X-linked hyper-immunoglobulin M (hyper-IgM) syndrome (XHIM) is a primary immunodeficiency due to mutations in CD40 ligand that affect immunoglobulin class-switch recombination and somatic ...hypermutation. The disease is amenable to gene therapy using retroviral vectors, but dysregulated gene expression results in abnormal lymphoproliferation in mouse models, highlighting the need for alternative strategies. Here, we demonstrate the ability of both the transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) platforms to efficiently drive integration of a normal copy of the CD40L cDNA delivered by Adeno-Associated Virus. Site-specific insertion of the donor sequence downstream of the endogenous CD40L promoter maintained physiologic expression of CD40L while overriding all reported downstream mutations. High levels of gene modification were achieved in primary human hematopoietic stem cells (HSCs), as well as in cell lines and XHIM-patient-derived T cells. Notably, gene-corrected HSCs engrafted in immunodeficient mice at clinically relevant frequencies. These studies provide the foundation for a permanent curative therapy in XHIM.
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•TALENs and CRISPRs allow targeted gene insertion at CD40LG in hematopoietic stem cells•Adenoviral helper proteins augment rates of gene modification in vitro, but not in vivo•Gene-modified HSCs engraft in mice at clinically relevant frequencies
Kuo et al. demonstrate that both TALEN and CRISPR/Cas9 platforms combined with a corrective AAV donor can mediate site-specific gene editing at the CD40 ligand locus. High levels of gene modification were achieved in human hematopoietic stem cells, which engraft in immunodeficient mice at clinically relevant frequencies.
Chloroplast development is an important determinant of plant productivity and is controlled by environmental factors including amounts of light and nitrogen as well as internal phytohormones ...including cytokinins and gibberellins (GA). The paralog GATA transcription factors GNC and CGA1/GNL up-regulated by light, nitrogen and cytokinin while also being repressed by GA signaling. Modifying the expression of these genes has previously been shown to influence chlorophyll content in Arabidopsis while also altering aspects of germination, elongation growth and flowering time. In this work, we also use transgenic lines to demonstrate that GNC and CGA1 exhibit a partially redundant control over chlorophyll biosynthesis. We provide novel evidence that GNC and CGA1 influence both chloroplast number and leaf starch in proportion to their transcript level. GNC and CGA1 were found to modify the expression of chloroplast localized GLUTAMATE SYNTHASE (GLU1/Fd-GOGAT), which is the primary factor controlling nitrogen assimilation in green tissue. Altering GNC and CGA1 expression was also found to modulate the expression of important chlorophyll biosynthesis genes (GUN4, HEMA1, PORB, and PORC). As previously demonstrated, the CGA1 transgenic plants demonstrated significantly altered timing to a number of developmental events including germination, leaf production, flowering time and senescence. In contrast, the GNC transgenic lines we analyzed maintain relatively normal growth phenotypes outside of differences in chloroplast development. Despite some evidence for partial divergence, results indicate that regulation of both GNC and CGA1 by light, nitrogen, cytokinin, and GA acts to modulate nitrogen assimilation, chloroplast development and starch production. Understanding the mechanisms controlling these processes is important for agricultural biotechnology.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Starlight subtraction algorithms based on the method of Karhunen–Loève eigenimages have proved invaluable to exoplanet direct imaging. However, they scale poorly in runtime when paired with ...differential imaging techniques. In such observations, reference frames and frames from which starlight is to be subtracted are drawn from the same set of data, requiring a new subset of references (and eigenimages) for each frame processed to avoid self-subtraction of the signal of interest. The data rates of extreme adaptive optics instruments are such that the only way to make this computationally feasible has been to downsample the data. We develop a technique that updates a precomputed singular value decomposition of the full data set to remove frames (i.e., a “downdate”) without a full recomputation, yielding the modified eigenimages. This not only enables analysis of much larger data volumes in the same amount of time, but also exhibits near-linear scaling in runtime as the number of observations increases. We apply this technique to archival data and investigate its scaling behavior for very large numbers of frames
N
. The resulting algorithm provides speed improvements of 2.6× (for 200 eigenimages at
N
= 300) to 140× (at
N
= 10
4
) with the advantage only increasing as
N
grows. This algorithm has allowed us to substantially accelerate Karhunen–Loève image projection (KLIP) even for modest
N
, and will let us quickly explore how KLIP parameters affect exoplanet characterization in large-
N
data sets.
Abstract
The detection of emission lines associated with accretion processes is a direct method for studying how and where gas giant planets form, how young planets interact with their natal ...protoplanetary disk, and how volatile delivery to their atmosphere takes place. H
α
(
λ
= 0.656
μ
m) is expected to be the strongest accretion line observable from the ground with adaptive optics systems, and is therefore the target of specific high-contrast imaging campaigns. We present MagAO-X and Hubble Space Telescope (HST) data obtained to search for H
α
emission from the previously detected protoplanet candidate orbiting AS209, identified through Atacama Large Millimeter/submillimeter Array observations. No signal was detected at the location of the candidate, and we provide limits on its accretion. Our data would have detected an H
α
emission with
F
H
α
> 2.5 ± 0.3 × 10
−16
erg s
−1
cm
−2
, a factor 6.5 lower than the HST flux measured for PDS70 b. The flux limit indicates that if the protoplanet is currently accreting it is likely that local extinction from circumstellar and circumplanetary material strongly attenuates its emission at optical wavelengths. In addition, the data reveal the first image of the jet north of the star as expected from previous detections of forbidden lines. Finally, this work demonstrates that current ground-based observations with extreme adaptive optics systems can be more sensitive than space-based observations, paving the way to the hunt for small planets in reflected light with extremely large telescopes.
Abstract
We use observations with the infrared-optimized Magellan Adaptive Optics (MagAO) system and Clio camera in 3.9
μ
m light to place stringent mass constraints on possible undetected companions ...to Sirius A. We suppress the light from Sirius A by imaging it through a grating vector-apodizing phase plate coronagraph with a 180° dark region (gvAPP-180). To remove residual starlight in postprocessing, we apply a time-domain principal-components-analysis-based algorithm we call PCA-Temporal, which uses eigen time series rather than eigenimages to subtract starlight. By casting the problem in terms of eigen time series, we reduce the computational cost of postprocessing the data, enabling the use of the fully sampled data set for improved contrast at small separations. We also discuss the impact of retaining fine temporal sampling of the data on final contrast limits. We achieve postprocessed contrast limits of 1.5 × 10
−6
–9.8 × 10
−6
outside of 0.″75, which correspond to planet masses of 2.6–8.0
M
J
. These are combined with values from the recent literature of high-contrast imaging observations of Sirius to synthesize an overall completeness fraction as a function of mass and separation. After synthesizing these recent studies and our results, the final completeness analysis rules out 99% of ≥9
M
J
planets from 2.5 to 7 au.
The multiligand receptors megalin (
) and cubilin (
) and their endocytic adaptor protein Dab2 (
) play essential roles in maintaining the integrity of the apical endocytic pathway of proximal tubule ...(PT) cells and have complex and poorly understood roles in the development of chronic kidney disease. Here, we used RNA-sequencing and CRISPR/Cas9 knockout (KO) technology in a well-differentiated cell culture model to identify PT-specific transcriptional changes that are directly consequent to the loss of megalin, cubilin, or Dab2 expression. KO of
had the greatest transcriptional effect, and nearly all genes whose expression was affected in
KO and
KO cells were also changed in
KO cells. Pathway analysis and more granular inspection of the altered gene profiles suggested changes in pathways with immunomodulatory functions that might trigger the pathological changes observed in KO mice and patients with Donnai-Barrow syndrome. In addition, differences in transcription patterns between
and
KO cells suggested the possibility that altered spatial signaling by aberrantly localized receptors contributes to transcriptional changes upon the disruption of PT endocytic function. A reduction in transcripts encoding sodium-glucose cotransporter isoform 2 was confirmed in
KO mouse kidney lysates by quantitative PCR analysis. Our results highlight the role of megalin as a master regulator and coordinator of ion transport, metabolism, and endocytosis in the PT. Compared with the studies in animal models, this approach provides a means to identify PT-specific transcriptional changes that are directly consequent to the loss of these target genes.
Megalin and cubilin receptors together with their adaptor protein Dab2 represent major components of the endocytic machinery responsible for efficient uptake of filtered proteins by the proximal tubule (PT). Dab2 and megalin expression have been implicated as both positive and negative modulators of kidney disease. We used RNA sequencing to knock out CRISPR/Cas9 cubilin, megalin, and Dab2 in highly differentiated PT cells to identify PT-specific changes that are directly consequent to knockout of each component.
Cultured cell models are an essential complement to dissecting kidney proximal tubule (PT) function in health and disease but do not fully recapitulate key features of this nephron segment. We ...recently determined that culture of opossum kidney (OK) cells under continuous orbital shear stress (OSS) significantly augments their morphological and functional resemblance to PTs
. Here we used RNASeq to identify temporal transcriptional changes upon cell culture under static or shear stress conditions. Comparison of gene expression in cells cultured under static or OSS conditions with a database of rat nephron segment gene expression confirms that OK cells cultured under OSS are more similar to the PT
compared with cells maintained under static conditions. Both improved oxygenation and mechanosensitive stimuli contribute to the enhanced differentiation in these cells, and we identified temporal changes in gene expression of known mechanosensitive targets. We observed changes in mRNA and protein levels of membrane trafficking components that may contribute to the enhanced endocytic capacity of cells cultured under OSS. Our data reveal pathways that may be critical for PT differentiation
and validate the utility of this improved cell culture model as a tool to study PT function.
Unlike many other hematologic malignancies, Richter syndrome (RS), an aggressive B cell lymphoma originating from indolent chronic lymphocytic leukemia, is responsive to PD-1 blockade. To discover ...the determinants of response, we analyze single-cell transcriptome data generated from 17 bone marrow samples longitudinally collected from 6 patients with RS. Response is associated with intermediate exhausted CD8 effector/effector memory T cells marked by high expression of the transcription factor ZNF683, determined to be evolving from stem-like memory cells and divergent from terminally exhausted cells. This signature overlaps with that of tumor-infiltrating populations from anti-PD-1 responsive solid tumors. ZNF683 is found to directly target key T cell genes (TCF7, LMO2, CD69) and impact pathways of T cell cytotoxicity and activation. Analysis of pre-treatment peripheral blood from 10 independent patients with RS treated with anti-PD-1, as well as patients with solid tumors treated with anti-PD-1, supports an association of ZNF683high T cells with response.
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•Richter syndrome PD-1 checkpoint blockade responders have ZNF683high CD8 T cells•ZNF683 regulates pathways of T cell activation and cytotoxicity•ZNF683high solid tumor infiltrating lymphocytes associate with better outcomes•ZNF683high signatures identified in blood of RS and solid tumor anti-PD-1 responders
Parry et al. conduct single-cell RNA-sequencing of bone marrow samples from patients with Richter syndrome and identify a ZNF683-high T cell population associated with anti-PD-1 response. ZNF683 regulates pathways of T cell activation and cytotoxicity and ZNF683high signatures could be identified across diverse malignancy settings, including blood of anti-PD-1 responders.
ABSTRACT
Improving direct detection capability close to the star through improved star subtraction and post-processing techniques is vital for discovering new low-mass companions and characterizing ...known ones at longer wavelengths. We present results of 17 binary star systems observed with the Magellan adaptive optics system (MagAO) and the Clio infrared camera on the Magellan Clay Telescope using binary differential imaging (BDI). BDI is an application of reference differential imaging (RDI) and angular differential imaging (ADI) applied to wide binary star systems (2 arcsec <Δρ < 10 arcsec) within the isoplanatic patch in the infrared. Each star serves as the point spread function (PSF) reference for the other, and we performed PSF estimation and subtraction using principal component analysis. We report contrast and mass limits for the 35 stars in our initial survey using BDI with MagAO/Clio in L′ and 3.95 µm bands. Our achieved contrasts varied between systems, and spanned a range of contrasts from 3.0 to 7.5 magnitudes and a range of separations from 0.2 to 2 arcsec. Stars in our survey span a range of masses, and our achieved contrasts correspond to late-type M-dwarf masses down to ∼10 MJup. We also report detection of a candidate companion signal at 0.2 arcsec (18 au) around HIP 67506 A (SpT G5V, mass ∼1.2 M⊙), which we estimate to be $\sim 60-90 \, \rm{M_{Jup}}$. We found that the effectiveness of BDI is highest for approximately equal brightness binaries in high-Strehl conditions.
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