Circle-seq based method for eccDNA synthesis and its application as a canonical promoter independent vector for robust microRNA overexpression.

Extrachromosomal circular DNA (eccDNA) has recently gained increasing attention due to its significant role in cancer and other pathophysiologic states. The majority of circular DNAs detected by Circle-seq are small-size eccDNAs with enigmatic functions. One major technical hurdle is to synthesize eccDNA for functional identification. Here, we describe CAES (Circle-seq based Artificial EccDNA Synthesis), a promising and reliable method for artificial eccDNA synthesis. Eight eccDNAs carrying different microRNA genes (eccMIR) found in gastric cancer tissues, ranging from 329 bp to 2189 bp in size, were created utilizing the CAES method. Exonuclease V and single restriction-endonuclease digestion identified the circular structure of synthetic eccDNAs. The DNA circularization efficiency afforded by CAES ranged from 15.6% to 31.1%, which was negatively correlated with the eccDNA length. In addition, we demonstrated that CAES-synthesized eccMIRs can express both miRNA-3p and - 5p molecules efficiently independent of a canonical promoter in human cell lines. Further assays proved that these eccMIRs were functional as they were able to repress the luciferase gene containing a miRNA-target sequence in the 3'UTR as well as the endogenous mRNA targets. Finally, kinetics study revealed that eccDNA exhibited a decay rate similar to the standard plasmids and linear DNA in cultured cells. Together, this study offers a rapid and convenient method for Circle-seq users to synthesize artificial eccDNAs. It also demonstrates the promising potential of eccMIR as a bacterial DNA-free vector for safe and robust miRNA overexpression in both basic research and therapeutic applications.

Genome-wide characterization of extrachromosomal circular DNA in gastric cancer and its potential role in carcinogenesis and cancer progression.

Extrachromosomal circular DNAs (eccDNAs) carrying random genomic segments are broadly found across different cancer types, but their molecular functions and impact in gastric cancer (GC) are rarely known. In this study, we aimed to investigate the potential role of eccDNA in GC. Using the Circle-seq strategy, we observed the eccDNA abundance in gastric cancer tissues (GCT) was aberrantly higher than that of normal adjacent tissues (NAT). The high abundance of eccDNAs carrying oncogene-segments in GCT may represent the DNA damage products of amplified oncogenes. Analysis of GCT over-represented eccDNA carrying enhancer (eccEnhancer) based on data from FANTOM5 project combined with TCGA database suggested the GC over-represented eccEnhancers may contribute to development of GC. GC over-represented eccDNAs carrying pre-miRNA (eccMIR) were enriched to multiple cancer-relevant signal pathways by KEGG analysis. We then synthesized the top six GC over-represented eccMIRs and found four of them enabled high expression of miRNAs and down-regulation of miRNA-target genes in MGC803 cells. Furthermore, we observed the inheritance of GC over-represented eccMIRs benefited host cell proliferation and promoted the aggressive features of host cells. Altogether, this study revealed the GC over-represented eccDNAs carrying functional genomic segments were related to the carcinogenesis of GC and presented the capability to facilitate cancer progression, suggesting the cancerous eccDNAs may serve as a dynamic reservoir for genome plasticity and rapid adaptive evolution of cancer. Therefore, blocking the pathways for eccDNAs generation may provide a novel therapeutic strategy for the treatment of gastric cancer.

Variation of choroidal thickness during the waking period over three consecutive days in different degrees of myopia and emmetropia using optical coherence tomography.

Purpose: To investigate the diurnal variation in subfoveal choroidal thickness (SFCT) during the waking period over three consecutive days in different degrees of myopia and emmetropia. Methods: A total of 60 adult volunteers were grouped into low, moderate, high myopia, and emmetropia subgroups. SFCT, axial length (AL), anterior chamber depth (ACD), and intraocular pressure (IOP) were measured every 2 h from 8 AM to 8 PM for three successive days. Results: The mean values of daily change amplitude were 3.18 mmHg (IOP), 0.05 mm (AL), 0.17 mm (ACD), and 13.51 µm (SFCT). The values of AL and ACD increased simultaneously with spherical equivalent refraction (SER), but SFCT was the opposite. IOP had a diurnal variation, and there was no difference among the four groups. AL of the high myopia group, ACD of the emmetropia group, and SFCT of each myopia group had diurnal variation over three consecutive days. AL had a high mean value at noon every day, and SFCT had a low mean value at noon every day. Conclusion: The choroid thickness of subjects with different degrees of myopia had a significant diurnal variation. The change of diurnal variation between emmetropic and myopic subjects may be one of the causes of myopia.

Changes in Retinal Vessel Flow after Small Incision Lenticule Extraction.

Objective: In order to analyze changes in retinal vessel flow after small incision lenticule extraction (SMILE). Methods: A total of 32 patients (62 eyes) who underwent SMILE were enrolled in this prospective study. Optical parameters, including vessel density (VD), and perfusion density (PD) of foveal, parafoveal, and perifoveal regions, respectively, were measured before surgery and at 1 day, 1 week, 1 month, and 3 months postoperation. Preoperative parameters and surgical parameters were recorded. Results: Significant decreases in VD and PD on postoperative day 1 were detected in all quadrants, both in 3 mm and in 6 mm regions (P < 0.001). One month after surgery, VD returned to preoperative levels. None of the preoperative and surgical parameters were significantly correlated with the VD and PD fluctuations (all P > 0.05). Conclusion. VD may decrease significantly with regional disparity 1 day after SMILE while recovering at 1 month. Elevation of intraocular pressure due to suction may account for such changes.

Proportion and characteristic of emmetropia in schoolchildren aged 6-11y: the Shenzhen elementary school eye study.

AIM: To investigate the proportion and characteristic of emmetropia in schoolchildren aged 6-11, especially estimate the normal value of ocular biometric parameters of emmetropia. METHODS: A population-based cross-sectional study was conducted on children aged 6-11y in Shenzhen. Totally, 2386 schoolchildren from two primary schools were involved. The axial length (AL) and the corneal radius of curvature (CRC) were measured by partial coherence laser interferometry. Noncycloplegic refraction and refractive astigmatism (RA) was measured using autorefraction. The axial length-to-corneal radius of curvature ratio (AL/CRC), corneal astigmatism (CA) and spherical equivalent refraction (SER) were calculated. RESULTS: The proportion of emmetropia in elementary school students was 41.30%. This percentage decreased gradually from 6 to 11 years of age and decreased rapidly after 9 years of age. The mean and 95%CI of each parameter were provided for boys and girls aged 6 to 11 years of age with emmetropia according to each age group. The change trend of parameters of boys and girls are similar. After 7 years of age, the AL of non-emmetropia started to increase faster than that of emmetropia. The change trend of AL/CRC was the same as that of AL. The other parameters tend to be stable after 7 years of age. CONCLUSION: The age of 7-9 is an important period for the changes of refractive state and ocular biometric parameters of primary school students, and it is a special focus period for children myopia prevention. The normal value and variation of ocular biometric parameters of emmetropia can provide the basis for the clinical judgment of whether or not children's ocular biometric parameters obtained by single measurement and changes obtained by multiple measurements are abnormal.

Neural stem cells induce M2 polarization of macrophages through the upregulation of interleukin-4.

Macrophages are divided into two types: M1- and M2-type macrophages. Both types of macrophages serve important roles during the process of inflammation. M1-type macrophages release various pro-inflammatory cytokines, such as IL-1, IFN-γ and other inflammatory mediators, such as nitric oxide, glutamate and reactive oxygen species to generate inflammation. In contrast, M2-type macrophages counteract the pro-inflammatory M1 conditions and promote tissue repair by secreting anti-inflammatory cytokines, such as IL-10. In spinal cord injury (SCI), an imbalance in M1/M2 macrophages leads to irreversible tissue destruction. Thus, it is crucial to increase the number of M2-type macrophages and promote M2 polarization of macrophages in SCI. Accordingly, in this study an in vitro co-culture system was established to investigate the effect of neural stem cells (NSCs) on macrophages. The results of the present study demonstrated that NSCs induced M2 polarization and suppressed M1 polarization of macrophages in an interleukin (IL)-4-dependent manner. Furthermore, the nuclear factor (NF)-κB/p65 signaling pathway was involved in the M1 polarization of macrophages and NSCs suppressed the activation of the NF-κB/p65 pathway in an IL-4-dependent manner to induce M2 macrophage polarization. These findings provide more insight into SCI and help to identify novel treatment strategies.

Multimodal imaging of aberrant macular microvessel crossing the foveal avascular zone in two young adults.

BACKGROUND: The traditional view is that there are no vessels in the foveal avascular zone. The two cases we report show microvessels crossing the foveal avascular zone. CASE PRESENTATION: A man and a woman, both 25 years old, were both incidentally found on optical coherence tomography angiography (OCTA) to have unilateral aberrant microvessels crossing the foveal avascular zone in their left eyes. Visual acuity was preserved in both patients. The vessel density (VD) and perfusion density (PD) of the eyes with the aberrant microvessels were all higher than those of the contralateral eyes. Nevertheless, measurements of foveal avascular zone (FAZ) dimensions, including its area, perimeter and circularity, were smaller in the left eyes than in the right eyes. No complications were recorded. CONCLUSIONS: To date, aberrant microvessels crossing the foveal avascular zone have not been found to impair visual function. OCTA is a non-invasive and quick method that does not require dilation or the use of fluorescein dye. It is a reliable tool for the detection of aberrant microvessels crossing the foveal avascular zone.

[A clinical study of actual corneal ablation depth in laser in situ keratomileusis].

PURPOSE: To assess the predictability of corneal ablation depth in LASIK using NIDEK EC5000 excimer laser. METHODS: Standard LASIK surgery was performed in 79 myopic patients with or without astigmatism with the NIDEK EC5000 excimer laser system.Ultrasonic corneal pachymetry was performed immediately after flap creation and after laser ablation during LASIK procedure,by which the actual corneal ablation depth was calculated.The values of actual and predicted ablation depth were compared. RESULTS: The actual ablation depth was (92.32+/-29.86)µm, the predicted ablation depth was (74.16+/-25.95)µm.The differences between them(18.16+/-14.71)µm were statistically significant(p<0.001).Linear regression suggested that the actual ablation depth correlated closely with the predicted ablation depth(r=0.87,p<0.001).The regression model was Y=18.06+1.001X.The differences remained statistically significant and were independent of the levels of preoperative corneal keratometry,absolute preoperative spherical equivalent and the preoperative central cornea thickness. CONCLUSIONS: The actual ablation depth was about(18.16+/-14.71)µm thicker than the predicted ablation depth in the NIDEK EC5000 excimer laser system. We may have to take into account this deviation in order to ensure sufficient thickness of residual stromal bed.