BCL11A Knockdown Using Targeted Endonucleases.

We examined the efficiency, specificity, and mutational signatures of zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 systems designed to target the gene encoding the transcriptional repressor BCL11A, in human K562 cells and human CD34+ progenitor cells. ZFNs and TALENs were delivered as in vitro transcribed mRNA through electroporation; CRISPR/Cas9 was codelivered by Cas9 mRNA with plasmid-encoded guideRNA (gRNA) (pU6.g1) or in vitro transcribed gRNA (gR.1). Analyses of efficacy revealed that for these specific reagents and the delivery methods used, the ZFNs gave rise to more allelic disruption in the targeted locus compared to the TALENs and CRISPR/Cas9, which was associated with increased levels of fetal hemoglobin in erythroid cells produced in vitro from nuclease-treated CD34+ cells. Genome-wide analysis to evaluate the specificity of the nucleases revealed high specificity of this specific ZFN to the target site, while specific TALENs and CRISPRs evaluated showed off-target cleavage activity. ZFN gene-edited CD34+ cells had the capacity to engraft in NOD-PrkdcSCID-IL2Rγnull mice, while retaining multi-lineage potential, in contrast to TALEN gene-edited CD34+ cells. CRISPR engraftment levels mirrored the increased relative plasmid-mediated toxicity of pU6.g1/Cas9 in hematopoietic stem/progenitor cells (HSPCs), highlighting the value for the further improvements of CRISPR/Cas9 delivery in primary human HSPCs

A Satellite-Based Multi-Pollutant Index of Global Air Quality

Air pollution is a major health hazard that is responsible formillions of annual excess deaths worldwide. Simpleindicators are useful for comparative studies and to asses strends over time. The development of global indicators hasbeen impeded by the lack of ground-based observations in vast regions of the world. Recognition is growing of the need for amultipollutant approach to air quality to better represent human exposure. Here we introduce the prospect of amultipollutant air quality indicator based on observations from satellite remote sensing

The human ankyrin 1 promoter insulator sustains gene expression in a β-globin lentiviral vector in hematopoietic stem cells.

Lentiviral vectors designed for the treatment of the hemoglobinopathies require the inclusion of regulatory and strong enhancer elements to achieve sufficient expression of the β-globin transgene. Despite the inclusion of these elements, the efficacy of these vectors may be limited by transgene silencing due to the genomic environment surrounding the integration site. Barrier insulators can be used to give more consistent expression and resist silencing even with lower vector copies. Here, the barrier activity of an insulator element from the human ankyrin-1 gene was analyzed in a lentiviral vector carrying an antisickling human β-globin gene. Inclusion of a single copy of the Ankyrin insulator did not affect viral titer, and improved the consistency of expression from the vector in murine erythroleukemia cells. The presence of the Ankyrin insulator element did not change transgene expression in human hematopoietic cells in short-term erythroid culture or in vivo in primary murine transplants. However, analysis in secondary recipients showed that the lentiviral vector with the Ankyrin element preserved transgene expression, whereas expression from the vector lacking the Ankyrin insulator decreased in secondary recipients. These studies demonstrate that the Ankyrin insulator may improve long-term β-globin expression in hematopoietic stem cells for gene therapy of hemoglobinopathies

What did HERA teach us about the structure of the proton?

Starting in 2008 the H1 and ZEUS experiments have been combining their data in order to provide the most complete and accurate set of deep-inelastic data as the legacy of HERA. The present review presents these combinations, both published and preliminary, and explores how they have been used to give information on the structure of the proton. The HERAPDF parton distribution functions (PDFs) are presented and compared with other current PDFs and with data from the Tevatron and LHC colliders.Comment: 49 pages, 49 figures, to be published in J.Phys.

Van der Waals density functional: Self-consistent potential and the nature of the van der Waals bond

We derive the exchange-correlation potential corresponding to the nonlocal van der Waals density functional [M. Dion, H. Rydberg, E. Schroder, D. C. Langreth, and B. I. Lundqvist, Phys. Rev. Lett. 92, 246401 (2004)]. We use this potential for a self-consistent calculation of the ground state properties of a number of van der Waals complexes as well as crystalline silicon. For the latter, where little or no van der Waals interaction is expected, we find that the results are mostly determined by semilocal exchange and correlation as in standard generalized gradient approximations (GGA), with the fully nonlocal term giving little effect. On the other hand, our results for the van der Waals complexes show that the self-consistency has little effect at equilibrium separations. This finding validates previous calculations with the same functional that treated the fully nonlocal term as a post GGA perturbation. A comparison of our results with wave-function calculations demonstrates the usefulness of our approach. The exchange-correlation potential also allows us to calculate Hellmann-Feynman forces, hence providing the means for efficient geometry relaxations as well as unleashing the potential use of other standard techniques that depend on the self-consistent charge distribution. The nature of the van der Waals bond is discussed in terms of the self-consistent bonding charge.Comment: submitted to Phys. Rev.

Reliable and accurate diagnostics from highly multiplexed sequencing assays

Scalable, inexpensive, and secure testing for SARS-CoV-2 infection is crucial for control of the novel coronavirus pandemic. Recently developed highly multiplexed sequencing assays (HMSAs) that rely on high-throughput sequencing can, in principle, meet these demands, and present promising alternatives to currently used RT-qPCR-based tests. However, reliable analysis, interpretation, and clinical use of HMSAs requires overcoming several computational, statistical and engineering challenges. Using recently acquired experimental data, we present and validate a computational workflow based on kallisto and bustools, that utilizes robust statistical methods and fast, memory efficient algorithms, to quickly, accurately and reliably process high-throughput sequencing data. We show that our workflow is effective at processing data from all recently proposed SARS-CoV-2 sequencing based diagnostic tests, and is generally applicable to any diagnostic HMSA

The Lick AGN Monitoring Project 2011: Dynamical Modeling of the Broad-Line Region

We present models of the H$\beta$-emitting broad-line region (BLR) in seven Seyfert 1 galaxies from the Lick AGN (Active Galactic Nucleus) Monitoring Project 2011 sample, drawing inferences on the BLR structure and dynamics as well as the mass of the central supermassive black hole. We find that the BLR is generally a thick disk, viewed close to face-on, with preferential emission back toward the ionizing source. The dynamics in our sample range from near-circular elliptical orbits to inflowing or outflowing trajectories. We measure black hole masses of $\log_{10}(M_{\rm BH}/M_\odot) = 6.48^{+0.21}_{-0.18}$ for PG 1310$-$108, $7.50^{+0.25}_{-0.18}$ for Mrk 50, $7.46^{+0.15}_{-0.21}$ for Mrk 141, $7.58^{+0.08}_{-0.08}$ for Mrk 279, $7.11^{+0.20}_{-0.17}$ for Mrk 1511, $6.65^{+0.27}_{-0.15}$ for NGC 4593, and $6.94^{+0.14}_{-0.14}$ for Zw 229$-$015. We use these black hole mass measurements along with cross-correlation time lags and line widths to recover the scale factor $f$ used in traditional reverberation mapping measurements. Combining our results with other studies that use this modeling technique, bringing our sample size to 16, we calculate a scale factor that can be used for measuring black hole masses in other reverberation mapping campaigns. When using the root-mean-square (rms) spectrum and using the line dispersion to measure the line width, we find $\log_{10}(f_{{\rm rms},\sigma})_{\rm pred} = 0.57 \pm 0.19$. Finally, we search for correlations between $f$ and other AGN and BLR parameters and find marginal evidence that $f$ is correlated with $M_{\rm BH}$ and the BLR inclination angle, but no significant evidence of a correlation with the AGN luminosity or Eddington ratio.Comment: 26 pages, 14 figures. Accepted for publication in Ap