Connexin43 (Cx43) is a major gap junction protein in glial cells. Mutations have been found in the gap-junction alpha 1 gene encoding Cx43 in glaucomatous human retinas, suggestive of the involvement of Cx43 in the pathogenesis of glaucoma. However, how Cx43 is involved in glaucoma is still unknown. We showed that increased intraocular pressure in a glaucoma mouse model of chronic ocular hypertension (COH) downregulated Cx43, which was mainly expressed in retinal astrocytes. Astrocytes in the optic nerve head where they gather and wrap the axons (optic nerve) of retinal ganglion cells (RGCs) were activated earlier than neurons in COH retinas and the alterations in astrocytes plasticity in the optic nerve caused a reduction in Cx43 expression. A time course showed that reductions of Cx43 expression were correlated with the activation of Rac1, a member of the Rho family. Co-immunoprecipitation assays showed that active Rac1, or the downstream signaling effector PAK1, negatively regulated Cx43 expression, Cx43 hemichannel opening and astrocyte activation. Pharmacological inhibition of Rac1 stimulated Cx43 hemichannel opening and ATP release, and astrocytes were identified to be one of the main sources of ATP. Furthermore, conditional knockout of Rac1 in astrocytes enhanced Cx43 expression and ATP release, and promoted RGC survival by upregulating the adenosine A3 receptor in RGCs. Our study provides new insight into the relationship between Cx43 and glaucoma, and suggests that regulating the interaction between astrocytes and RGCs via the Rac1/PAK1/Cx43/ATP pathway may be used as part of a therapeutic strategy for managing glaucoma.

BACKGROUND Hepatocellular carcinoma (HCC) is one of the most common malignancies around the world. It has been verified that the expression of SOX18 is correlated to poor clinical prognosis in patients with ovarian cancer, non-small cell lung cancer, or breast invasive ductal carcinoma. However, the expression pattern and biological function of SOX18 in HCC tissues remains unclear. In this study, we set out to investigate the associated biological function and potential molecular mechanism of the SOX18 gene in HCC cells. MATERIAL AND METHODS The mRNA and protein expression levels of experimental related genes were detected by real-time polymerase chain reaction and western blotting assay, respectively. The MTT method was used to assess cell viability, and cell apoptosis analysis was performed by means of FACScan flow cytometry. Wound-healing assay and Transwell analysis were performed to evaluate the ability of cell migration and invasiveness, respectively. RESULTS SOX18 was highly expressed in various HCC cell lines. In addition, SOX18 promoted cell viability, migration, and invasion and simultaneously induce cell apoptosis. SOX18 promoted epithelial-to-mesenchymal transition (EMT) progression, and SOX18 downregulation activated the autophagy signaling pathway AMPK/mTOR in HCC cells. CONCLUSIONS SOX18 downregulation in HCC cells suppressed cell viability and metastasis, induced cell apoptosis and hindered the occurrence and progression of tumor cells by participating in the EMT process and regulating the autophagy signaling pathway AMPK/mTOR.

The rodent retina does not exhibit a positive OFF-response in the electroretinogram (ERG), which makes it difficult to evaluate its OFF-pathway functions in vivo. We studied the rod-driven OFF pathway responses by using a dark-adapted 10-Hz flicker ERG procedure in mouse.

The novel anti-VEGF drug conbercept has been used in the treatment of several retinal neovascular diseases. Owning to the alteration of the structure, the newest drug is capable of combining more molecular targets and present higher affinity to the angiogenesis promoting factors. However, it is unknown whether it will cause any unwanted effects like other anti-VEGF agents. We studied the short-term safety of high concentration and high frequency intravitreal injection of conbercept in rabbits. Intraocular pressure, fundus-photography, ERGs were applied. Retinal morphology, the amount of apoptotic cells and protein levels of IL-6, IL-8 and TNF-α in the aqueous humor were determined. Retinal proteomics was detected using tandem mass tags (TMTs) quantitative mass spectrometry. The difference of IOP, ERGs, protein levels of inflammatory factors among rabbits received conbercept and PBS was not significant (P > 0.05). Fundus photographs and retinal morphology of animals in the conbercept-injected groups mimic those observed in the PBS-injected groups. No TUNEL-positive cell was seen in the retinal ganglion cell layer in the conbercept-injected groups. Proteomics did not show significant changes of inflammation or apoptosis associated proteins in the conbercept-injected eyes. We conclude that intravitreal injection of high concentration and high frequency conbercept is well tolerated at least in a short-term in rabbits.

Amyloid β (Aβ) is a pathogenic peptide associated with many neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. The retinal inflammation in response to Aβ is implicated in the pathogenesis of several ocular diseases including age-related macular degeneration, Alzheimer's-related optic neuropathy and glaucoma. In the present study, we found that a single intravitreal injection of oligomeric Aβ1-40 in mouse activated the NLRP3 inflammasome and the NF-κB signaling, induced the production of inflammatory cytokines including TNF-α and IL-6. In addition, Aβ1-40 caused retinal function impairment while no noticeable morphological changes were observed under light microscope. Furthermore, immunohistochemical results showed that Aβ1-40 enhanced the number of Iba1-positive cells in the inner retina. The mRNA expressions of LXRα and LXRβ decreased in the neuroretina of the Aβ1-40-injected mice. No significant difference was found on the protein expressions of LXRs and ABCA1 in both neuroretina and RPE/choroid complex between the Aβ1-40-injected group and the control group. A synthetic LXR ligand, TO901317 (TO90), enhanced the expressions of LXRα and ABCA1 at both mRNA and protein levels in the Aβ1-40-injected mice, while the LXRβ expression was unchanged. TO90 preserved ERG a- and b-wave amplitudes and reduced the number of Iba1-positive cells in the Aβ1-40-treated retina. Furthermore, TO90 down-regulated the mRNA levels of TNF-α and IL-6, as well as the expressions of p-IκBα, NLRP3, caspase-1 and IL-1β in the Aβ1-40-injected animals. We suggest that activation of LXRα and its target gene ABCA1 exerts potent anti-inflammatory effect on the Aβ-treated retina.

Monocytic leukemia-associated antigen-34 (MLAA-34) is a novel monocytic leukemia-associated antigen and a candidate oncogene. The aim of the present study was to investigate the involvement of the MLAA-34 gene in acute myeloid leukemia (AML). MLAA-34 expression level, chromosome location, gene copy number and single nucleotide polymorphisms (SNPs) of 40 patients with AML and 5 healthy volunteers were analyzed by reverse transcription-polymerase chain reaction, fluorescence in situ hybridization and DNA sequencing. The effects of MLAA-34 mutation on overall survival (OS) and progression-free survival (PFS) of patients with AML were also analyzed. MLAA-34 was significantly upregulated in patients with AML when compared with volunteer controls, and this upregulation was associated with a C59T SNP site located in the second exon of MLAA-34. MLAA-34 was mapped to 13q14.2 and no translocation was observed in patients with AML. In addition, this SNP site is affinitive to the well-known molecular markers of AML, including Fms-like tyrosine kinase 3 and DNA methyltransferase 3A, as well as extramedullary lesions, periphery leukocyte numbers, remission and cytogenetic abnormalities of patients with AML. Patients with AML with MLAA-34 C59T mutations had significantly shorter OS and PFS times compared with that of patients without C59T mutations. The present findings indicated that the MLAA-34 C59T mutation was a high-risk factor for recurrence of AML, and may be a candidate target for AML therapy.

TGF-β1-induced epithelial-mesenchymal transition (EMT) has been proved to be associated with metastasis of breast cancer cells. We attempted to detect a novel mechanism that microRNAs mediated the TGF-β1-induced EMT in the process of breast cancer metastasis. Here we reported that the expression of miR-23a was higher in breast cancer cells with high metastasis ability and patients with lymph node metastasis and the treatment of TGF-β1 significantly upregulated the expression of miR-23a in breast cancer cells. We found that miR-23a was upregulated by TGF-β1 post-transcriptionally and Smads directly bound the RNA Smad binding element (R-SBE) of miR-23a. Functional studies showed that inhibition of miR-23a suppressed the TGF-β1-induced EMT, migration, invasion and metastasis of breast cancer both in vitro and in vivo. In addition, we determined that miR-23a directly targeted and suppressed CDH1, one important gene in EMT phenomenon. Notably, Wnt/β-catenin signaling was activated by the suppression of CDH1 in the miR-23a mediated process of TGF-β1-induced EMT and tumor invasion. These results demonstrate that miR-23a promotes TGF-β1-induced tumor metastasis in breast cancer by targeting CDH1 and activating Wnt/β-catenin signaling. Taken together, our results indicate a novel regulatory mechanism of TGF-β1-induced EMT and suggest that miR-23a might be a potential target in breast cancer therapy.

We previously showed that adeno-associated virus 2 (AAV2) mediated subretinal delivery of human interferon-alpha (IFN-α) could effectively inhibit experimental autoimmune uveoretinitis (EAU). In this study we investigated whether subretinal injection of both AVV2.IFN-α and AAV2.IL-4 had a stronger inhibition on EAU activity. B10RIII mice were subretinally injected with AAV2.IFN-α alone (1.5×10(7) vg), AAV2.IL-4 alone (3.55×10(7) vg), and AAV2.IFN-α combined with AAV2.IL-4. PBS, AAV2 vector encoding green fluorescent protein (AAV2.GFP) (5×10(7) vg) was subretinally injected as a control. IFN-α and IL-4 were effectively expressed in the eyes from three weeks to three months following subretinal injection of AAV2 vectors either alone or following combined administration and significantly attenuated EAU activity clinically and histopathologically. AAV2.IL-4 showed a better therapeutic effect as compared to AAV2.IFN-α. The combination of AAV2.IL-4 and AAV2.IFN-α was not significantly different as compared to AAV2.IL-4 alone. There was no difference concerning DTH (delayed-type hypersensitivity) reaction, lymphocyte proliferation and IL-17 production among the investigated treatment groups, suggesting that local retinal gene delivery did not affect the systemic immune response.

Although the leaf is the most important photosynthetic organ in most plants, many of the molecular mechanisms underlying leaf developmental dynamics remain to be explored. To better understand the transcriptional regulatory mechanisms involved in leaf development, we conducted comparative transcriptomic and metabolomic analysis of leaves from seven positions on tobacco (Nicotiana tabacum) plants. A total of 35,622 unique differentially expressed genes and 79 metabolites were identified. A time-series expression analysis detected two interesting transcriptional profiles, one comprising 10,197 genes that displayed continual up-regulation during leaf development and another comprising 4696 genes that displayed continual down-regulation. Combining these data with co-expression network results identified four important regulatory networks involved in photorespiration and the tricarboxylic acid cycle; these networks may regulate carbon/nitrogen balance during leaf development. We also found that the transcription factor NtGATA5 acts as a hub associated with C and N metabolism and chloroplast development during leaf development through regulation of phytohormones. Furthermore, we investigated the transcriptional dynamics of genes involved in the auxin, cytokinin, and jasmonic acid biosynthesis and signaling pathways during tobacco leaf development. Overall, our study greatly expands the understanding of the regulatory network controlling developmental dynamics in plant leaves.

MLAA-34 is a novel leukemia-associated gene closely related to the carcinogenesis of acute monocytic leukemia (AML). MLAA-34 over expression has been observed to inhibit apoptosis in vitro. JAK2/STAT3 pathway plays an important role in cell proliferation, differentiation and inhibition of apoptosis in number of cancers. However, the relationship and interaction between MLAA-34 and JAK2/STAT3 has never been investigated in AML. This study investigates and reports a novel relationship between MLAA-34 and JAK2/STAT3 pathway in AML both in vitro and in vivo. We constructed MLAA-34 knockdown vector and transfected U937 cells to observe its apoptotic activities in relation to JAK2/STAT3 signaling pathway in vitro and then in vivo in mouse model. Levels of expression of MLAA-34 and JAK2/STAT3 and its downstream targets were also measured in AML patients and a few volunteers. We found that MLAA-34 knockdown increased U937 apoptosis in vitro and inhibited tumor growth in vivo. Components of the canonical JAK2/STAT3 pathway or its downstream targets, including c-myc, bcl-2, Bax, and caspase-3, were shown to be involved in the carcinogenesis of AML. We also found that the JAK2/STAT3 pathway positively regulated MLAA-34 expression. We additionally identified a STAT3 binding site in the MLAA-34 promoter where STAT3 binds directly and activates MLAA-34 expression. In addition, MLAA-34 was found to form a complex with JAK2 and was enhanced by JAK2 activation. Correlation of MLAA-34 and JAK2/STAT3 was further confirmed in AML patients. In conclusion, MLAA-34 is a novel regulator for JAK2/STAT3 signaling, and in turn, is regulated by this interaction in a positive feedback loop. Thus we report a novel model of interaction mechanism between MLAA-34 and JAK2/STAT3 which can be utilized as a potential target for a novel therapeutic approach in AML.

Excessive reactive oxygen species (ROS) in the injured skin may impede the wound repair and skin regeneration. Herein, we develop an injectable self-healing ceria-based nanocomposite hydrogel with ROS-scavenging activity to accelerate wound healing. The nanocomposite hydrogels were successfully prepared by coating cerium oxide nanorods with polyethylenimine and crosslinked with benzaldehyde-terminated F127 (F127-CHO) through the dynamic Schiff-base reaction (FVEC hydrogel). The results showed that the FVEC hydrogel possessed the good thermosensitivity, injectability, self-healing ability and ROS scavenging activity. The subcutaneous implantation experiments in mice confirmed that FVEC hydrogels are biocompatible and biodegradable in vivo. The full-thickness skin wound studies showed that FVEC hydrogel could significantly enhance the wound healing and epithelium regeneration with the formation of hair follicle and adipocyte tissue. This work provides a new strategy for the development of multifunctional Ce-based nanocomposite hydrogel for full-thickness skin wound healing and regeneration.

Forgetting is a universal feature for most types of memories. The best-defined and extensively characterized behaviors that depict forgetting are natural memory decay and interference-based forgetting [1, 2]. Molecular mechanisms underlying the active forgetting remain to be determined for memories in vertebrates. Recent progress has begun to unravel such mechanisms underlying the active forgetting [3-11] that is induced through the behavior-dependent activation of intracellular signaling pathways. In Drosophila, training-induced activation of the small G protein Rac1 mediates natural memory decay and interference-based forgetting of aversive conditioning memory [3]. In mice, the activation of photoactivable-Rac1 in recently potentiated spines in a motor learning task erases the motor memory [12]. These lines of evidence prompted us to investigate a role for Rac1 in time-based natural memory decay and interference-based forgetting in mice. The inhibition of Rac1 activity in hippocampal neurons through targeted expression of a dominant-negative Rac1 form extended object recognition memory from less than 72 hr to over 72 hr, whereas Rac1 activation accelerated memory decay within 24 hr. Interference-induced forgetting of this memory was correlated with Rac1 activation and was completely blocked by inhibition of Rac1 activity. Electrophysiological recordings of long-term potentiation provided independent evidence that further supported a role for Rac1 activation in forgetting. Thus, Rac1-dependent forgetting is evolutionarily conserved from invertebrates to vertebrates.

Glaucoma, the leading cause of irreversible blindness, is a retinal neurodegenerative disease, which results from progressive apoptotic death of retinal ganglion cells (RGCs). Although the mechanisms underlying RGC apoptosis in glaucoma are extremely complicated, an abnormal cross-talk between retinal glial cells and RGCs is generally thought to be involved. However, how interaction of Müller cells and microglia, two types of glial cells, contributes to RGC injury is largely unknown.

In age-related macular degeneration (AMD) or diabetic retinopathy (DR), hypoxia and inflammatory processes lead to an upregulation of the vascular endothelial growth factor (VEGF) expression and thereby to pathological neovascularisation with incorrectly formed vessels prone to damage, thus increasing the vascular permeability and the risk of bleeding and oedema in the retina. State of the art treatment is the repeated intraocular injection of anti-VEGF molecules. For developing improved individualized treatment approaches, a minimally invasive, repeatable method for in vivo quantification of VEGF in the eye is necessary. Therefore, we designed single molecule eBRET2 VEGF biosensors by directly fusing a Renilla luciferase mutant (Rluc8) N-terminal and a green fluorescent protein (GFP2) C-terminal to a VEGF binding domain. In total, 10 different VEGF biosensors (Re01- Re10) were generated based on either single domains or full length of VEGF receptor 1 or 2 extracellular regions as VEGF binding domains. Full length expression of the biosensors in HEK293-T cells was verified via Western Blot employing an anti-Rluc8-IgG. Expression of alternative splice variants was eliminated through the deletion of the donor splice site by introduction of a silent point mutation. In all ten biosensors the energy transfer from the Rluc8 to the GFP2 occurs and generates a measurable eBRET2 ratio. Four biosensors show a relevant change of the BRET ratio (ΔBR) after VEGF binding. Furthermore, each biosensor shows a unique detection range for VEGF quantification and especially Re06 and Re07 have a high sensitivity in the range of in vivo VEGF concentrations in the eye, previously measured by invasive methods. In conclusion, we generated several eBRET2 biosensors that are suitable for VEGF quantification in vitro and could identify two eBRET2 biosensors, which may be suitable for non-invasive in vivo VEGF quantification with an implantable device.

Complete skeletal muscle repair and regeneration due to severe large injury or disease is still a challenge. Biochemical cues are critical to control myoblast cell function and can be utilized to develop smart biomaterials for skeletal muscle engineering. Citric acid-based biodegradable polymers have received much attention on tissue engineering, however, their regulation on myoblast cell differentiation and mechanism was few investigated. Here, we find that citrate-based polycitrate-polyethylene glycol-polyethylenimine (POCG-PEI600) nanoclusters can significantly enhance the in vitro myoblast proliferation by probably reinforcing the mitochondrial number, promote the myotube formation and full-thickness skeletal muscle regeneration in vivo by activating the myogenic biomarker genes expression of Myod and Mhc. POCG-PEI600 nanoclusters could also promote the phosphorylation of p38 in MAP kinases (MAPK) signaling pathway, which led to the promotion of the myoblast differentiation. The in vivo skeletal muscle loss rat model also confirmed that POCG-PEI600 nanoclusters could significantly improve the angiogenesis, myofibers formation and complete skeletal muscle regeneration. POCG-PEI600 nanocluster could be also biodegraded into small molecules and eliminated in vivo, suggesting their high biocompatibility and biosafety. This study could provide a bioactive biomaterial-based strategy to repair and regenerate skeletal muscle tissue.

To determine the risk state distribution, risk level, and risk evolution situation of agricultural non-point source pollution (AGNPS), we built an 'Input-Translate-Output' three-dimensional evaluation (ITO3dE) model that involved 12 factors under the support of GIS and analyzed the spatiotemporal evolution characteristics of AGNPS risks from 2005 to 2015 in Chongqing by using GIS space matrix, kernel density analysis, and Getis-Ord Gi* analysis. Land use changes during the 10 years had a certain influence on the AGNPS risk. The risk values in 2005, 2010, and 2015 were in the ranges of 0.40-2.28, 0.41-2.57, and 0.41-2.28, respectively, with the main distribution regions being the western regions of Chongqing (Dazu, Jiangjin, etc.) and other counties such as Dianjiang, Liangping, Kaizhou, Wanzhou, and Zhongxian. The spatiotemporal transition matrix could well exhibit the risk transition situation, and the risks generally showed no changes over time. The proportions of 'no-risk no-change', 'low-risk no-change', and 'medium-risk no-change' were 10.86%, 33.42%, and 17.25%, respectively, accounting for 61.53% of the coverage area of Chongqing. The proportions of risk increase, risk decline, and risk fluctuation were 13.45%, 17.66%, and 7.36%, respectively. Kernel density analysis was suitable to explore high-risk gathering areas. The peak values of kernel density in the three periods were around 1110, suggesting that the maximum gathering degree of medium-risk pattern spots basically showed no changes, but the spatial positions of high-risk gathering areas somehow changed. Getis-Ord Gi* analysis was suitable to explore the relationships between hot and cold spots. Counties with high pollution risks were Yongchuan, Shapingba, Dianjiang, Liangping, northwestern Fengdu, and Zhongxian, while counties with low risks were Chengkou, Wuxi, Wushan, Pengshui, and Rongchang. High-value hot spot zones gradually dominated in the northeast of Chongqing, while low-value cold spot zones gradually dominated in the Midwest. Our results provide a scientific base for the development of strategies to prevent and control AGNPS in Chongqing.

Epstein-Barr virus nuclear antigen 2 (EBNA2) is a transactivator of viral and cellular gene expression, which plays a critical role in the Epstein-Barr virus-associated diseases. It was reported that EBNA2 regulates gene expression by reorganizing chromatin and manipulating epigenetics. Recent studies showed that liquid-liquid phase separation plays an essential role in epigenetic and transcriptional regulation. Here we show that EBNA2 reorganized chromatin topology to form accessible chromatin domains (ACDs) of the host genome by phase separation. The N-terminal region of EBNA2, which is necessary for phase separation, is sufficient to induce ACDs. The C-terminal domain of EBNA2 promotes the acetylation of accessible chromatin regions by recruiting histone acetylase p300 to ACDs. According to these observations, we proposed a model of EBNA2 reorganizing chromatin topology for its acetylation through phase separation to explain the mechanism of EBNA2 hijacking the host genome by controlling its epigenetics.

The choice of surgical treatment for chronic, persistent and large full-thickness macular holes (FTMH) continues to be undefined and challenging, as some of these cases remain refractory to the treatment. We report the efficacy of combination of inverted internal limiting membrane flap technique (IILMFT) and subretinal application of the fluid (SR fluid application) technique for treatment of refractory FTMHs.

Circular RNAs (circRNAs) are a large class of noncoding RNAs that have potential regulatory roles in disease pathogenesis and progression. Recently, circRNAs have been found to be expressed in hepatocellular carcinoma (HCC) tissues and involved in the development and metastasis of HCC. However, the significance of circRNAs in peripheral blood mononuclear cells (PBMCs) of HCC patients remains unclear. In this study, RNA sequencing analysis was performed to identify circRNAs from four HCC patients and three healthy controls to determine the expression pattern of circRNAs in the PBMCs and the circRNAs' molecular regulatory networks in HCC pathogenesis. A total of 58 circRNAs were found to be significantly changed (≥2 or ≤0.5-fold) in the PBMCs of HCC patients compared with those of the healthy cases. Six random representative circRNAs (three up- and three down-regulated) were further validated by real-time RT-PCR in 72 samples of PBMCs from HCC patients and 30 control subjects. Chi-square test indicated that one of the up-regulated circRNA candidates-circ_0000798-was correlated with clinical variables. Highly expressed circ_0000798 was associated with poor overall survival of HCC patients. Receiver operating characteristic curve analysis further revealed that circ_0000798 was discriminating HCC patients from healthy controls. Finally, the predicted competing endogenous RNA network of circ_0000798 showed that it might act as a "sponge" of target microRNAs, that would subsequently regulate the expression of target genes in PBMCs. In summary, this is the first study to comprehensively identify dysregulated circRNAs in PBMCs of HCC patients, and its findings suggest that dysregulated circ_0000798 in PBMCs has potential as a convenient biomarker for diagnosing or prognosticating HCC.

Chimeric antigen receptor (CAR) T cell therapy has demonstrated proven efficacy in some hematologic cancers. We evaluated the safety and efficacy of LCAR-B38M, a dual epitope-binding CAR T cell therapy directed against 2 distinct B cell maturation antigen epitopes, in patients with relapsed/refractory (R/R) multiple myeloma (MM).