Vision rescue via unconstrained in vivo prime editing in degenerating neural retinas.

Retinitis pigmentosa (RP) is an inherited retinal dystrophy causing progressive and irreversible loss of retinal photoreceptors. Here, we developed a genome-editing tool characterized by the versatility of prime editors (PEs) and unconstrained PAM requirement of a SpCas9 variant (SpRY), referred to as PESpRY. The diseased retinas of Pde6b-associated RP mouse model were transduced via a dual AAV system packaging PESpRY for the in vivo genome editing through a non-NGG PAM (GTG). The progressing cell loss was reversed once the mutation was corrected, leading to substantial rescue of photoreceptors and production of functional PDE6ß. The treated mice exhibited significant responses in electroretinogram and displayed good performance in both passive and active avoidance tests. Moreover, they presented an apparent improvement in visual stimuli-driven optomotor responses and efficiently completed visually guided water-maze tasks. Together, our study provides convincing evidence for the prevention of vision loss caused by RP-associated gene mutations via unconstrained in vivo prime editing in the degenerating retinas.

Microglia: The breakthrough to treat neovascularization and repair blood-retinal barrier in retinopathy.

Microglia are the primary resident retinal macrophages that monitor neuronal activity in real-time and facilitate angiogenesis during retinal development. In certain retinal diseases, the activated microglia promote retinal angiogenesis in hypoxia stress through neurovascular coupling and guide neovascularization to avascular areas (e.g., the outer nuclear layer and macula lutea). Furthermore, continuously activated microglia secrete inflammatory factors and expedite the loss of the blood-retinal barrier which causes irreversible damage to the secondary death of neurons. In this review, we support microglia can be a potential cellular therapeutic target in retinopathy. We briefly describe the relevance of microglia to the retinal vasculature and blood-retinal barrier. Then we discuss the signaling pathway related to how microglia move to their destinations and regulate vascular regeneration. We summarize the properties of microglia in different retinal disease models and propose that reducing the number of pro-inflammatory microglial death and conversing microglial phenotypes from pro-inflammatory to anti-inflammatory are feasible for treating retinal neovascularization and the damaged blood-retinal barrier (BRB). Finally, we suppose that the unique properties of microglia may aid in the vascularization of retinal organoids.

Roles of Histone Acetyltransferases and Deacetylases in the Retinal Development and Diseases.

The critical role of epigenetic modification of histones in maintaining the normal function of the nervous system has attracted increasing attention. Among these modifications, the level of histone acetylation, modulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), is essential in regulating gene expression. In recent years, the research progress on the function of HDACs in retinal development and disease has advanced remarkably, while that regarding HATs remains to be investigated. Here, we overview the roles of HATs and HDACs in regulating the development of diverse retinal cells, including retinal progenitor cells, photoreceptor cells, bipolar cells, ganglion cells, and Müller glial cells. The effects of HATs and HDACs on the progression of various retinal diseases are also discussed with the highlight of the proof-of-concept research regarding the application of available HDAC inhibitors in treating retinal diseases.

Prenatal phthalate exposure reduction through an integrated intervention strategy.

Pregnancy represents a sensitive susceptibility window to phthalate esters (PAEs). In this study, we develop an intervention strategy for reducing the exposure of pregnant women to phthalates. Thirty-five pregnant women, who initially underwent maternity examination, were recruited from an ongoing longitudinal prospective prenatal cohort study. The intervention strategy integrates diet, lifestyle, and environmental factors. Participants were encouraged to modify their behaviors and habits according to the intervention strategy at three different periods. Urine samples were collected from the participants after antenatal examination every month, for 8 months, to measure ten PAE metabolites. Mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono-n-butyl phthalate (MnBP), and mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP) declined significantly after the 1st intervention, while mono-isobutyl phthalate (MiBP) and mono-methyl phthalate (MMP) noticeably decreased after the 2nd intervention. The sum of the molar concentrations of MEHP, MEHHP, MEOHP, and MECPP reduced by 20 to 40% during subsequent intervention. In addition, the sum of the molar concentrations of MEP, MnBP, MMP, and MiBP as well as the sum of the molar concentrations of the ten metabolites also reduced. Our findings suggest that intervention through written recommendations can effectively reduce the burden of phthalates during pregnancy.

Notch Signaling Activation in Cervical Cancer Cells Induces Cell Growth Arrest with the Involvement of the Nuclear Receptor NR4A2.

Cervical cancer is a second leading cancer death in women world-wide, with most cases in less developed countries. Notch signaling is highly conserved with its involvement in many cancers. In the present study, we established stable cervical cell lines with Notch activation and inactivation and found that Notch activation played a suppressive role in cervical cancer cells. Meanwhile, the transient overexpression of the active intracellular domain of all four Notch receptors (ICN1, 2, 3, and 4) also induced the suppression of cervical cancer Hela cell growth. ICN1 also induced cell cycle arrest at phase G1. Notch1 signaling activation affected the expression of serial genes, especially the genes associated with cAMP signaling, with an increase of genes like THBS1, VCL, p63, c-Myc and SCG2, a decrease of genes like NR4A2, PCK2 and BCL-2. Particularly, The nuclear receptor NR4A2 was observed to induce cell proliferation via MTT assay and reduce cell apoptosis via FACS assay. Furthermore, NR4A2's activation could reverse ICN1-induced suppression of cell growth while erasing ICN1-induced increase of tumor suppressor p63. These findings support that Notch signaling mediates cervical cancer cell growth suppression with the involvement of nuclear receptor NR4A2. Notably, Notch/NR4A2/p63 signaling cascade possibly is a new signling pathway undisclosed.

Valproic acid exhibits different cell growth arrest effect in three HPV-positive/negative cervical cancer cells and possibly via inducing Notch1 cleavage and E6 downregulation.

We investigated the effect of valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, and the mechanism of VPA-induced growth inhibition on three cervical cancer cell lines with different molecular and genetic background. We found that VPA induced proliferation suppression, cell apoptosis and cell cycle arrest in all tested cell lines, with an increase of Notch1 active form ICN1 as a tumor suppressor and its target gene HES1. Noteworthy, blocking of Notch signaling with DAPT resulted in growth inhibition in ICN1-overexpressing CaSki and HT-3 cells. Thus, endogenous Notch signaling may be necessary for survival of ICN1-overexpressing cervical cancer cell lines. Furthermore, G1 phase arrest was induced in HeLa and CaSki cells by VPA while G2 phase arrest was induced in HT-3 cells, suggesting different mechanism in this cycle arrest. We also found VPA suppressed oncogene E6 in a Notch-independent manner, and induced significant apoptosis in E6-overexpressing HPV positive CaSki cells. Cell morphological change was also observed in HeLa and HT-3 cell lines after VPA treatment with an upregulation of EMT transcription factor Snail1. Notch signaling inhibitor DAPT partly reversed VPA-induced Snail1 upregulation in HeLa cells. This discovery supports that VPA may induce EMT at least partly via Notch activation.

[Expression of epidermal growth factor receptor and the correlation with HPV16/18 infection in cervical intraepithelial neoplasia and cervical carcinoma].

OBJECTIVE: The aim of this study was to elucidate the role of EGFR expression and HPV infection and their relationship in the genesis and progression of cervical carcinoma. METHODS: This analysis included 60 cases of cervical carcinoma, 40 cases of CIN and 30 cases of control group. Patients of cervical carcinoma group were all subjected to radical hysterectomy with bilateral pelvic lymphadenectomy in Tumour Hospital, Zhongshan University from 1997 to 2001. The FIGO stage was I a - II b. EGFR protein was stained by S-P immunohistochemistry, and HPV16/18 DNA was detected by PCR. RESULTS: The moderate/ strong expression of EGFR was observed in 0, 42.5%, 76.7% of normal cervical epithelium, CIN and cervical tumor tissue, respectively, with a significant difference among them (P < 0.05). The infection of HPV16/18 was observed in 6.7%, 67.5%, 58.3% of normal cervical tissue, CIN and cervical tumors, respectively. The infection rate of CIN or cervical carcinoma was significantly higher than that in normal cervicaltissue (P = 0.000), but no statistically significant difference was observed between cervical carcinoma and CIN. The moderate/strong expression of EGFR demonstrated an association with the cervical invasion depth. The EGFR expression increased significantly as the invasion depth progressed from less than or equal to a half cervical stroma to deeper than that (89.2% vs. 56.5%, P = 0.004). The infection of HPV16/18 demonstrated a correlation with the cervical canal invasion. The infection increased significantly in the cases with cervical invasion than that in the cases without invasion (88.2% vs. 46.5%, P = 0.003). But no significant correlation was observed between EGFR and HPV. Neither EGFR nor HPV had a significant association with carcinoma prognosis. CONCLUSION: EGFR and HPV demonstrate a significant correlation with genesis and progression of cervical carcinoma. In our study, neither EGFR nor HPV demonstrates a significant association with tumor prognosis, and no significant correlation is observed between EGFR and HPV.

[Risk factors and prognosis of node-positive cervical carcinoma].

BACKGROUND & OBJECTIVE: Pelvic lymph node metastasis is an important prognostic factor of cervical carcinoma, but few have been reported on both risk factors and prognostic factors of node-positive cervical carcinoma. This study was to evaluate risk factors, distribution pattern, and prognosis of node-positive cervical carcinoma, and explore the forecast of lymph node metastasis and appropriate treatment. METHODS: Clinical data of 205 cervical cancer patients who had undergone radical hysterectomy and bilateral pelvic lymphadenectomy were analyzed retrospectively. RESULTS: The overall prevalence of lymph node metastasis was 24.4% (50/205). Univariate analysis showed that risk factors of node metastasis were serum level of squamous cell carcinoma antigen (SCC-Ag) before treatment, clinical stage, invasive depth of cervical canal or vaginal portion of the cervix, and uterine ligaments involvement. SCC values exceeding 4 microg/L increased the risk of nodal metastasis by 4.2 folds (P<0.001, OR=4.212). Multivariate analysis showed that clinical stage and invasive depth of cervical canal were the major risk factors. The obturator and obturator fossae lymph nodes were the most frequently involved, with a rate of 48.0%. Moreover, 60.0% node-positive patients had multiple sites lymph node metastases, and saltatory metastasis was found. Lymph node metastasis was closely related to deep muscularis involvement of the cervix and parametrial involvement; 72.0% nodal metastases were accompanied with deep muscularis involvement of the cervix, 90.9% uterine ligament invasions were accompanied with lymph node metastasis. The 5-year survival rate was significantly higher in the patients received postoperative radiation than in the patients didn't receive radiation (89.1% vs. 45.5%, P=0.012). CONCLUSIONS: Serum level of SCC-Ag before treatment exceeding 4 microg/L, deep muscularis involvement of vaginal portion of the cervix, uterine ligaments involvement, especially advanced stage and deep muscularis involvement of the cervical canal, are risk factors of pelvic lymph node metastasis of cervical cancer. The standard type III radical hysterectomy and bilateral pelvic lymphadenectomy should be performed to the patients with high risk of lymph node metastasis to ensure enough amplitude of parametrectomy and excision of positive nodes. When lymph node metastasis is confirmed after surgery, postoperative radiation can improve the prognosis.