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The Sox family of transcription factors plays a crucial role in the development of the vertebrate nervous system. In the zebrafish embryo,sox1genes are expressed in neural progenitor cells and neurons of the ventral spinal cord. We recently reported that the loss of function ofsox1aandsox1bleads to a significant decline in a subtype of V2 neurons, called V2s, in zebrafish. Here, a single-cell RNA sequencing approach was used to analyse the transcriptome ofsox1alineage progenitors and neurons in the zebrafish spinal cord at four different time points during the first five days of embryonic development, using the Tg(sox1a:eGFP) line. In addition to the previously describedsox1a-expressing neurons, we found thatsox1ais also expressed in late-developing intraspinal serotonergic neurons (ISNs). Analysis of developmental trajectories from single-cell data and depletion of lateral floor plate (LFP) cells bynkx2.9morpholino knockdown suggest that ISNs arise from LFP precursor cells. Pharmacological inhibition of the Notch signalling pathway indicates that this pathway is required for the negative regulation of the development of LFP progenitor cells into ISN populations. Our results show that the zebrafish LFP is a precursor domain that longitudinally gives rise to ISNs in addition to the previously described KA” and V3 interneurons.

Tumor-associated macrophages (TAMs) are crucially associated with tumor development and progression; however, it remains unclear how the tumor microenvironment (TME) rewires the metabolic circuits and preferentially induces TAMs to polarize toward a protumoral phenotype. Here, we report that polyunsaturated fatty acids (PUFAs) in malignant ascites promote protumoral M2-like TAMs deposition and facilitate peritoneal metastases of epithelial ovarian cancer (EOC). We demonstrated that PUFAs in the lipid-enriched malignant ascites inactivate RhoA, reducing nuclear YAP1 in macrophages and promoting protumoral M2-like TAMs polarization with OXPHOS metabolism. Conditional Yap1 depletion in murine MΦs leads to skew macrophage polarization toward protumoral M2-like TAMs that, in turn, suppress CD8+ T cell infiltration and aggravate tumor colonization in vivo. Noticeably, the significance of nuclear YAP1 depletion was evinced in the infiltrating TAMs in tumor spheroids of malignant ascites from EOC patients. In contrast, restored nuclear YAP1 expression in TAMs by pharmacological suppression of MST1/2 enhances tumoricidal M1-like TAMs population and CD8+ T cells infiltration, restricting EOC peritoneal metastasis. These results indicate that PUFAs are a key player in promoting tumor-infiltrated TAMs polarization that, in turn, facilitates EOC tumor growth and metastasis.

Refractive disorder is the most prevalent cause of visual impairment worldwide. While treatment of refractive errors can bring improvement to quality of life and socio-economic benefits, there is a need for individualization, precision, convenience, and safety with the chosen method. Herein, we propose using pre-designed refractive lenticules based on poly-NAGA-GelMA (PNG) bio-inks photo-initiated by digital light processing (DLP)-bioprinting for correcting refractive errors. DLP-bioprinting allows PNG lenticules to have individualized physical dimensions with precision achievable to 10 µm (μm). Material characteristics of PNG lenticules in tests included optical and biomechanical stability, biomimetical swelling and hydrophilic capability, nutritional and visual functionality, supporting its suitability as stromal implants. Cytocompatibility distinguished by morphology and function of corneal epithelial, stromal, and endothelial cells on PNG lenticules suggested firm adhesion, over 90% viability, phenotypic maintenance instead of excessive keratocyte-myofibroblast transformation. In-vitro immune response analyzed by illumina RNA sequencing in human peripheral blood mononuclear cells indicated that PNG lenticules activated type-2 immunity, facilitating tissue regeneration and suppressing inflammation. In-vivo performance assessed using intrastromal keratoplasty models in New Zealand white rabbits illustrated that implantation of PNG lenticules maintained stable optical pathway, induced controlled stromal bio-integration and regeneration, avoided complications such as stromal melt, interface scarring, etc, but exerted no adverse effects on the host. Postoperative follow-up examination on intraocular pressure, corneal sensitivity, and tear production remained unaffected by surgery up to 1-month post-implantation of PNG lenticules. DLP-bioprinted PNG lenticule is a bio-safe and functionally effective stromal implants with customizable physical dimensions, providing potential therapeutic strategies in correction of refractive errors.

Malignant ascites in peritoneal metastases is a lipid-enriched microenvironment and is frequently involved in the poor prognosis of epithelial ovarian cancer (EOC). However, the detailed mechanisms underlying ovarian cancer (OvCa) cells dictating their lipid metabolic activities in promoting tumor progression remain elusive. Here, we report that two critical fatty acid desaturases, stearoyl-CoA desaturase-1 (SCD1) and acyl-CoA 6-desaturase (FADS2), are aberrantly upregulated, accelerating lipid metabolic activities and tumor aggressiveness of ascites-derived OvCa cells. Lipidomic analysis revealed that the elevation of unsaturated fatty acids (UFAs) is positively associated with SCD1/FADS2 levels and the oncogenic capacities of OvCa cells. In contrast, pharmaceutical inhibition and genetic ablation of SCD1/FADS2 retarded tumor growth, suppressed cancer stem cell (CSC) formation and reduced platinum resistance in OvCa cells. Mechanistically, inhibition of SCD1/FADS2 directly downregulated GPX4 and the GSH/GSSG ratio, causing disruption of the cellular redox balance and subsequent iron-mediated lipid peroxidation in ascites-derived OvCa cells. Hence, combinational treatment with SCD1/FADS2 inhibitors and cisplatin synergistically repressed tumor cell dissemination, providing a promising chemotherapeutic strategy against EOC platinum resistance and peritoneal metastases.

Simple Summary Omental metastasis and peritoneal dissemination are frequently observed in ovarian cancer peritoneal metastases and are associated with high mortality and poor prognosis. The tumor microenvironment is known to influence cancer epigenomics, which plays an essential role in promoting tumor development and metastatic progression. Therefore, investigation of the epigenetic mechanisms underlying the growth of ovarian cancer cells in the omental metastatic microenvironment is of great importance. Here, we report that miR-33b is significantly silenced by DNA hypermethylation in metastatic ovarian cancer cells to adapt to a lipid-rich microenvironment. Restoration of miR-33b was shown to impair lipid metabolic activities and reduce the oncogenic properties of ovarian cancer cells by negatively regulating the TAK1/FASN/CPT1A/NF-κB pathway, indicating that targeting this signaling cascade may be a molecular therapeutic choice for ovarian cancer metastatic progression. Abstract Peritoneal metastases are frequently found in high-grade serous carcinoma (HGSOC) patients and are commonly associated with a poor prognosis. The tumor microenvironment (TME) is a complex milieu that plays a critical role in epigenetic alterations driving tumor development and metastatic progression. However, the impact of epigenetic alterations on metastatic ovarian cancer cells in the harsh peritoneal microenvironment remains incompletely understood. Here, we identified that miR-33b is frequently silenced by promoter hypermethylation in HGSOC cells derived from metastatic omental tumor tissues. Enforced expression of miR-33b abrogates the oncogenic properties of ovarian cancer cells cocultured in omental conditioned medium (OCM), which mimics the ascites microenvironment, and in vivo tumor growth. Of note, restoration of miR-33b inhibited OCM-upregulated de novo lipogenesis and fatty acid β-oxidation in ovarian cancer cells, indicating that miR-33b may play a novel tumor suppressor role in the lipid-mediated oncogenic properties of metastatic ovarian cancer cells found in the omentum. Mechanistic studies demonstrated that miR-33b directly targets transforming growth factor beta-activated kinase 1 (TAK1), thereby suppressing the activities of fatty acid synthase (FASN) and carnitine palmitoyltransferase 1A (CPT1A) in modulating lipid metabolic activities and simultaneously inhibiting the phosphorylation of NF-κB signaling to govern the oncogenic behaviors of ovarian cancer cells. Thus, our data suggest that a lipid-rich microenvironment may cause epigenetic silencing of miR-33b, which negatively modulates ovarian cancer peritoneal metastases, at least in part, by suppressing TAK1/FASN/CPT1A/NF-κB signaling.

Background In contrast to stable genetic events, epigenetic changes are highly plastic and play crucial roles in tumor evolution and development. Epithelial ovarian cancer (EOC) is a highly heterogeneous disease that is generally associated with poor prognosis and treatment failure. Profiling epigenome-wide DNA methylation status is therefore essential to better characterize the impact of epigenetic alterations on the heterogeneity of EOC. Methods An epigenome-wide association study was conducted to evaluate global DNA methylation in a retrospective cohort of 80 mixed subtypes of primary ovarian cancers and 30 patients with high-grade serous ovarian carcinoma (HGSOC). Three demethylating agents, azacytidine, decitabine, and thioguanine, were tested their anti-cancer and anti-chemoresistant effects on HGSOC cells. Results Global DNA hypermethylation was significantly associated with high-grade tumors, platinum resistance, and poor prognosis. We determined that 9313 differentially methylated probes (DMPs) were enriched in their relative gene regions of 4938 genes involved in small GTPases and were significantly correlated with the PI3K-AKT, MAPK, RAS, and WNT oncogenic pathways. On the other hand, global DNA hypermethylation was preferentially associated with recurrent HGSOC. A total of 2969 DMPs corresponding to 1471 genes were involved in olfactory transduction, and calcium and cAMP signaling. Co-treatment with demethylating agents showed significant growth retardation in ovarian cancer cells through differential inductions, such as cell apoptosis by azacytidine or G2/M cell cycle arrest by decitabine and thioguanine. Notably, azacytidine and decitabine, though not thioguanine, synergistically enhanced cisplatin-mediated cytotoxicity in HGSOC cells. Conclusions This study demonstrates the significant association of global hypermethylation with poor prognosis and drug resistance in high-grade EOC and highlights the potential of demethylating agents in cancer treatment. Graphic abstract

The central nervous system of adult zebrafish displays an extraordinary neurogenic and regenerative capacity. In the zebrafish adult brain, this regenerative capacity relies on neural stem cells (NSCs) and the careful management of the NSC pool. However, the mechanisms controlling NSC pool maintenance are not yet fully understood. Recently, Bone Morphogenetic Proteins (BMPs) and their downstream effector Id1 (Inhibitor of differentiation 1) were suggested to act as key players in NSC maintenance under constitutive and regenerative conditions. Here, we further investigated the role of BMP/Id1 signaling in these processes, using different genetic and pharmacological approaches. Our data show that BMPs are mainly expressed by neurons in the adult telencephalon, while id1 is expressed in NSCs, suggesting a neuron-NSC communication via the BMP/Id1 signaling axis. Furthermore, manipulation of BMP signaling by conditionally inducing or repressing BMP signaling via heat-shock, lead to an increase or a decrease of id1 expression in the NSCs, respectively. Induction of id1 was followed by an increase in the number of quiescent NSCs, while knocking down id1 expression caused an increase in NSC proliferation. In agreement, genetic ablation of id1 function lead to increased proliferation of NSCs, followed by depletion of the stem cell pool with concomitant failure to heal injuries in repeatedly injured mutant telencephala. Moreover, pharmacological inhibition of BMP and Notch signaling suggests that the two signaling systems cooperate and converge onto the transcriptional regulator her4.1. Interestingly, brain injury lead to a depletion of NSCs in animals lacking BMP/Id1 signaling despite an intact Notch pathway. Taken together, our data demonstrate how neurons feedback on NSC proliferation and that BMP1/Id1 signaling acts as a safeguard of the NSC pool under regenerative conditions.

Purpose To study the in vitro effect of vitamin D3 on the healing response of human Tenon's fibroblasts (HTF) and its possible role in preventing excessive postoperative subconjunctival fibrosis. Methods Effect of vitamin D3 on cytotoxicity and cell survival of primary cultured HTF was measured by lactate dehydrogenase and PrestoBlue assays, respectively. Proliferation and migration of vitamin D3-treated HTF (D3-HTF) was determined by CyQUANT proliferation and scratch assay, respectively. The mRNA expression profiles of control-HTF and D3-HTF from six subjects (three with glaucoma and long-term use of topical medications, three with primary pterygium) were assessed by RNA sequencing analyses to identify potential biomarkers for the inhibitory effect on HTF by vitamin D3. Validation of these biomarkers and their potential pathways were performed by quantitative real-time polymerase chain reaction (qRT-PCR) detection. Results Pure monolayers of HTF from controls (retinal detachment or squint surgeries), pterygium, and glaucoma subjects were successfully prepared and passaged. Proliferation and migration of pterygium and glaucoma HTF were inhibited by vitamin D3 in a dose-dependent manner, and without cytotoxicity or decrease in cellular viability with concentrations up to 10 µM. The qRT-PCR results were consistent with the transcriptome analyses, vitamin D3 appears to enhance CYP24A1, SHE, KRT16 but suppresses CILP expression in HTF. Conclusions Vitamin D3 can inhibit the in vitro activity of HTF without compromising cellular survivability at concentration up to 10 µM. This has potential clinical application for improving the outcome of pterygium and filtering surgeries. Translational relevance Vitamin D3 can suppress the in vitro proliferation, migration, and transdifferentiation of human Tenon's fibroblasts, without the cytotoxicity of mitomycin-C, the current standard antifibrotic agent in clinical use.

Motivation: DNA methylation is an important epigenetic modification that has essential role in gene regulation, cell differentiation and cancer development. Bisulfite sequencing is a widely used technique to obtain genome-wide DNA methylation profiles, and one of the key tasks of analyzing bisulfite sequencing data is to detect differentially methylated regions (DMRs) among samples under different treatment conditions. Although numerous tools have been proposed to detect differentially methylated single CpG site (DMC) between samples, methods for direct DMR detection, especially for complex study designs, are largely limited. Results: We present a new software, GetisDMR, for direct DMR detection. We use beta-binomial regression to model the whole-genome bisulfite sequencing data, where variations in methylation levels and confounding effects have been accounted for. We employ a region-wise test statistic, which is derived from local Getis-Ord statistics and considers the spatial correlation between nearby CpG sites, to detect DMRs. Unlike existing methods, that attempt to infer DMRs from DMCs based on empirical criteria, we provide statistical inference for direct DMR detection. Through extensive simulations and an application to two mouse datasets, we demonstrate that GetisDMR achieves better sensitivities, positive predictive values, more exact locations and better agreement of DMRs with current biological knowledge. Availability and Implementation: It is available at https://github.com/DMU-lilab/GetisDMR. Contacts: y.wen@auckland.ac.nz or zhiguangli@dlmedu.edu.cn Supplementary information: Supplementary data are available at Bioinformatics online.

The relationship between CpG content and DNA methylation has attracted considerable interest in recent years. Direct or indirect methods have been developed to investigate their regulatory functions based on various hypotheses, large cohort studies, and meta-analyses. However, all of these analyses were performed at units of CpG blocks and, thus, the influence of finer genome structure has been neglected. Herein, we present a novel algorithm of base-pair resolution to systematically investigate the relationship between CpG contents and DNA methylation. By introducing the concept of 'complementary index' we examined the methylomes of 34 adult and 7 embryonic tissues and successfully fitted the relationship of DNA methylation and CpG density into a nonlinear mathematical model. A further algorithm was developed to locate the regions where CpG density does not match expectations from the model, termed 'conflict of gap' (COG) regions. Interestingly, COGs are highly concordant in human and mouse and their distributions display a tissue-specific pattern. Based on COG methylation patterns we correctly classified tissues according to their function or origin. We demonstrate that COGs based on our method can reveal more and deeper information than traditional differential methylation region (DMR) approaches. We also found that when COGs are located near to transcription start site (TSS), these regions can determine which promoters will be utilized for initiating gene transcription. Furthermore, COGs located far from the TSS perform as enhancers in terms of histone modification, sequence conservation, transcription factor binding, and DNase I-hypersensitivity.