TFIIH, Mediator, and RNAPII (Figure S1). Experiments were completed with the native human HSP70 promoter (HSPA1B gene), because others have shown that it is a quintessential model for promoter-proximal RNAPII pausing (Core et al., 2012). Because chromatin per se does not appear to be an essential regulator of RNAPII pausing in Drosophila or mammalian cells Lai and Pugh, 2017;Li et al., 2013), the in vitro transcription assays were completed on naked DNA templates (also see below).Using purified PIC factors, primer extension assays established that transcription initiation occurred at the annotated HSPA1B start site in vitro (Figure S2A), as expected. An overview of the transcription assay is shown in Figure 1A, which was based in part upon in vitro pausing assays with nuclear extracts (Marshall and Price, 1992;Qiu and Gilmour, 2017;Renner et al., 2001). Following PIC assembly, transcription was initiated by adding ATP, GTP, and UTP at physiologically relevant concentrations, with a low concentration of CTP, primarily 32 P-CTP. After one minute, reactions were chased with a physiologically relevant concentration of cold CTP and transcription was allowed to proceed for an additional nine minutes. These "pulse-chase" assays allow better detection of short (potentially paused) transcripts, which otherwise would be drowned out by elongated transcripts that invariably possess more incorporated 32 P-C bases. By directly labeling all transcripts with 32 P-CTP, the method is highly sensitive and allowed detection of transcripts of varied lengths; furthermore, the 32 P-CTP pulse-chase protocol ensured that 32 P-labeled transcripts resulted almost exclusively from single-round transcription (see Methods). Control experiments confirmed that transcripts detected were driven by the HSP70 promoter (e.g. not any contaminating nucleic acid) and that transcription was dependent on added PIC factors, as expected (Figure S2B).A variety of methods have established that RNAPII pauses after transcribing 20-80 bases in Drosophila and mammalian cells (
TFIIH, Mediator, and RNAPII (Figure S1). Experiments were completed with the native human HSP70 promoter (HSPA1B gene), because others have shown that it is a quintessential model for promoter-proximal RNAPII pausing (Core et al., 2012). Because chromatin per se does not appear to be an essential regulator of RNAPII pausing in Drosophila or mammalian cells Lai and Pugh, 2017;Li et al., 2013), the in vitro transcription assays were completed on naked DNA templates (also see below).Using purified PIC factors, primer extension assays established that transcription initiation occurred at the annotated HSPA1B start site in vitro (Figure S2A), as expected. An overview of the transcription assay is shown in Figure 1A, which was based in part upon in vitro pausing assays with nuclear extracts (Marshall and Price, 1992;Qiu and Gilmour, 2017;Renner et al., 2001). Following PIC assembly, transcription was initiated by adding ATP, GTP, and UTP at physiologically relevant concentrations, with a low concentration of CTP, primarily 32 P-CTP. After one minute, reactions were chased with a physiologically relevant concentration of cold CTP and transcription was allowed to proceed for an additional nine minutes. These "pulse-chase" assays allow better detection of short (potentially paused) transcripts, which otherwise would be drowned out by elongated transcripts that invariably possess more incorporated 32 P-C bases. By directly labeling all transcripts with 32 P-CTP, the method is highly sensitive and allowed detection of transcripts of varied lengths; furthermore, the 32 P-CTP pulse-chase protocol ensured that 32 P-labeled transcripts resulted almost exclusively from single-round transcription (see Methods). Control experiments confirmed that transcripts detected were driven by the HSP70 promoter (e.g. not any contaminating nucleic acid) and that transcription was dependent on added PIC factors, as expected (Figure S2B).A variety of methods have established that RNAPII pauses after transcribing 20-80 bases in Drosophila and mammalian cells (
Diabetes mellitus (DM), a disorder rapidly growing in prevalence, is linked to the retinal microvasculature complication diabetic retinopathy (DR). As one of the leading global causes of vision impairment and loss, imaging techniques to detect and monitor DR must continue to improve in order to address this growing burden. Optical coherence tomography angiography (OCTA) is a nascent imaging modality that generates three-dimensional visualizations of the retinal and choroidal microvasculature. Compared to fluorescein angiography, the gold-standard imaging modality for retinal vessels, OCTA offers the advantages of being non-invasive, quick, and able to resolve the multiple plexuses within the retina. Quantitative OCTA studies have explored parameters such as vessel density (VD), foveal avascular zone (FAZ), acircularity index, vessel tortuosity (VT), and fractal dimension (FD) amongst DR patients. This review synthesizes the main trends emerging from quantitative OCTA-based studies of DR and interrogates them within the context of DR pathophysiology. We offer a glimpse into how analysis techniques have shifted in the years since OCTA came into existence, while speculating on its future role in clinical practice.
Computerized texture analysis uses higher-order mathematics to identify patterns beyond what the naked eye can recognize. We tested its feasibility in optical coherence tomography angiography imaging of choriocapillaris. Our objective was to determine sets of parameters that provide coherent and consistent output when applied to a homogeneous, healthy group of patients. This observational cross-sectional study involved 19 eyes of 10 young and healthy Caucasian subjects. En-face macular optical coherence tomography angiography of superficial choriocapillaris was obtained by the RTVue-XR Avanti system. Various algorithms were used to extract texture features. The mean and standard deviation were used to assess the distribution and dispersion of data points in each metric among eyes, which included: average gray level, gray level yielding 70% threshold and 30% threshold, balance, skewness, energy, entropy, contrast, edge mean gradient, root-mean-square variation, and first moment of power spectrum, which was compared between images, showing a highly concordant homology between all eyes of participants. We conclude that computerized texture analysis for en-face optical coherence tomography angiography images of choriocapillaris is feasible and provides values that are coherent and tightly distributed around the mean in a homogenous, healthy group of patients. Homology of blob size among subjects may represent a “repeat pattern” in signal density and thus a perfusion in the superficial choriocapillaris of healthy young individuals of the same ethnic background.
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