Dynamic transcriptional activity and chromatin remodeling of regulatory T cells after varied duration of interleukin-2 receptor signaling.

Regulatory T (Treg) cells require (interleukin-2) IL-2 for their homeostasis by affecting their proliferation, survival and activation. Here we investigated transcriptional and epigenetic changes after acute, periodic and persistent IL-2 receptor (IL-2R) signaling in mouse peripheral Treg cells in vivo using IL-2 or the long-acting IL-2-based biologic mouse IL-2-CD25. We show that initially IL-2R-dependent STAT5 transcription factor-dependent pathways enhanced gene activation, chromatin accessibility and metabolic reprogramming to support Treg cell proliferation. Unexpectedly, at peak proliferation, less accessible chromatin prevailed and was associated with Treg cell contraction. Restimulation of IL-2R signaling after contraction activated signature IL-2-dependent genes and others associated with effector Treg cells, whereas genes associated with signal transduction were downregulated to somewhat temper expansion. Thus, IL-2R-dependent Treg cell homeostasis depends in part on a shift from more accessible chromatin and expansion to less accessible chromatin and contraction. Mouse IL-2-CD25 supported greater expansion and a more extensive transcriptional state than IL-2 in Treg cells, consistent with greater efficacy to control autoimmunity.

Molecular mechanism of high glucose-induced mitochondrial DNA damage in retinal ganglion cells.

Mitochondrial DNA damage in retinal ganglion cells (RGCs) may be closely related to lesions of glaucoma. RGCs were cultured with different concentrations of glucose and grouped into 3 groups, namely normal control (NC) group, Low-Glu group, and High-Glu group. Cell viability was measured with cell counting kit-8, and cell apoptosis was measured using flow cytometry. The DNA damage was measured with comet assay, and the morphological changes of damaged mitochondria in RGCs were observed using TEM. Western blot analyzed the expression of MRE11, RAD50, and NBS1 protein. Cell viability of RGCs in Low-Glu and High-Glu groups were lower than that of NC group in 48 and 96 h. The cell apoptosis in NC group was 4.9%, the Low-Glu group was 12.2% and High-Glu group was 24.4%. The comet imaging showed that NC cells did not have tailings, but the low-Glu and high-Glu group cells had tailings, indicating that the DNA of RGCs had been damaged. TEM, mitochondrial membrane potential, ROS, mitochondrial oxygen consumption, and ATP content detection results showed that RGCs cultured with high glucose occurred mitochondrial morphology changes and dysfunction. MRE11, RAD50, and NBS1 protein expression associated with DNA damage repair pathway in High-Glu group declined compared with Low-Glu group. Mitochondrial DNA damage caused by high glucose will result in apoptosis of retinal ganglion cells in glaucoma.

IS-Seq: a bioinformatics pipeline for integration sites analysis with comprehensive abundance quantification methods.

BACKGROUND: Integration site (IS) analysis is a fundamental analytical platform for evaluating the safety and efficacy of viral vector based preclinical and clinical Gene Therapy (GT). A handful of groups have developed standardized bioinformatics pipelines to process IS sequencing data, to generate reports, and/or to perform comparative studies across different GT trials. Keeping up with the technological advances in the field of IS analysis, different computational pipelines have been published over the past decade. These pipelines focus on identifying IS from single-read sequencing or paired-end sequencing data either using read-based or using sonication fragment-based methods, but there is a lack of a bioinformatics tool that automatically includes unique molecular identifiers (UMI) for IS abundance estimations and allows comparing multiple quantification methods in one integrated pipeline. RESULTS: Here we present IS-Seq a bioinformatics pipeline that can process data from paired-end sequencing of both old restriction sites-based IS collection methods and new sonication-based IS retrieval systems while allowing the selection of different abundance estimation methods, including read-based, Fragment-based and UMI-based systems. CONCLUSIONS: We validated the performance of IS-Seq by testing it against the most popular  analytical workflow available in the literature (INSPIIRED) and using different scenarios. Lastly, by performing extensive simulation studies and a comprehensive wet-lab assessment of our IS-Seq pipeline we could show that in clinically relevant scenarios, UMI quantification provides better accuracy than the currently most widely used sonication fragment counts as a method for IS abundance estimation.

High-throughput analysis of hematopoietic stem cell engraftment after intravenous and intracerebroventricular dosing.

Hematopoietic stem/progenitor cell gene therapy (HSPC-GT) has shown clear neurological benefit in rare diseases, which is achieved through the engraftment of genetically modified microglia-like cells (MLCs) in the brain. Still, the engraftment dynamics and the nature of engineered MLCs, as well as their potential use in common neurogenerative diseases, have remained largely unexplored. Here, we comprehensively characterized how different routes of administration affect the biodistribution of genetically engineered MLCs and other HSPC derivatives in mice. We generated a high-resolution single-cell transcriptional map of MLCs and discovered that they could clearly be distinguished from macrophages as well as from resident microglia by the expression of a specific gene signature that is reflective of their HSPC ontogeny and irrespective of their long-term engraftment history. Lastly, using murine models of Parkinson's disease and frontotemporal dementia, we demonstrated that MLCs can deliver therapeutically relevant levels of transgenic protein to the brain, thereby opening avenues for the clinical translation of HSPC-GT to the treatment of major neurological diseases.

Clinical Significance of Visual Training in Improving Visual Function After Retinal Detachment in Adolescents.

Context: Epidemiological data has shown that retinal detachment (RD) can occur at any age but has a poor prognosis in the adolescent population, which can cause huge obstacles to their life and learning. Although medications can achieve some curative effect, they have potential side effects and differences in individual efficacy. Objective: • The study intended to explore the clinical significance of visual training in improving recovery of postoperative visual function after external reduction of retinal detachment in adolescent patients. Design: The research team designed a prospective randomized controlled study. Setting: The study took place at Guiyang First People's Hospital in Guiyang, Guizhou, China. Participants: Participants were 110 adolescents with retinal detachments who underwent external reduction surgery, each on one eye for 110 eyes in total, and who were patients at the hospital between June 2015 and June 2019. Intervention: The research team assigned 52 participants to the visual-function training group, the intervention group, and 58 participants to the control group, according to the random-number-table method. Each group participated in training method for six months. Outcome Measures: To compare the groups, the research team measured visual function using the Visual Function scale (VF), binocular fusion function using the Worth Four Light Test (W4LT), and stereoscopic vision using the Titmus Stereo Test. The team obtained preoperative and postoperative data-at baseline, one day before surgery and postoperatively at one month and 3 months, and postintervention at 6 months. Results: Both groups had visual impairment after surgery. For visual function, the intervention group's scores after surgery increased gradually and were significantly higher than those of the control group at each follow-up time (P < .05). No significant difference in binocular fusion function existed between the groups at baseline or at one month after surgery (P > .05). At 3 months after surgery and postintervention, the proportions of participants in the intervention group with normal binocular fusion function were 86.54% and 88.46%, respectively, compared to that of the control group, at 68.97% and 70.69%, respectively. The intervention group's recovery was significantly better than that of the control group at 3 months after surgery and postintervention, at P < .028 and P < .022, respectively. No significant difference existed between the groups in stereoscopic vision at baseline, at 9.62% and 12.07%, respectively (P > .05). The proportion of participants in the intervention group with normal, binocular, stereoscopic vision increased gradually, and at one and 3 months after surgery and postintervention was 67.31%, 82.69%, and 88.46%, respectively. The intervention group's recovery was significantly better than that of the control group at one and 3 months after surgery and postintervention, at P < .028, P < .010, and P < .013, respectively. Conclusions: Visual training can effectively promote the recovery of visual function after external reduction of RD in adolescents and improve patients' prognoses. Moreover, long-term persistence can achieve significant effects, making the training worthy of clinical promotion.

Two contrasting classes of nucleolus-associated domains in mouse fibroblast heterochromatin.

In interphase eukaryotic cells, almost all heterochromatin is located adjacent to the nucleolus or to the nuclear lamina, thus defining nucleolus-associated domains (NADs) and lamina-associated domains (LADs), respectively. Here, we determined the first genome-scale map of murine NADs in mouse embryonic fibroblasts (MEFs) via deep sequencing of chromatin associated with purified nucleoli. We developed a Bioconductor package called NADfinder and demonstrated that it identifies NADs more accurately than other peak-calling tools, owing to its critical feature of chromosome-level local baseline correction. We detected two distinct classes of NADs. Type I NADs associate frequently with both the nucleolar periphery and the nuclear lamina, and generally display characteristics of constitutive heterochromatin, including late DNA replication, enrichment of H3K9me3, and little gene expression. In contrast, Type II NADs associate with nucleoli but do not overlap with LADs. Type II NADs tend to replicate earlier, display greater gene expression, and are more often enriched in H3K27me3 than Type I NADs. The nucleolar associations of both classes of NADs were confirmed via DNA-FISH, which also detected Type I but not Type II probes enriched at the nuclear lamina. Type II NADs are enriched in distinct gene classes, including factors important for differentiation and development. In keeping with this, we observed that a Type II NAD is developmentally regulated, and present in MEFs but not in undifferentiated embryonic stem (ES) cells.

Genomic Characterization of Endothelial Enhancers Reveals a Multifunctional Role for NR2F2 in Regulation of Arteriovenous Gene Expression.

RATIONALE: Significant progress has revealed transcriptional inputs that underlie regulation of artery and vein endothelial cell fates. However, little is known concerning genome-wide regulation of this process. Therefore, such studies are warranted to address this gap. OBJECTIVE: To identify and characterize artery- and vein-specific endothelial enhancers in the human genome, thereby gaining insights into mechanisms by which blood vessel identity is regulated. METHODS AND RESULTS: Using chromatin immunoprecipitation and deep sequencing for markers of active chromatin in human arterial and venous endothelial cells, we identified several thousand artery- and vein-specific regulatory elements. Computational analysis revealed that NR2F2 (nuclear receptor subfamily 2, group F, member 2) sites were overrepresented in vein-specific enhancers, suggesting a direct role in promoting vein identity. Subsequent integration of chromatin immunoprecipitation and deep sequencing data sets with RNA sequencing revealed that NR2F2 regulated 3 distinct aspects related to arteriovenous identity. First, consistent with previous genetic observations, NR2F2 directly activated enhancer elements flanking cell cycle genes to drive their expression. Second, NR2F2 was essential to directly activate vein-specific enhancers and their associated genes. Our genomic approach further revealed that NR2F2 acts with ERG (ETS-related gene) at many of these sites to drive vein-specific gene expression. Finally, NR2F2 directly repressed only a small number of artery enhancers in venous cells to prevent their activation, including a distal element upstream of the artery-specific transcription factor, HEY2 (hes related family bHLH transcription factor with YRPW motif 2). In arterial endothelial cells, this enhancer was normally bound by ERG, which was also required for arterial HEY2 expression. By contrast, in venous endothelial cells, NR2F2 was bound to this site, together with ERG, and prevented its activation. CONCLUSIONS: By leveraging a genome-wide approach, we revealed mechanistic insights into how NR2F2 functions in multiple roles to maintain venous identity. Importantly, characterization of its role at a crucial artery enhancer upstream of HEY2 established a novel mechanism by which artery-specific expression can be achieved.

Distinct features of nucleolus-associated domains in mouse embryonic stem cells.

Heterochromatin in eukaryotic interphase cells frequently localizes to the nucleolar periphery (nucleolus-associated domains (NADs)) and the nuclear lamina (lamina-associated domains (LADs)). Gene expression in somatic cell NADs is generally low, but NADs have not been characterized in mammalian stem cells. Here, we generated the first genome-wide map of NADs in mouse embryonic stem cells (mESCs) via deep sequencing of chromatin associated with biochemically purified nucleoli. As we had observed in mouse embryonic fibroblasts (MEFs), the large type I subset of NADs overlaps with constitutive LADs and is enriched for features of constitutive heterochromatin, including late replication timing and low gene density and expression levels. Conversely, the type II NAD subset overlaps with loci that are not lamina-associated, but in mESCs, type II NADs are much less abundant than in MEFs. mESC NADs are also much less enriched in H3K27me3 modified regions than are NADs in MEFs. Additionally, comparision of MEF and mESC NADs revealed enrichment of developmentally regulated genes in cell-type-specific NADs. Together, these data indicate that NADs are a developmentally dynamic component of heterochromatin. These studies implicate association with the nucleolar periphery as a mechanism for developmentally regulated gene expression and will facilitate future studies of NADs during mESC differentiation.

Dual-energy phase retrieval algorithm for inline phase sensitive x-ray imaging system.

Phase retrieval is vital for quantitative x-ray phase contrast imaging. This work presents an iterative method to simultaneously retrieve the x-ray absorption and phase images from a single x-ray exposure. The proposed approach uses the photon-counting detectors' energy-resolving capability in providing multiple spectrally resolved phase contrast images from a single x-ray exposure. The retrieval method is derived, presented, and experimentally tested with a multi-material phantom in an inline phase contrast imaging setup. By separating the contributions of photoelectric absorption and Compton scattering to the attenuation, the authors divide the phase contrast image into two portions, the attenuation map arises from photoelectric absorption and a pseudo phase contrast image generated by electron density. This way one can apply the Phase Attenuation Dualiby (PAD) algorithm and Fresnel propagation for the iteration. The retrieval results from the experimental images show that this iterative method is fast, accurate, robust against noise, and thus yields noticeable enhancement in contrast to noise ratios.

Predicting fringe visibility in dual-phase grating interferometry with polychromatic X-ray sources.

Dual phase grating X-ray interferometry is radiation dose-efficient as compared to common Talbot-Lau grating interferometry. The authors developed a general quantitative theory to predict the fringe visibility in dual-phase grating X-ray interferometry with polychromatic X-ray sources. The derived formulas are applicable to setups with phase gratings of any phase modulation and with either monochromatic or polychromatic X-rays. Numerical simulations are presented to validate the derived formulas. The theory provides useful tools for design optimization of dual-phase grating X-ray interferometers.

PathwaySplice: an R package for unbiased pathway analysis of alternative splicing in RNA-Seq data.

Summary: Pathway analysis of alternative splicing would be biased without accounting for the different number of exons or junctions associated with each gene, because genes with higher number of exons or junctions are more likely to be included in the 'significant' gene list in alternative splicing. We present PathwaySplice, an R package that (i) Performs pathway analysis that explicitly adjusts for the number of exons or junctions associated with each gene; (ii) visualizes selection bias due to different number of exons or junctions for each gene and formally tests for presence of bias using logistic regression; (iii) supports gene sets based on the Gene Ontology terms, as well as more broadly defined gene sets (e.g. MSigDB) or user defined gene sets; (iv) identifies the significant genes driving pathway significance and (v) organizes significant pathways with an enrichment map, where pathways with large number of overlapping genes are grouped together in a network graph. Availability and implementation: https://bioconductor.org/packages/release/bioc/html/PathwaySplice.html. Supplementary information: Supplementary data are available at Bioinformatics online.

Sample phase gradient and fringe phase shift in dual phase grating X-ray interferometry.

One of the key tasks in grating based x-ray phase contrast imaging is to accurately retrieve local phase gradients of a sample from measured intensity fringe shifts. To fulfill this task in dual phase grating interferometry, one needs to know the exact mathematical relationship between the two. In this work, using intuitive analysis of the sample-generated fringe shifts based on the beat pattern formation mechanism, the authors derived the formulas relating sample phase gradients to fringe phase shifts. These formulas provide also a design optimization tool for dual phase grating interferometry.

Clarification on generalized Lau condition for X-ray interferometers based on dual phase gratings.

To implement dual phase grating x-ray interferometry with x-ray tubes, one needs to incorporate an absorbing source grating. In order to attain good fringe visibility, the period of a source grating should be subject to a stringent condition. In literature some authors claim that the Lau-condition in Talbot-Lau interferometry can be literally transferred to dual phase grating interferometry. In this work we show that this statement in literature is incorrect. Instead, through an intuitive geometrical analysis of fringe formation, we derived a new generalized Lau-condition that provides a useful design tool for implementation of dual phase grating interferometry.

Multiple-image encryption based on angular-multiplexing holography with quick response code and spiral phase keys.

In this research, a novel optical multiple-image encryption method based on angular-multiplexing holography, quick response (QR) code, and spiral phase keys is proposed. With this method, images are transformed into QR codes and subsequently encrypted into a series of encrypted holograms using an angular-multiplexing technique. The encrypted holograms can only be decrypted when the hologram is illuminated with a duplicate of the reference beam and correct fingerprint and spiral phase plate (SPP) keys. The multiplexing performance and key sensitivity of fingerprint and SPP order were both analyzed, showing the high strength of the security of our proposed method.

Quantitative theory of X-ray interferometers based on dual phase grating: Fringe period and visibility.

In most of grating x-ray interferometry one needs an absorbing grating as the analyzer to measure high-resolution interference fringes. Dual phase grating interferometry is a technique to get rid of the absorbing grating for radiation dose reduction. The authors present a quantitative theory of dual grating x-ray interferometry. The theory elucidates the fringe formation mechanism. The derived formulas of fringe period and fringe visibility provide useful tools for design optimization of dual phase grating interferometers.

Beam hardening correction in polychromatic x-ray grating interferometry.

The beam hardening is one of the two causes of the fringe shift distortion in polychromatic X-ray grating interferometry. Based on the assumption of the uniform energy dependence, we developed a novel analytic approach to accurately retrieve the monochromatic attenuation function and fringe phase shift from the polychromatic measurement. This approach provides a useful tool for precise measurement of sample electron density distribution in X-ray grating interferometry.

Polychromatic X-ray effects on fringe phase shifts in grating interferometry.

In order to quantitatively determine the projected electron densities of a sample, one needs to extract the monochromatic fringe phase shifts from the polychromatic fringe phase shifts measured in the grating interferometry with incoherent X-ray sources. In this work the authors propose a novel analytic approach that allows to directly compute the monochromatic fringe shifts from the polychromatic fringe shifts. This approach is validated with numerical simulations of several grating interferometry setups. This work provides a useful tool in quantitative imaging for biomedical and material science applications.

The Sol Genomics Network (SGN)--from genotype to phenotype to breeding.

The Sol Genomics Network (SGN, http://solgenomics.net) is a web portal with genomic and phenotypic data, and analysis tools for the Solanaceae family and close relatives. SGN hosts whole genome data for an increasing number of Solanaceae family members including tomato, potato, pepper, eggplant, tobacco and Nicotiana benthamiana. The database also stores loci and phenotype data, which researchers can upload and edit with user-friendly web interfaces. Tools such as BLAST, GBrowse and JBrowse for browsing genomes, expression and map data viewers, a locus community annotation system and a QTL analysis tools are available. A new tool was recently implemented to improve Virus-Induced Gene Silencing (VIGS) constructs called the SGN VIGS tool. With the growing genomic and phenotypic data in the database, SGN is now advancing to develop new web-based breeding tools and implement the code and database structure for other species or clade-specific databases.

Predicting visibility of interference fringes in X-ray grating interferometry.

The interference fringe visibility is a common figure of merit in designs of x-ray grating-based interferometers. Presently one has to resort to laborious computer simulations to predict fringe visibility values of interferometers with polychromatic x-ray sources. Expanding the authors' previous work on Fourier expansion of the intensity fringe pattern, in this work the authors developed a general quantitative theory to predict the intensity fringe pattern in closed-form formulas, which incorporates the effects of partial spatial coherence, spectral average and detector pixel re-binning. These formulas can be used to predict the fringe visibility of a Talbot-Lau interferometer with any geometry configuration and any source spectrum.

Novel optical scanning cryptography using Fresnel telescope imaging.

We propose a new method called modified optical scanning cryptography using Fresnel telescope imaging technique for encryption and decryption of remote objects. An image or object can be optically encrypted on the fly by Fresnel telescope scanning system together with an encryption key. For image decryption, the encrypted signals are received and processed with an optical coherent heterodyne detection system. The proposed method has strong performance through use of secure Fresnel telescope scanning with orthogonal polarized beams and efficient all-optical information processing. The validity of the proposed method is demonstrated by numerical simulations and experimental results.