The stability of mRNA influences the temporal order of the induction of genes encoding inflammatory molecules.

The inflammatory response plays out over time in a reproducible and organized way after an initiating stimulus. Here we show that genes activated in cultured mouse fibroblasts in response to the cytokine tumor necrosis factor could be categorized into roughly three groups, each with different induction kinetics. Although differences in transcription were important in determining the grouping of these genes, differences in mRNA stability also exerted a strong influence on the temporal order of gene expression, in some cases overriding that of transcriptional control elements. Transcripts of mRNA expressed early had abundant AU-rich elements in their 3' untranslated regions, whereas those expressed later had fewer. Thus, mRNA stability and transcriptional control, two intrinsic characteristics of genes, control the kinetics of gene expression induced by proinflammatory cytokines.

Population-based 3D genome structure analysis reveals driving forces in spatial genome organization.

Conformation capture technologies (e.g., Hi-C) chart physical interactions between chromatin regions on a genome-wide scale. However, the structural variability of the genome between cells poses a great challenge to interpreting ensemble-averaged Hi-C data, particularly for long-range and interchromosomal interactions. Here, we present a probabilistic approach for deconvoluting Hi-C data into a model population of distinct diploid 3D genome structures, which facilitates the detection of chromatin interactions likely to co-occur in individual cells. Our approach incorporates the stochastic nature of chromosome conformations and allows a detailed analysis of alternative chromatin structure states. For example, we predict and experimentally confirm the presence of large centromere clusters with distinct chromosome compositions varying between individual cells. The stability of these clusters varies greatly with their chromosome identities. We show that these chromosome-specific clusters can play a key role in the overall chromosome positioning in the nucleus and stabilizing specific chromatin interactions. By explicitly considering genome structural variability, our population-based method provides an important tool for revealing novel insights into the key factors shaping the spatial genome organization.

RNA splicing regulates the temporal order of TNF-induced gene expression.

When cells are induced to express inflammatory genes by treatment with TNF, the mRNAs for the induced genes appear in three distinct waves, defining gene groups I, II, and III, or early, intermediate, and late genes. To examine the basis for these different kinetic classes, we have developed a PCR-based procedure to distinguish pre-mRNAs from mRNAs. It shows that the three groups initiate transcription virtually simultaneously but that delays in splicing characterize groups II and III. We also examined the elongation times, concluding that pre-mRNA synthesis is coordinate but splicing differences directly regulate the timing of mRNA production.

The features of serous retinal detachment in preeclampsia viewed on spectral-domain optical coherence tomography.

OBJECTIVE: To evaluate the characteristics of serous retinal detachment on spectral-domain optical coherence tomography in preeclampsia. METHODS: In this retrospective case-series study, clinical characteristics of retinal damage were evaluated using spectral-domain optical coherence tomography (SD-OCT) imaging. RESULTS: Thirty affected eyes from 16 pregnant women with preeclampsia were included. The features of serous retinal detachment, observed using SD-OCT, consisted of lesions located in the macular or peripapillary region; the presence of intraretinal or subretinal fluid (intraretinal fluid, IRF; subretinal fluid, SRF); ellipsoid zone integrity (normal/abnormal); intraretinal hyper-reflective dots; and Elschnig spots (retinal pigment epithelium lesions). Of the 30 affected eyes, 25 (83.33%) had lesions located in the macular region, 19 (63.33%) outside the macula (in the peripapillary region), and 14 (46.67%) in both. SD-OCT showed IRF in 2 eyes (6.67%), SRF in 30 eyes (100.00%), and both in 2 eyes (6.67%). The ellipsoid zone was disrupted in 20 eyes (66.67%), intraretinal hyper-reflective dots were observed in 4 eyes (13.33%), and Elschnig spots were observed in 20 eyes (66.67%). CONCLUSION: Spectral-domain optical coherence tomography is a non-invasive, reliable imaging tool for the assessment of retinal pathologies in preeclampsia.

EHMT1 protein binds to nuclear factor-κB p50 and represses gene expression.

Transcriptional homeostasis relies on the balance between positive and negative regulation of gene transcription. Methylation of histone H3 lysine 9 (H3K9) is commonly correlated with gene repression. Here, we report that a euchromatic H3K9 methyltransferase, EHMT1, functions as a negative regulator in both the NF-κB- and type I interferon-mediated gene induction pathways. EHMT1 catalyzes H3K9 methylation at promoters of NF-κB target genes. Moreover, EHMT1 interacts with p50, and, surprisingly, p50 appears to repress the expression of type I interferon genes and genes activated by type I interferons by recruiting EHMT1 to catalyze H3K9 methylation at their promoter regions. Silencing the expression of EHMT1 by RNA interference enhances expression of a subset NF-κB-regulated genes, augments interferon production, and augments antiviral immunity.

Evaluating the role of the nuclear microenvironment in gene function by population-based modeling.

The nuclear folding of chromosomes relative to nuclear bodies is an integral part of gene function. Here, we demonstrate that population-based modeling-from ensemble Hi-C data-provides a detailed description of the nuclear microenvironment of genes and its role in gene function. We define the microenvironment by the subnuclear positions of genomic regions with respect to nuclear bodies, local chromatin compaction, and preferences in chromatin compartmentalization. These structural descriptors are determined in single-cell models, thereby revealing the structural variability between cells. We demonstrate that the microenvironment of a genomic region is linked to its functional potential in gene transcription, replication, and chromatin compartmentalization. Some chromatin regions feature a strong preference for a single microenvironment, due to association with specific nuclear bodies in most cells. Other chromatin shows high structural variability, which is a strong indicator of functional heterogeneity. Moreover, we identify specialized nuclear microenvironments, which distinguish chromatin in different functional states and reveal a key role of nuclear speckles in chromosome organization. We demonstrate that our method produces highly predictive three-dimensional genome structures, which accurately reproduce data from a variety of orthogonal experiments, thus considerably expanding the range of Hi-C data analysis.

The integrated transcriptome bioinformatics analysis identifies key genes and cellular components for proliferative diabetic retinopathy.

Proliferative Diabetic Retinopathy (PDR) is a chronic complication of Diabetes and the main cause of blindness among the world's working population at present. While there have been many studies on the pathogenesis of PDR, its intrinsic molecular mechanisms have not yet been fully elucidated. In recent years, several studies have employed bulk RNA-sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq) to profile differentially expressed genes (DEGs) and cellular components associated with PDR. This study adds to this expanding body of work by identifying PDR's target genes and cellular components by conducting an integrated transcriptome bioinformatics analysis. This study integrately examined two public bulk RNA-seq datasets(including 11 PDR patients and 7 controls) and one single-cell RNA-seq datasets(including 5 PDR patients) of Fibro (Vascular) Membranes (FVMs) from PDR patients and control. A total of 176 genes were identified as DEGs between PDR patients and control among both bulk RNA-seq datasets. Based on these DEGs, 14 proteins were identified in the protein overlap within the significant ligand-receptor interactions of retinal FVMs and Protein-Protein Interaction (PPI) network, three of which were associated with PDR (CD44, ICAM1, POSTN), and POSTN might act as key ligand. This finding may provide novel gene signatures and therapeutic targets for PDR.

Actin depolymerization transduces the strength of B-cell receptor stimulation.

Polymerization of the actin cytoskeleton has been found to be essential for B-cell activation. We show here, however, that stimulation of BCR induces a rapid global actin depolymerization in a BCR signal strength-dependent manner, followed by polarized actin repolymerization. Depolymerization of actin enhances and blocking actin depolymerization inhibits BCR signaling, leading to altered BCR and lipid raft clustering, ERK activation, and transcription factor activation. Furthermore actin depolymerization by itself induces altered lipid raft clustering and ERK activation, suggesting that F-actin may play a role in separating lipid rafts and in setting the threshold for cellular activation.

A kinase independent function for Tec kinase ITK in regulating antigen receptor induced serum response factor activation.

The Tec family kinases are critical downstream regulators of antigen receptor signals in lymphocytes. As kinases, they act on critical substrates to regulate signals such as calcium increase leading to activation of transcription factors such as NFAT, NFkappaB and SRF. We now show here that ITK, a member of the Tec family of tyrosine kinases, has a kinase independent function. Mutants of ITK that lack kinase activity or a kinase domain can rescue cells lacking Tec family kinases for antigen receptor induced SRF activation, but not for NFAT, AP-1 or NFkappaB activation. Furthermore, expression of these mutants in WT cells enhanced SRF activation. This kinase independent function required the SH2 domain since a mutant lacking both the kinase and SH2 domains was much less effective at rescuing SRF activation. This kinase-deleted mutant could partially rescue ERK activation, and interact with multiple tyrosine phosphorylated proteins during antigen receptor signaling, suggesting that ITK uses a scaffolding function that regulates signals leading to specific regulation of SRF activation.

Mining 3D genome structure populations identifies major factors governing the stability of regulatory communities.

Three-dimensional (3D) genome structures vary from cell to cell even in an isogenic sample. Unlike protein structures, genome structures are highly plastic, posing a significant challenge for structure-function mapping. Here we report an approach to comprehensively identify 3D chromatin clusters that each occurs frequently across a population of genome structures, either deconvoluted from ensemble-averaged Hi-C data or from a collection of single-cell Hi-C data. Applying our method to a population of genome structures (at the macrodomain resolution) of lymphoblastoid cells, we identify an atlas of stable inter-chromosomal chromatin clusters. A large number of these clusters are enriched in binding of specific regulatory factors and are therefore defined as 'Regulatory Communities.' We reveal two major factors, centromere clustering and transcription factor binding, which significantly stabilize such communities. Finally, we show that the regulatory communities differ substantially from cell to cell, indicating that expression variability could be impacted by genome structures.

The Tec family of tyrosine kinases in T cells, amplifiers of T cell receptor signals.

ITK and Rlk/Txk are the predominant Tec family of tyrosine kinases expressed in T cells, and are involved in T cell antigen receptor mediated activation of T cells. These kinases require prior activation of Lck, Zap-70 and PI3-kinase for efficient activation. They share major substrates with both Lck and Zap-70, however the pathways they regulate are unclear. Recent evidence suggests that these kinases may not activate unique pathways, but instead serve as amplifiers for the upstream kinases Lck and Zap-70. This review will discuss the evidence for this view.

The proline rich region of the Tec homology domain of ITK regulates its activity.

Inducible T-cell kinase (ITK) is a member of the Tec family of tyrosine kinases that are involved in signals emanating from cytokine receptors, antigen receptors and other lymphoid cell surface receptors. Stimulation of tyrosine phosphorylation and activation of ITK by the T-cell antigen receptor, CD28 and CD2 requires the presence of the Src family kinase Lck in T-cells. We have previously demonstrated that the activation of ITK by Src family kinases uses a phosphatidylinositol 3-kinase pathway, which recruits ITK to the membrane via its pleckstrin homology (PH) domain where it is acted upon by Src. We have further explored the mechanism of this requirement for Src family kinases in the activation of ITK. We found that deletion of the proline rich sequence found in the Tec homology domain of ITK results in reduced basal activity of ITK approximately 50%. These differences in the basal activity of ITK were observed when the PH domain was deleted or when the kinase was membrane targeted. Furthermore, this deletion reduces the ability of the Src family kinase Lck to activate ITK, as well as to induce the ITK mediated tyrosine phosphorylation of its substrate PLCgamma1. By contrast, deletion of the SH3 domain of ITK resulted in a two-fold increase in the basal activity of ITK, and allowed this mutant to have an enhanced response to Lck. These results suggest that the proline rich region within the Tec homology domain of ITK regulates its basal activity and its response to Src family kinase signals.

Late postoperative opacification of the hydrophobic acrylic intraocular lens in both eyes.

PURPOSE: To report on the clinical findings of a case of late opacification of the hydrophobic acrylic intraocular lens (IOLs) in both eyes after cataract surgery. METHODS: A 79-year-old man with a history of decreased visual acuity and complaints of glare and blurred vision in both eyes over the past 3 years. He had received an uneventful phacoemulsification combined with posterior chamber IOL implantation for senile cataract in both eyes 13 years ago, and had undergone neodymium:YAG(Nd:YAG) laser posterior capsulotomy on both eyes 6 years ago, for posterior capsular opacification (PCO). The optical portion of the IOLs showed uniform gray haze. Binocular posterior capsular laser holes were clearly noted. RESULTS: IOL exchange was performed by the same surgeon on both eyes, three days apart. Postoperatively, the patient's visual acuity was elevated to 6/12 (OD) and 6/9 (OS) at 6 months, and intraocular pressure (IOP) reached 16.8 mmHg (OD) and 18.4 mmHg (OS). Neither glare or blurred vision were observed in either eye. CONCLUSION: IOL exchange can be used to effectively manage clinically significant optic opacification.

Optical coherence tomographic features in a case of bilateral macular coloboma with strabismus.

PURPOSE: To report the optical coherence tomography (OCT) findings in a patient with bilateral macular coloboma with strabismus. METHODS: A 21-year-old male presented with macular coloboma in both eyes.Fundus photography, fundus fluorescence angiography (FFA) and OCT were performed. RESULTS: Color fundus photography showed a sharply-demarcated, round macular defect, approximately 4×4 disc diameters with bare sclera at the base and pigment clumping in both eyes. FFA showed hypofluorescence at the macula corresponding to the size of the lesion bilaterally. OCT revealed a crater-like depression at the macula, demonstrating atrophic neurosensory retina, and an absence of retinal pigment epithelium (RPE) and choroid in the lesion. CONCLUSION: OCT can be a beneficial tool to confirm the diagnosis of macular coloboma.

Differential regulation of NFAT and SRF by the B cell receptor via a PLCgamma-Ca(2+)-dependent pathway.

NFAT and SRF are important in the regulation of proliferation and cytokine production in lymphocytes. NFAT activation by the B cell receptor (BCR) occurs via the PLCgamma-Ca(2+)-calcineurin pathway, however how the BCR activates SRF is unclear. We show here that like NFAT, BCR regulation of SRF occurs via an Src-Syk-Tec-PLCgamma-Ca(2+) (Lyn-Syk-Btk-PLCgamma-Ca(2+)) pathway. However, SRF responds to lower Ca(2+) and is less dependent on IP(3)R expression than NFAT. Ca(2+)-regulated calcineurin plays a partial role in SRF activation, in combination with diacylglycerol (DAG), while is fully required for NFAT activation. Signals from the DAG effectors protein kinase C, Ras and Rap1, and the downstream MEK-ERK pathway are required for both SRF and NFAT; however, NFAT but not SRF is dependent on JNK signals. Both SRF and NFAT were also dependent on Rac, Rho, CDC42 and actin. Finally, we show that Ca(2+) is not required for ERK activation, but instead for its association with nuclear areas of the cell. These data suggest that combinatorial assembly of signaling pathways emanating from the BCR differentially regulate NFAT and SRF, to activate gene expression.