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Background: Human amniotic membrane (AM) transplantation has been applied to treat ocular surface diseases, including corneal trauma. The focus of much deliberation is to balance the mechanical strength of the amniotic membrane, its resistance to biodegradation, and its therapeutic efficacy. It is commonly observed that the crosslinked human decellularized amniotic membranes lose the functional human amniotic epithelial cells (hAECs), which play a key role in curing the injured tissues., Methods and Results: In this study, we crosslinked human decellularized amniotic membranes (dAM) with genipin and re-planted the hAECs onto the genipin crosslinked AM. The properties of the AM were evaluated based on optical clarity, biodegradation, cytotoxicity, and ultrastructure. The crosslinked AM maintained its transparency. The color of crosslinked AM deepened with increasing concentrations of genipin. And the extracts from low concentrations of genipin crosslinked AM had no toxic effect on human corneal epithelial cells (HCECs), while high concentrations of genipin exhibited cytotoxicity. The microscopic observation and H&E staining revealed that 2 mg/mL genipin-crosslinked dAM (2 mg/mL cl-dAM) was more favorable for the attachment, migration, and proliferation of hAECs. Moreover, the results of the CCK-8 assay and the transwell assay further indicated that the living hAECs' tissue-engineered amniotic membranes could facilitate the proliferation and migration of human corneal stromal cells (HCSCs) in vitro., Conclusions: In conclusion, the cl-dAM with living hAECs demonstrates superior biostability and holds significant promise as a material for ocular surface tissue repair in clinical applications., (© 2024. The Author(s).)

Purpose: This study aimed to explore the impact of HSPA13 on epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells and proliferative vitreoretinopathy (PVR) development, along with its associated molecular mechanisms., Methods: HSPA13 expression was evaluated in epiretinal membranes (ERMs) from patients with PVR using immunohistochemistry. The effects of HSPA13 knockdown on TGFβ1-induced EMT in hESC-RPE cells were studied through quantitative PCR (qPCR), Western blot, and wound healing assays. Intracellular Ca2+ levels were measured using Fluo-8/AM incubation. A rat PVR model was induced by the intravitreal injection of RPE cells combined with platelet-rich plasma (PRP). RNA-seq was applied to study the molecular mechanism of HSPA13 knockdown-mediated EMT inhibition., Results: HSPA13 was found in human ERMs and its expression increased with TGFβ1 treatment in hESC-RPE cells. Knockdown of HSPA13 inhibited TGFβ1-induced EMT and migration. In the PVR rat model, HSPA13 was expressed in the ERMs and its knockdown in RPE cells reduced the development of PVR. Consistent with these observations, RNA-seq showed a global suppression of TGFβ1-induced EMT and migration by shHSPA13 in RPE cells. Mechanistically, TGFβ1 treatment increased intracellular Ca2+ levels, leading to an upregulation of HSPA13 expression. Downregulation of HSPA13 hindered the phosphorylation of PI3K/Akt in TGFβ1-induced RPE cells., Conclusions: Our study revealed the involvement of HSPA13 in PVR development, as well as in TGFβ1-induced EMT of RPE through the PI3K/Akt signaling pathway. Targeting HSPA13-related pathways involved in regulating EMT in RPE cells could serve as a novel therapeutic approach for patients with PVR.

Introduction: Individualized treatment of colorectal cancer liver metastases (CRLM) remains challenging due to differences in the severity of metastatic disease and tumour biology. Exploring specific prognostic risk subgroups is urgently needed. The current study aimed to investigate the prognostic value of chromosomal instability (CIN) in patients with initially resectable CRLM and the predictive value of CIN for the efficacy of bevacizumab., Methods: Ninety-one consecutive patients with initially resectable CRLM who underwent curative liver resection from 2006 to 2018 at Sun Yat-sen University Cancer Center were selected for analysis. CIN was evaluated by automated digital imaging systems. Immunohistochemistry (IHC) was performed to detect interleukin-6 (IL-6), vascular endothelial growth factor A (VEGFA) and CD31 expression in paraffin-embedded specimens. Recurrence-free survival (RFS) and overall survival (OS) were analysed using the Kaplan-Meier method and Cox regression models., Results: Patients with high chromosomal instability (CIN-H) had a worse 3-year RFS rate (HR, 1.953; 95% CI, 1.001-3.810; p  = 0.049) and a worse 3-year OS rate (HR, 2.449; 95% CI, 1.150-5.213; p  = 0.016) than those with low chromosomal instability (CIN-L). CIN-H was identified as an independent prognostic factor for RFS (HR, 2.569; 95% CI, 1.078-6.121; p  = 0.033) and OS (HR, 3.852; 95% CI, 1.173-12.645; p  = 0.026) in the multivariate analysis. The protein levels of IL-6, VEGFA and CD31 were upregulated in patients in the CIN-H group compared to those in the CIN-L group in both primary tumour and liver metastases tissues. Among them, 22 patients with recurrent tumours were treated with first-line bevacizumab treatment and based on the clinical response assessment, disease control rates were adversely associated with chromosomal instability ( p  = 0.043)., Conclusions: Our study showed that high chromosomal instability is a negative prognostic factor for patients with initially resectable CRLM after liver resection. CIN may have positive correlations with angiogenesis through expression of IL-6-VEGFA axis and be used as a potential predictor of efficacy of bevacizumab.

Background: To develop and validate a serum protein nomogram for colorectal cancer (CRC) screening., Methods: The serum protein characteristics were extracted from an independent sample containing 30 colorectal cancer and 12 polyp tissues along with their paired samples, and different serum protein expression profiles were validated using RNA microarrays. The prediction model was developed in a training cohort that included 1345 patients clinicopathologically confirmed CRC and 518 normal participants, and data were gathered from November 2011 to January 2017. The lasso logistic regression model was employed for features selection and serum nomogram building. An internal validation cohort containing 576 CRC patients and 222 normal participants was assessed., Results: Serum signatures containing 27 secreted proteins were significantly differentially expressed in polyps and CRC compared to paired normal tissue, and REG family proteins were selected as potential predictors. The C-index of the nomogram1 (based on Lasso logistic regression model) which contains REG1A, REG3A, CEA and age was 0.913 (95% CI, 0.899 to 0.928) and was well calibrated. Addition of CA199 to the nomogram failed to show incremental prognostic value, as shown in nomogram2 (based on logistic regression model). Application of the nomogram1 in the independent validation cohort had similar discrimination (C-index, 0.912 [95% CI, 0.890 to 0.934]) and good calibration. The decision curve (DCA) and clinical impact curve (ICI) analysis demonstrated that nomogram1 was clinically useful., Conclusions: This study presents a serum nomogram that included REG1A, REG3A, CEA and age, which can be convenient for screening of colorectal cancer., (© 2024. The Author(s), under exclusive licence to Federación de Sociedades Españolas de Oncología (FESEO).)

Background: Glia maturation factor beta (GMFB) is a growth and differentiation factor that acts as an intracellular regulator of signal transduction pathways. The small ubiquitin-related modifier (SUMO) modification, SUMOylation, is a posttranslational modification (PTM) that plays a key role in protein subcellular localization, stability, transcription, and enzymatic activity. Recent studies have highlighted the importance of SUMOylation in the inflammation and progression of numerous diseases. However, the relationship between GMFB and SUMOylation is unclear., Results: Here, we report for the first time that GMFB and SUMO1 are markedly increased in retinal pigment epithelial (RPE) cells at the early stage of diabetes mellitus (DM) under hyperglycemia. The GMFΒ protein could be mono-SUMOylated by SUMO1 at the K20, K35, K58 or K97 sites. SUMOylation of GMFB led to its increased protein stability and subcellular translocation. Furthermore, deSUMOylation of GMFΒ downregulates multiple signaling pathways, including the Jak-STAT signaling pathway, p38 pathway and NF-kappa B signaling pathway., Conclusions: This work provides novel insight into the role of SUMOylated GMFB in RPE cells and provides a novel therapeutic target for diabetic retinopathy (DR)., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Retinal degeneration, characterized by Müller cell gliosis and photoreceptor apoptosis, is considered an early event in diabetic retinopathy (DR). Our previous study proposed that GMFB may mediate diabetic retinal degeneration. This study identified GMFB as a sensitive and functional gliosis marker for DR. Compared to the wild type (WT) group, Gmfb knockout (KO) significantly improved visual function, attenuated gliosis, reduced the apoptosis of neurons, and decreased the mRNA levels of tumor necrosis factor α (Tnf-α) and interleukin-1β (Il-1β) in diabetic retinas. Tgf-β3 was enriched by hub genes using RNA sequencing in primary WT and KO Müller cells. Gmfb KO significantly upregulated the transforming growth factor (TGF)-β3 protein level via the AKT pathway. The protective effect of TGF-β3 in the vitreous resulted in significantly improved visual function and decreased the number of apoptotic cells in the diabetic retina. The protection of Gmfb KO in primary Müller cells against high glucose (HG)-induced photoreceptor apoptosis was partially counteracted by TGF-β3 antibody and administration of TGFBR1/2 inhibitors. Nuclear receptor subfamily 3 group C member 1 (NR3C1) binds to the promoter region of Gmfb and regulates Gmfb mRNA at the transcriptional level. NR3C1 was increased in the retinas of early diabetic rats but decreased in the retinas of late diabetic rats. N'-[(1E)-(3-Methoxyphenyl)Methylene]-3-Methyl-1H-Pyrazole-5-Carbohydrazide (DS-5) was identified as an inhibitor of GMFB, having a protective role in DR. We demonstrated that GMFB/AKT/TGF-β3 mediated early diabetic retinal degeneration in diabetic rats. This study provides a novel therapeutic strategy for treating retinal degeneration in patients with DR., (© 2023 Wiley Periodicals LLC.)

Bevacizumab plays an important role in the first and second line treatment for metastatic colorectal cancer (CRC). And induction of hypoxia and the tumors response to it plays an important role in determining the efficacy of antiangiogenic therapy while the connection between them remains unclear. Here, we found that lactate accumulated in the tumor environment of CRC and acted as substrates for histone lactylation, and this process was further induced by cellular enhanced glycolysis in hypoxia. We determined that CRC patients resistant to bevacizumab treatment presented with elevated levels of histone lactylation and inhibition of histone lactylation efficiently suppressed CRC tumorigenesis, progression and survival in hypoxia. Histone lactylation promoted the transcription of RUBCNL/Pacer, facilitating autophagosome maturation through interacting with BECN1 (beclin 1) and mediating the recruitment and function of the class III phosphatidylinositol 3-kinase complex, which had a crucial role in hypoxic cancer cells proliferation and survival. Moreover, combining inhibition of histone lactylation and macroautophagy/autophagy with bevacizumab treatment demonstrated remarkable treatment efficacy in bevacizumab-resistance patients-derived pre-clinical models. These findings delivered a new exploration and important supplement of metabolic reprogramming-epigenetic regulation, and provided a new strategy for improving clinical efficacy of bevacizumab in CRC by inhibition of histone lactylation. Abbreviations: 2-DG: 2-deoxy-D-glucose; BECN1: beclin 1; CQ: chloroquine; CRC: colorectal cancer; DMOG: dimethyloxalylglycine; H3K18la: histone H3 lysine 18 lactylation; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; Nala: sodium lactate; PDO: patient-derived orgnoid; PDX: patient-derived xenograft; RUBCNL/Pacer: rubicon like autophagy enhancer; SQSTM1/p62: sequestosome 1.

Examining the association between dietary patterns and colorectal cancer (CRC) risk can provide valuable insights beyond the assessment of individual foods or nutrients. However, there is a lack of in-depth analysis of dietary patterns and CRC risk in Chinese populations, and few studies have compared dietary patterns derived from different posteriori methods with the aim of predicting disease risk. The aim of this study was to derive dietary patterns using both principal component analysis (PCA) and cluster analysis (CA) and to assess their respective associations with CRC risk. A large-scale case-control study was conducted in Guangdong Province, China, including 2799 incident colorectal cancer cases and an equal number of frequency-matched controls. Dietary intake information was gathered through the use of a validated food frequency questionnaire. PCA and CA were used to derive dietary patterns. A multivariable logistic regression model was used to calculate the adjusted odds ratio (aOR) and 95% confidence interval (CI). Four major dietary patterns were identified by PCA. CA identified two dietary patterns, referred to as the "Balanced dietary pattern" and the "Refined grain dietary pattern". Notably, there were significant inverse associations between the milk-egg-nut-soy dietary pattern (aOR, 0.51; 95% CI, 0.42, 0.62), the vegetable-fruit dietary pattern (aOR, 0.61; 95%CI, 0.51, 0.74), and the poultry-fish dietary pattern (aOR, 0.81; 95%CI, 0.68, 0.97) and CRC risk. However, the red meat-preserved food dietary pattern was associated with an increased risk of CRC (aOR, 2.99; 95%CI, 2.43, 3.67). When compared with the Refined grain dietary pattern, the Balanced dietary pattern showed a decreased risk of CRC (aOR, 0.59; 95%CI, 0.52, 0.66). The results from the comparison of the two methods indicate that both CA and PCA derived remarkably similar patterns. The combined use of PCA and CA identified consistent underlying patterns, showing comparable associations with CRC risk. These findings suggest that individuals who prefer dietary patterns characterized by a high intake of red meat, preserved food, and refined grains should be cautious about their increased CRC risk. Conversely, dietary patterns rich in fruits, vegetables, and high-quality protein sources are advisable for the prevention of CRC in the Chinese population.

Background: T helper 2 (Th2) cells are thought to play critical roles in allergic conjunctivitis (AC). They release inflammatory cytokines to promote an allergic response in AC. Due to individual heterogeneity and long-term chronic management, current therapies do not always effectively control AC. Mesenchymal stem cells (MSCs) have been shown to be effective in treating allergy-related disorders, but it is unclear how exactly the Th2-mediated allergic response is attenuated. This study aims to elucidate the therapeutic effect and mechanism of the human umbilical cord MSCs (hUCMSCs) in a mouse model of experimental AC (EAC)., Methods: A mouse EAC model was established by inoculating short ragweed (SRW) pollen. After the SRW pollen challenge, the mice received a single subconjunctival or tail vein injection of 2 × 10 6 hUCMSCs, or subconjunctival injection of hUCMSCs conditioned medium (hUCMSC-CM), and dexamethasone eye drops was used as positive control; subsequent scratching behavior and clinical symptoms were assessed. Immunostaining and flow cytometry were carried out to show allergic reactions and the activation of CD4 + T cell subsets in the conjunctiva and cervical lymph nodes (CLNs). Gene expression was determined by RNA-seq and further verified by qRT-PCR and Western blot. Co-culture assays were performed to explore the regulatory role of hUCMSCs in the differentiation of CD4 + naive T cells (Th0) into Th2 cells., Results: Subconjunctival administration of hUCMSCs resulted in fewer instances of scratching and lower inflammation scores in EAC mice compared to the tail vein delivery, hUCMSC-CM and control groups. Subconjunctival administration of hUCMSCs reduced the number of activated mast cells and infiltrated eosinophils in the conjunctiva, as well as decreased the number of Th2 cells in CLNs. After pretreatment with EAC mouse serum in vitro to mimic the in vivo milieu, hUCMSCs were able to inhibit the differentiation of Th0 into Th2 cells. Further evidence demonstrated that repression of Th2 cell differentiation by hUCMSCs is mediated by CRISPLD2 through downregulation of STAT6 phosphorylation. Additionally, hUMCSCs were able to promote the differentiation of Th0 cells into regulatory T cells in CLNs of EAC mice., Conclusions: Subconjunctival injection of hUCMSCs suppressed the Th2-allergic response and alleviated clinical symptoms. This study provides not only a potential therapeutic target for the treatment of AC but also other T cell-mediated diseases., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Introduction: Given the important roles of immune tolerance and inflammation in both preterm and term labor, some inflammation-related genes could be related to the initiation of labor, even preterm labor. Inspection of cell-free RNA (cfRNA) engaged in inflammation in maternal blood may represent the varied gestational age and may have significant implications for the development of noninvasive diagnostics for preterm birth., Methods: To identify potential biomarkers of preterm birth, we investigated the cfRNA and exosomal miRNA in the peripheral blood of pregnant women at different gestational ages that undergo term labor or preterm labor. 17 inflammatory initiation-related cfRNAs were screened by overlapping with the targets of decreasing miRNAs during gestation and highly expressed cfRNAs at late gestation in maternal blood. To reveal the origins and mechanisms of these screened cfRNAs, the datasets of single-cell RNA sequencing from peripheral blood mononuclear cells of pregnant women, the fetal lung, and the placenta across different gestational ages were analyzed., Results: During late gestation, TNFSF4 expression increased exclusively in pro-inflammatory macrophages of maternal blood, whereas its receptor, TNFRSF4, increased expression in T cells from the decidua, which suggested the potential cell-cell communication of maternally-originated pro-inflammatory macrophages with the decidual T cells and contributed to the initiation of labor. Additionally, the cfRNA of TNFSF4 was also increased in preterm labor compared to term labor in the validation cohorts. The EIF2AK2 and TLR4 transcripts were increased in pro-inflammatory macrophages from both fetal lung and placenta but not in those from maternal mononuclear cells at late gestation, suggesting these cfRNAs are possibly derived from fetal tissues exclusively. Moreover, EIF2AK2 and TLR4 transcripts were found highly expressed in the pro-inflammatory macrophages from decidua as well, which suggested these specific fetal-origin macrophages may function at the maternal-fetal interface to stimulate uterine contractions, which have been implicated as the trigger of parturition and preterm labor., Discussion: Taken together, our findings not only revealed the potential of peripheral TNFSF4 as a novel cfRNA biomarker for noninvasive testing of preterm labor but further illustrated how maternal and fetal signals coordinately modulate the inflammatory process at the maternal-fetal interface, causing the initiation of term or preterm labor., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wang, Ou and Gao.)

Excessive osteoclast activation, which depends on dramatic changes in actin dynamics, causes osteoporosis (OP). The molecular mechanism of osteoclast activation in OP related to type 1 diabetes (T1D) remains unclear. Glia maturation factor beta (GMFB) is considered a growth and differentiation factor for both glia and neurons. Here, we demonstrated that Gmfb deficiency effectively ameliorated the phenotype of T1D-OP in rats by inhibiting osteoclast hyperactivity. In vitro assays showed that GMFB participated in osteoclast activation rather than proliferation. Gmfb deficiency did not affect osteoclast sealing zone (SZ) formation but effectively decreased the SZ area by decreasing actin depolymerization. When GMFB was overexpressed in Gmfb-deficient osteoclasts, the size of the SZ area was enlarged in a dose-dependent manner. Moreover, decreased actin depolymerization led to a decrease in nuclear G-actin, which activated MKL1/SRF-dependent gene transcription. We found that pro-osteoclastogenic factors (Mmp9 and Mmp14) were downregulated, while anti-osteoclastogenic factors (Cftr and Fhl2) were upregulated in Gmfb KO osteoclasts. A GMFB inhibitor, DS-30, targeting the binding site of GMFB and Arp2/3, was obtained. Biocore analysis revealed a high affinity between DS-30 and GMFB in a dose-dependent manner. As expected, DS-30 strongly suppressed osteoclast hyperactivity in vivo and in vitro. In conclusion, our work identified a new therapeutic strategy for T1D-OP treatment. The discovery of GMFB inhibitors will contribute to translational research on T1D-OP., (© 2023. The Author(s).)

The association between circulating saturated fatty acids (SFAs) including very long-chain SFAs (VLCSFAs) and colorectal cancer (CRC) risk has not been clearly established. To investigate the association between serum SFAs and CRC risk in Chinese population, 680 CRC cases and 680 sex and age-matched (5-year interval) controls were recruited in our study. Serum levels of SFAs were detected by gas chromatography. Unconditional logistic regression models were used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for the association between serum SFAs and CRC risk. Results showed that total SFAs were positively associated with the risk of CRC (adjusted OR quartile 4 vs. 1 = 2.64, 95%CI: 1.47-4.74). However, VLCSFAs were inversely associated with CRC risk (adjusted OR quartile 4 vs. 1 = 0.51, 95%CI: 0.36-0.72). Specifically, lauric acid, myristic acid, palmitic acid, heptadecanoic acid, and arachidic acid were positively associated with CRC risk, while behenic acid and lignoceric acid were inversely associated with CRC risk. This study indicates that higher levels of total serum SFAs and lower levels of serum VLCSFAs were associated with an increased risk of CRC in Chinese population. To reduce the risk of CRC, we recommend reducing the intake of foods containing palmitic acid and heptadecanoic acid such as animal products and dairy products, and moderately increasing the intake of foods containing VLCSFAs such as peanuts and canola oil.

Background: Systemic sclerosis (SSc) is a rare autoimmune disease characterized by extensive skin fibrosis. There are no effective treatments due to the severity, multiorgan presentation, and variable outcomes of the disease. Here, integrated bioinformatics was employed to discover tissue-specific expressed hub genes associated with SSc, determine potential competing endogenous RNAs (ceRNA) regulatory networks, and identify potential targeted drugs., Methods: In this study, four datasets of SSc were acquired. To identify the genes specific to tissues or organs, the BioGPS web database was used. For differentially expressed genes (DEGs), functional and enrichment analyses were carried out, and hub genes were screened and shown in a network of protein-protein interactions (PPI). The potential lncRNA-miRNA-mRNA ceRNA network was constructed using the online databases. The specifically expressed hub genes and ceRNA network were validated in the SSc mouse and in normal mice. We also used the receiver operating characteristic (ROC) curve to determine the diagnostic values of effective biomarkers in SSc. Finally, the Drug-Gene Interaction Database (DGIdb) identified specific medicines linked to hub genes., Results: The pooled datasets identified a total of 254 DEGs. The tissue/organ-specifically expressed genes involved in this analysis are commonly found in the hematologic/immune system and bone/muscle tissue. The enrichment analysis of DEGs revealed the significant terms such as regulation of actin cytoskeleton, immune-related processes, the VEGF signaling pathway, and metabolism. Cytoscape identified six gene cluster modules and 23 hub genes. And 4 hub genes were identified, including Serpine1, CCL2, IL6, and ISG15. Consistently, the expression of Serpine1, CCL2, IL6, and ISG15 was significantly higher in the SSc mouse model than in normal mice. Eventually, we found that MALAT1-miR-206-CCL2, let-7a-5p-IL6, and miR-196a-5p-SERPINE1 may be promising RNA regulatory pathways in SSc. Besides, ten potential therapeutic drugs associated with the hub gene were identified., Conclusions: This study revealed tissue-specific expressed genes, SERPINE1, CCL2, IL6, and ISG15, as effective biomarkers and provided new insight into the mechanisms of SSc. Potential RNA regulatory pathways, including MALAT1-miR-206-CCL2, let-7a-5p-IL6, and miR-196a-5p-SERPINE1, contribute to our knowledge of SSc. Furthermore, the analysis of drug-hub gene interactions predicted TIPLASININ, CARLUMAB and BINDARIT as candidate drugs for SSc., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Jin, Liu, Tang, Yan, Jiang, Zhao, Chen, Jin, Ou and Zhao.)

Aim: To assess whether serum thymidine kinase 1 (STK1p), CEA and CA19.9 can be used as prognostic biomarkers in the primary tumor location (PTL) of colorectal carcinoma (CRC). Additional clinical factors of TNM stage, pathological grade, age and sex were also included., Methods: STK1p was determined by an ECL-dot-blot assay, and CEA/CA19.9 was determined by an automatic electrochemiluminescence analyzer in a retrospective presurgery of right-colon carcinoma (R-CC, n = 90), left-colon carcinoma (L-CC, n = 128) and rectal carcinoma (RC, n = 270). Prognostic factors were evaluated by COX and overall survival (OS)., Results: The multivariate-COX and OS in relation to the prognostic factors of PTL in CRC were different and complex. An elevated STK1p value was significantly associated with poor OS in RC (P = 0.002) and L-CC (P = 0.037) but not in R-CC (P > 0.05). Elevated CEA (P≈.000) and CA19.9 (P≈.000) were significantly associated with poor OS in RC but not in L-CC and R-CC. Multivariate-COX showed that STK1p (P = 0.02, HR = 1.779, 95%CI 1.30-7.582), CEA (P = 0.001, HR = 2.052, 95%CI 1.320-3.189), CA19.9 (P≈.000, HR = 2.574, 95%CI 1.592-4.162) and TNM-stage (P≈.000, HR = 2.368, 95%CI 1.518-3.694) were independent prognostic factors in RC, while TNM-stage was an independent prognostic factor only in R-CC (P = 0.011, HR = 3.139, 95% CI 1.30-7.582) and L-CC (P≈.000, HR = 4.168, 95%CI 1.980-8.852). Moreover, elevated STK1p was significantly more sensitive (P < .001) for predicting mortality than CEA and CA19.9. No correlation was found between STK1p, CEA or AFP., Conclusion: Combining TNM stage and suitable biomarkers, STK1p provides further reliable information on the survival of PTL of CRC., (© 2023. The Author(s).)

Age-related macular degeneration (AMD) is one of the most common leading causes of irreversible blindness, and there is no effective treatment for it. It has been reported that aging is the greatest risk factor for AMD, and epithelial-mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells plays an important role in the pathogenesis of AMD. To clarify the relationship between senescence and EMT in RPE cells, we used the replicative senescence model, H 2 O 2 - and/or Nutlin3a-induced senescence model, and low-density and/or TGF-β-induced EMT model to detect the expression of senescence-, RPE- and EMT-related genes, and assessed the motility of cells by using a scratch wound migration assay. The results showed that replicative senescence of RPE cells was accompanied by increased expression of EMT markers. However, senescent RPE cells themselves did not undergo EMT, as the H 2 O 2 and Nutlin3a treated cells showed no increase in EMT characteristics, including unchanged or decreased expression of EMT markers and decreased motility. Furthermore, conditioned medium (CM) from senescent cells induced EMT in presenescent RPE cells, and EMT accelerated the process of senescence. Importantly, dasatinib plus quercetin, which selectively eliminates senescent cells, inhibited low-density-induced EMT in RPE cells. These findings provide a better understanding of the interconnection between senescence and EMT in RPE cells. Removal of senescent cells by certain methods such as senolytics, might be a promising potential approach to prevent or delay the progression of RPE-EMT-related retinal diseases such as AMD., Competing Interests: Declarations of interest None., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Age-related macular degeneration (AMD) is a major vision-threatening disease. Although mesenchymal stem cells (MSCs) exhibit beneficial neural protective effects, their limited differentiation capacity in vivo attenuates their therapeutic function. Therefore, the differentiation of MSCs into retinal pigment epithelial (RPE) cells in vitro and their subsequent transplantation into the subretinal space is expected to improve the outcome of cell therapy. Here, we transdifferentiated human umbilical cord MSCs (hUCMSCs) into induced RPE (iRPE) cells using a cocktail of five transcription factors (TFs): CRX, NR2E1, C-MYC, LHX2, and SIX6. iRPE cells exhibited RPE specific properties, including phagocytic ability, epithelial polarity, and gene expression profile. In addition, high expression of PTPN13 in iRPE cells endows them with an epithelial-to-mesenchymal transition (EMT)-resistant capacity through dephosphorylating syntenin1, and subsequently promoting the internalization and degradation of transforming growth factor-β receptors. After grafting into the subretinal space of the sodium iodate-induced rat AMD model, iRPE cells demonstrated a better therapeutic function than hUCMSCs. These results suggest that hUCMSC-derived iRPE cells may be promising candidates to reverse AMD pathophysiology., (© 2022. The Author(s).)

To study the biological functions and applications of human amniotic epithelial cell-derived extracellular vesicles (hAEC-EVs), the cargos of hAEC-EVs were analyzed using miRNA sequencing and proteomics analysis. The hAECs and hAEC-EVs in this study had specific characteristics. Multi-omics analyses showed that extracellular matrix (ECM) reorganization, inhibition of excessive myofibroblasts, and promotion of target cell adhesion to the ECM were their primary functions. We evaluated the application of hAEC-EVs for corneal alkali burn healing in rabbits and elucidated the fundamental mechanisms. Slit-lamp images revealed that corneal alkali burns induced central epithelial loss, stromal haze, iris, and pupil obscurity in rabbits. Slit-lamp examination and histological findings indicated that hAEC-EVs facilitated re-epithelialization of the cornea after alkali burns, reduced scar formation and promoted the restoration of corneal tissue transparency. Significantly fewer α-SMA-positive myofibroblasts were observed in the hAEC-EV-treated group than the PBS group. HAEC-EVs effectively promoted the proliferation and migration of hCECs and hCSCs in vitro and activated the focal adhesion signaling pathway. We demonstrated that hAEC-EVs were excellent cell-free candidates for the treatment of ECM lesion-based diseases, including corneal alkali burns. HAEC-EVs promoted ECM reorganization and cell adhesion of target tissues or cells via orderly activation of the focal adhesion signaling pathway., Competing Interests: Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2022.)

The hostile microenvironment of the retina in patients with age-related macular degeneration (AMD) may trigger epithelial-to-mesenchymal transition (EMT) of grafted retinal pigment epithelial (RPE) cells, thus attenuating the therapeutic outcome. Here, we transformed human dedifferentiated induced pluripotent stem cell-derived RPE (iPSC-RPE) cells into induced RPE (iRPE) cells using a cocktail of four transcription factors (TFs)-CRX, MITF-A, NR2E1, and C-MYC. These critical TFs maintained the epithelial property of iRPE cells by regulating the expression of bmp7 , forkhead box f2, lin7a , and pard6b , and conferred resistance to TGF-β-induced EMT in iRPE cells by targeting ppm1a . The iRPE cells with Tet-on system-regulated c-myc expression exhibited EMT resistance and better therapeutic function compared with iPSC-RPE cells in rat AMD model. Our study demonstrates that endowing RPE cells with anti-EMT property avoids the risk of EMT after cells are grafted into the subretinal space, and it may provide a suitable candidate for AMD treatment., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

Kidney renal clear cell carcinoma (KIRC) has the highest mortality rate and potential for invasion among renal cancers. The diagnosis and treatment of KIRC are becoming challenging because of its diverse pathogenic mechanisms. Glia (GMFB) is a highly conserved growth and differentiation factor for glia cells and neurons, and it is closely associated with neurodegenerative diseases. However, its role in KIRC remains unknown. The present study integrated bioinformatics approaches with suitable meta-analyses to determine the position of GMFB in KIRC. There was a significant decrease in Gmfb expression in KIRC kidneys compared with normal controls. Gmfb expression was negatively associated with pathologic stage, T and M stages, and histologic grade. Univariate and multivariate analyses showed that elevated Gmfb expression was an independent factor for a favorable prognosis. Furthermore, the nomogram verified that Gmfb is a low-risk factor for KIRC. Knockdown of Gmfb in Caki-2 cells increased viability and decreased p21 and p27 levels. Overexpression of Gmfb inhibited Caki-2 cell proliferation, migration, and invasion and decreased mitochondrial membrane potential. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses considering Gmfb co-expressed differentially expressed genes (DEGs) showed that collecting duct acid secretion and mineral absorption ranked were the most important upregulated and downregulated DEGs, respectively. The upregulated hub genes for DEGs were mainly involved in nucleosome assembly, nucleosome organization, and chromatin assembly, and the downregulated hub genes were primarily associated with keratinization. The ratio of tumor-infiltrating immune cells in KIRC tissues was evaluated using CIBERSORTx. The results showed that the Gmfb expression was significantly positively correlated with macrophage M2 cells and mast resting cell infiltration levels and negatively correlated with T follicular helper, T regulatory, and B plasma cell infiltration levels. The former cell types were associated with a beneficial outcome, while the latter had a worse outcome in patients with KIRC. In summary, this study identified GMFB as a novel independent biomarker and therapeutic target for KIRC, and it provides a helpful and distinct individualized treatment strategy for KIRC with a combination of molecular targets and tumor microenvironment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhu, Wang, Feng, Gao, Zhang, Jin, Tian, Xu, Chen, Ou, Wang, Xu and Lu.)

RIO Kinase 1 (RIOK1) is involved in various pathologies, including cancer. However, the role of RIOK1 in radioresistance of colorectal cancer (CRC) remains largely unknown. In this study, we reported that RIOK1 was overexpressed in rectal cancer tissue with weaker tumor regression after neoadjuvant chemoradiotherapy (neoCRT). Moreover, higher RIOK1 expression predicted a poor prognosis in patients with rectal cancer. Blockade of RIOK1 using Toyocamycin, a pharmacological inhibitor of RIOK1, or by knocking down its expression, decreased the resistance of CRC cells to radiotherapy in vitro and in vivo. A mechanistic study revealed that RIOK1 regulates radioresistance by suppressing the p53 signaling pathway. Furthermore, we found that RIOK1 and Ras-GAP SH3 domain binding protein 2 (G3BP2) interact with each other. RIOK1 phosphorylates G3BP2 at Thr226, which increases the activity of G3BP2. RIOK1-mediated phosphorylation of G3BP2 facilitated ubiquitination of p53 by murine double minute 2 protein (MDM2). Altogether, our study revealed the clinical significance of RIOK1 in CRC, and therapies targeting RIOK1 might alleviate the CRC tumor burden in patients., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Diabetic retinopathy (DR) is one of the leading causes of blindness in the world, and timely prevention and treatment are very important. Previously, we found that a neurodegenerative factor, Glia maturation factor-β (GMFB), was upregulated in the vitreous at a very early stage of diabetes, which may play an important role in pathogenesis. Here, we found that in a high glucose environment, large amounts of GMFB protein can be secreted in the vitreous, which translocates the ATPase ATP6V1A from the lysosome, preventing its assembly and alkalinizing the lysosome in the retinal pigment epithelial (RPE) cells. ACSL4 protein can be recognized by HSC70, the receptor for chaperone-mediated autophagy, and finally digested in the lysosome. Abnormalities in the autophagy-lysosome degradation process lead to its accumulation, which catalyzes the production of lethal lipid species and finally induces ferroptosis in RPE cells. GMFB antibody, lysosome activator NKH477, CMA activator QX77, and ferroptosis inhibitor Liproxstatin-1 were all effective in preventing early diabetic retinopathy and maintaining normal visual function, which has powerful clinical application value. Our research broadens the understanding of the relationship between autophagy and ferroptosis and provides a new therapeutic target for the treatment of DR., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Corneal endothelial cells (CECs) play a major role in the maintenance of stromal hydration via the barrier and pump function for clear vision. Adult CECs cannot regenerate after injury. CECs cultured in vitro can undergo mitosis but may undergo corneal endothelial-to-mesenchymal transition (EnMT) and lose their endothelial characteristics. In this study, we examined the effects of CHIR99021 on transforming growth factor beta-1(TGFβ1)-induced EnMT in human CECs (hCECs) lines. CHIR99021 kept hCECs in the hexagonal shape and could downregulate the EnMT markers alpha-smooth muscle actin (α-SMA) and fibronectin (FN1), meanwhile maintained the hCECs function markers Na + /K + -ATPase and zonula occludens-1 (ZO-1) at levels comparable to those in the normal control. Interestingly, we found that the combination of CHIR99021 and TGFβ1 at appropriate concentrations would significantly promote the proliferation and migration of hCECs. These effects may be related to the inhibition of RhoA or Rac1, as well as the activation of Wnt and Erk pathway, with a calcium homeostasis. Our findings indicate that CHIR99021 inhibit EnMT and that the combination of CHIR99021 and TGFβ1 may provide new ideas for corneal endothelial regeneration and wound healing., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Purpose: To explore the function and regulatory mechanism of IFITM3 in mouse neural retinal progenitor cells (mNRPCs), which was found to be very important not only in the development of the retina in embryos but also in NRPCs after birth., Methods: Published single-cell sequencing data were used to analyze IFITM3 expression in mNRPCs. RNA interference was used to knock down the expression of IFITM3. CCK-8 assays were used to analyze cell viability. RNA-seq was used to assess mRNA expression, as confirmed by real-time quantitative PCR, and immunofluorescence assays and western blots were used to validate the levels of relative proteins, and autophagy flux assay. Lysosomal trackers were used to track the organelle changes., Results: The results of single-cell sequencing data showed that IFITM3 is highly expressed in the embryo, and after birth, RNA-seq showed high IFITM3 expression in mNRPCs. Proliferation and cell viability were greatly reduced after IFITM3 was knocked down. The cell membrane system and lysosomes were dramatically changed, and lysosomes were activated and evidently agglomerated in RAMP-treated cells. The expression of LAMP1 was significantly increased with lysosome agglomeration after treatment with rapamycin (RAMP). Further detection showed that SQSTM1/P62, HSC70 and LAMP-2A were upregulated, while no significant difference in LC3A/B expression was observed; no autophagic flux was generated., Conclusion: IFITM3 regulates mNRPC viability and proliferation mainly through chaperone-mediated autophagy (CMA) but not macroautophagy (MA). IFITM3 plays a significant role in maintaining the homeostasis of progenitor cell self-renewal by sustaining low-level activation of CMA to eliminate deleterious factors in cells., (© 2022. The Author(s).)

Diabetic kidney disease (DKD) is a long-term major microvascular complication of uncontrolled hyperglycemia and one of the leading causes of end-stage renal disease (ESDR). The pathogenesis of DKD has not been fully elucidated, and effective therapy to completely halt DKD progression to ESDR is lacking. This study aimed to identify critical molecular signatures and develop novel therapeutic targets for DKD. This study enrolled 10 datasets consisting of 93 renal samples from the National Center of Biotechnology Information (NCBI) Gene Expression Omnibus (GEO). Networkanalyst, Enrichr, STRING, and Cytoscape were used to conduct the differentially expressed genes (DEGs) analysis, pathway enrichment analysis, protein-protein interaction (PPI) network construction, and hub gene screening. The shared DEGs of type 1 diabetic kidney disease (T1DKD) and type 2 diabetic kidney disease (T2DKD) datasets were performed to identify the shared vital pathways and hub genes. Strepotozocin-induced Type 1 diabetes mellitus (T1DM) rat model was prepared, followed by hematoxylin & eosin (HE) staining, and Oil Red O staining to observe the lipid-related morphological changes. The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was conducted to validate the key DEGs of interest from a meta-analysis in the T1DKD rat. Using meta-analysis, 305 shared DEGs were obtained. Among the top 5 shared DEGs, Tmem43, Mpv17l, and Slco1a1, have not been reported relevant to DKD. Ketone body metabolism ranked in the top 1 in the KEGG enrichment analysis. Coasy, Idi1, Fads2, Acsl3, Oxct1, and Bdh1, as the top 10 down-regulated hub genes, were first identified to be involved in DKD. The qRT-PCR verification results of the novel hub genes were mostly consistent with the meta-analysis. The positive Oil Red O staining showed that the steatosis appeared in tubuloepithelial cells at 6 w after DM onset. Taken together, abnormal ketone body metabolism may be the key factor in the progression of DKD. Targeting metabolic abnormalities of ketone bodies may represent a novel therapeutic strategy for DKD. These identified novel molecular signatures in DKD merit further clinical investigation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Diao, Wu, Yang, Liu, Xu, Jin, Wang, Zhang, Gao, Jin, Tian, Xu, Ou, Li, Xu and Lu.)

Purpose: Corneal endothelial cells (CECs) serve as a barrier and foothold for the corneal stroma to maintain the function and transparency of the cornea. Loss of CECs during aging or disease states leads to blindness, and cell replacement therapy using either donated or artificially differentiated CECs remains the only curative approach. Methods: Human induced pluripotent stem cells (hiPSCs) that were cultured in chemically defined medium were induced with dual-SMAD inhibition to differentiate into neural crest cells (NCCs). A small-molecule library was screened to differentiate the NCCs into corneal endothelial-like cells. The characteristics of these cells were identified with real-time PCR and immunofluorescence. Western blotting was applied to detect the signaling pathways and key factors regulated by the small molecules. Results: We developed an effective protocol to differentiate hiPSCs into CECs with defined small molecules. The hiPSC-CECs were characterized by ZO-1, AQP1, Vimentin and Na + /K + -ATPase. Based on our small-molecule screen, we identified a small-molecule combination, A769662 and AT13148, that enabled the most efficient production of CECs. The combination of A769662 and AT13148 upregulated the PKA/AKT signaling pathway, FOXO1 and PITX2 to promote the conversion of NCCs to CECs. Conclusion: We established an efficient small molecule-based method to differentiate hiPSCs into corneal endothelial-like cells, which might facilitate drug discovery and the development of cell-based therapies for corneal diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chen, Ou, Wang, Liu, Hu, Liu, Tian, Xu, Gao, Lu, Jin, Xu and Cui.)

Hepatocellular carcinoma (HCC) is one of the most common types of cancer. The novel sensitive biomarkers and therapeutic targets are urgently needed for the early diagnosis of HCC and improvement of clinical outcomes. Glia maturation factor-β (GMFB) is a growth and differentiation factor for both glia and neurons and has been found to be tightly involved in inflammation and neurodegeneration conditions. In our study, the expression level of GMFB was significantly up-regulated in patients with HCC and positively co-expression with tumor node metastases (TNM) stage and histopathological grade of HCC. The high expression level of GMFB was remarkably associated with poor overall survival, which mainly occurred in males rather than females. Multivariate analysis revealed GMFB to be an independent prognostic factor for overall survival in patients with HCC. Results of Gene Ontology (GO) and KEGG pathways analysis showed that down-regulation of pathways related to protein translation and mitochondria function were enriched. Protein-protein interaction analysis revealed the central role of mitochondria protein in HCC. The downregulation of genes involved in glycolysis and gluconeogenesis was observed among the co-expression genes of GMFB. Knockdown of GMFB in Hep3B significantly inhibited proliferation, migration, and invasion of Hep3B cells, and also downregulated the expression levels of some of metal matrix proteinase (MMP), increased mtDNA copy number and loss of mitochondrial transmembrane potential. GMFB influences the malignancy rate of HCC possibly through regulation of the expression of MMPs, mtDNA function and glycolysis. We proposed that GMFB was a promising HCC diagnostic and prognostic biomarker and therapeutic target in HCC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sun, Hu, Wang, Wang, Zhang, Gao, Ou, Tian, Jin, Xu, Zhang, Xu and Lu.)

Deep mining of the molecular mechanisms underlying diabetic retinopathy (DR) is critical for the development of novel therapeutic targets. This study aimed to identify key molecular signatures involved in experimental DR on the basis of integrated bioinformatics analysis. Four datasets consisting of 37 retinal samples were downloaded from the National Center of Biotechnology Information Gene Expression Omnibus. After batch-effect adjustment, bioinformatics tools such as Networkanalyst, Enrichr, STRING, and Metascape were used to evaluate the differentially expressed genes (DEGs), perform enrichment analysis, and construct protein-protein interaction networks. The hub genes were identified using Cytoscape software. The DEGs of interest from the meta-analysis were confirmed by quantitative reverse transcription-polymerase chain reaction in diabetic rats and a high-glucose-treated retinal cell model, respectively. A total of 743 DEGs related to lens differentiation, insulin resistance, and high-density lipoprotein (HDL) cholesterol metabolism were obtained using the meta-analysis. Alterations of dynamic gene expression in the chloride ion channel, retinol metabolism, and fatty acid metabolism were involved in the course of DR in rats. Importantly, H3K27m3 modifications regulated the expression of most DEGs at the early stage of DR. Using an integrated bioinformatics approach, novel molecular signatures were obtained for different stages of DR progression, and the findings may represent distinct therapeutic strategies for DR patients., (© 2021 International Union of Biochemistry and Molecular Biology.)

Background: Systemic sclerosis (SSc) is a disease that features severe fibrosis of the skin and lacks effective therapy. Bone marrow mesenchymal stem cell (BMSC)-derived extracellular vesicles (EVs) are potential stem cell-based tools for the treatment of SSc., Methods: BMSCs were isolated from the bone marrow of mice and identified with surface markers according to multilineage differentiation. EVs were isolated from the BMSC culture medium by ultracentrifugation and identified with a Nanosight NS300 particle size analyzer, transmission electron microscopy (TEM), and western blot. The microRNAs (miRNAs) of BMSC-derived EVs (BMSC-EVs) were studied via miRNA sequencing (miRNA-seq) and bioinformatic analysis. An SSc mouse model was established via subcutaneous bleomycin (BLM) injection, and the mice were treated with BMSCs or BMSC-derived EVs. Skin tissues were dissociated and analyzed with H&E staining, RNA sequencing (RNA-seq), western blot, and immunohistochemical staining., Results: Evident pathological changes, like fibrosis and inflammation, were induced in the skin of BLM-treated mice. BMSCs and BMSC-EVs effectively intervened such pathological manifestations and disease processes in a very similar way. The effects of the BMSC-EVs were found to be caused by the miRNAs they carried, which were proven to be involved in regulating the proliferation and differentiation of multiple cell types and in multiple EV-related biological processes. Furthermore, TGF-β1-positive cells and α-SMA-positive myofibroblasts were significantly increased in the scleroderma skin of BLM-treated mice but evidently reduced in the scleroderma skin of the EV-treated SSc group. In addition, the numbers of mast cells and infiltrating macrophages and lymphocytes were evidently increased in the skin of BLM-treated mice but significantly reduced by EV treatment. In line with these observations, there were significantly higher mRNA levels of the inflammatory cytokines Il6, Il10, and Tnf-α in SSc mice than in control mice, but the levels decreased following EV treatment. Through bioinformatics analysis, the TGFβ and WNT signaling pathways were revealed to be closely involved in the pathogenic changes seen in mouse SSc, and these pathways could be therapeutic targets for treating the disease., Conclusions: BMSC-derived EVs could be developed as a potential therapy for treating skin dysfunction in SSc, especially considering that they show similar efficacy to BMSCs but have fewer developmental regulatory requirements than cell therapy. The effects of EVs are generated by the miRNAs they carry, which alleviate SSc pathogenic changes by regulating the WNT and TGFβ signaling pathways.

Resistance to chemotherapy remains the major cause of treatment failure in patients with colorectal cancer (CRC). Here, we identified TRIM25 as an epigenetic regulator of oxaliplatin (OXA) resistance in CRC. The level of TRIM25 in OXA-resistant patients who experienced recurrence during the follow-up period was significantly higher than in those who had no recurrence. Patients with high expression of TRIM25 had a significantly higher recurrence rate and worse disease-free survival than those with low TRIM25 expression. Downregulation of TRIM25 dramatically inhibited, while overexpression of TRIM25 increased, CRC cell survival after OXA treatment. In addition, TRIM25 promoted the stem cell properties of CRC cells both in vitro and in vivo. Importantly, we demonstrated that TRIM25 inhibited the binding of E3 ubiquitin ligase TRAF6 to EZH2, thus stabilizing and upregulating EZH2, and promoting OXA resistance. Our study contributes to a better understanding of OXA resistance and indicates that inhibitors against TRIM25 might be an excellent strategy for CRC management in clinical practice.

Purpose: Epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is a key pathological event in proliferative retinal diseases such as proliferative vitreoretinopathy (PVR). This study aimed to explore a new method to reverse EMT in RPE cells to develop an improved therapy for proliferative retinal diseases., Methods: In vitro, human embryonic stem cell-derived RPE cells were passaged and cultured at low density for an extended period of time to establish an EMT model. At different stages of EMT after treatment with known molecules or combinations of molecules, the morphology was examined, transepithelial electrical resistance (TER) was measured, and expression of RPE- and EMT-related genes were examined with RT-PCR, Western blotting, and immunofluorescence. In vivo, a rat model of EMT in RPE cells was established via subretinal injection of dispase. Retinal function was examined by electroretinography (ERG), and retinal morphology was examined., Results: EMT of RPE cells was effectively induced by prolonged low-density culture. After EMT occurred, only the combination of the Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor Y27632 and the TGF-β receptor inhibitor RepSox (RY treatment) effectively suppressed and reversed the EMT process, even in cells in an intermediate state of EMT. In dispase-treated Sprague-Dawley rats, RY treatment maintained the morphology of RPE cells and the retina and preserved retinal function., Conclusions: RY treatment might promote mesenchymal-epithelial transition (MET), the inverse process of EMT, to maintain the epithelial-like morphology and function of RPE cells. This combined RY therapy could be a new strategy for treating proliferative retinal diseases, especially those involving EMT of RPE cells.

Na v 1.5, encoded by SCN5A, has been associated with metastasis in colorectal cancer (CRC). Here, we investigated the mechanism by which Na v 1.5 regulates tumor progression and whether Na v 1.5 influences chemosensitivity to 5-fluorouracil (5-FU) in CRCs. CRC cases were evaluated for Na v 1.5 expression. Elevated Na v 1.5 expression was associated with poor prognosis in CRCs, whereas stage II/III patients with upregulated SCN5A expression could have better survival after receiving 5-FU-based adjuvant chemotherapy. In CRC cells, SCN5A knockdown reduced the proliferation, migration and invasion. According to RNA sequencing, SCN5A knockdown inhibited both the cell cycle and epithelial-mesenchymal transition. In addition, Na v 1.5 stabilized the KRas-calmodulin complex to modulate Ras signaling, promoting Ca 2+ influx through the Na + -Ca 2+ exchanger and Ca 2+ release-activated calcium channel. Meanwhile, SCN5A knockdown increased the 50% inhibitory concentration to 5-FU by upregulating 5-FU-stimulated apoptosis in CRCs. In conclusion, Na v 1.5 could progress to proliferation and metastasis through Ca 2+ /calmodulin-dependent Ras signaling in CRC, and it could also enhance 5-FU-stimulated apoptosis. Clinically, patients with stage II/III CRCs with elevated SCN5A expression demonstrated poor prognosis, yet those patients could benefit more from 5-FU-based chemotherapy than patients with lower SCN5A expression., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Photoreceptor (PR) dysfunction or death is the key pathological change in retinal degeneration (RD). The death of PRs might be due to a primary change in PRs themselves or secondary to the dysfunction of the retinal pigment epithelium (RPE). Poly(ADP-ribose) polymerase (PARP) was reported to be involved in primary PR death, but whether it plays a role in PR death secondary to RPE dysfunction has not been determined. To clarify this question and develop a new therapeutic approach, we studied the changes in PAR/PARP in the RCS rat, a RD model, and tested the effect of PARP intervention when given alone or in combination with RPE cell transplantation. The results showed that poly(ADP-ribosyl)ation of proteins was increased in PRs undergoing secondary death in RCS rats, and this result was confirmed by the observation of similar changes in sodium iodate (SI)-induced secondary RD in SD rats. The increase in PAR/PARP was highly associated with increased apoptotic PRs and decreased visual function, as represented by lowered b-wave amplitudes on electroretinogram (ERG). Then, as we expected, when the RCS rats were treated with subretinal injection of the PARP inhibitor PJ34, the RD process was delayed. Furthermore, when PJ34 was given simultaneously with subretinal ARPE-19 cell transplantation, the therapeutic effects were significantly improved and lasted longer than those of ARPE-19 or PJ34 treatment alone. These results provide a potential new approach for treating RD., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Dickkopf 1 (DKK1) is associated with tumor progression. However, whether DKK1 influences the tumor response to programmed cell death protein 1 (PD-1) blockade in colorectal cancers (CRCs) with deficient mismatch repair (dMMR) or microsatellite instability (MSI) has never been clarified., Methods: Tumor tissues from 80 patients with dMMR CRC were evaluated for DKK1 expression and immune status via immunohistochemistry. Serum DKK1 was measured in another set of 43 patients who received PD-1 blockade therapy. CT26 cells and dMMR CRC organoids were cocultured with T cells, and CT26-grafted BALB/c mice were also constructed. T-cell cytotoxicity was assessed by apoptosis assays and flow cytometry. The pathway through which DKK1 regulates CD8+ T cells was investigated using RNA sequencing, and chromatin immunoprecipitation and luciferase reporter assays were conducted to determine the downstream transcription factors of DKK1., Results: Elevated DKK1 expression was associated with recurrence and decreased CD8+ T-cell infiltration in dMMR CRCs, and patients with high-serum DKK1 had a poor response to PD-1 blockade. RNA interference or neutralization of DKK1 in CRC cells enhanced CD8+ T-cell cytotoxicity, while DKK1 decreased T-bet expression and activated GSK3β in CD8+ T cells. In addition, E2F1, a downstream transcription factor of GSK3β, directly upregulated T-bet expression. In organoid models, the proportion of apoptotic cells was elevated after individual neutralization of PD-1 or DKK1 and was further increased on combined neutralization of PD-1 and DKK1., Conclusions: DKK1 suppressed the antitumor immune reaction through the GSK3β/E2F1/T-bet axis in CD8+ T cells. Elevated serum DKK1 predicted poor tumor response to PD-1 blockade in dMMR/MSI CRCs, and DKK1 neutralization may restore sensitivity to PD-1 blockade., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: The aim of this study was to assess the prognostic value of CD8+ tumor infiltrating lymphocytes (TIL) combined with programmed cell death-ligand 1 (PD-L1) expression for patients with solitary colorectal cancer liver metastasis (SCLM) undergoing R0 resection., Methods: Patients undergoing curative hepatectomy for SCLM were reviewed. Immunohistochemical multiplex technique was used for quantifying CD8+ TIL, and immunohistochemical staining was used for assessing PD-L1 expression. The tumor immune microenvironment (TIME) was classified as strong for high CD8+ TIL and low PD-L1, weak for low CD8+ TIL and high PD-L1, and mild for the rest. Recurrence-free survival (RFS) and overall survival (OS) was compared between these groups., Results: Among the 94 patients included, a high CD8+ TIL and high PD-L1 expression was observed in 51 (54.3%) and 47 (50.0%) patients, respectively. Strong, mild, and weak TIME was observed in 24 (25.5%), 42 (44.7%), and 28 (29.8%) patients, respectively. Patients with a high CD8+ TIL had a significant longer RFS than patients with a low CD8+ TIL (3-year RFS rate, 71.6% vs. 55.3%, P=0.018). The 3-year RFS rate in the strong TIME group was significantly higher than that in the mild and weak TIME groups (89.5% vs. 71.7% and 28.8%, P<0.001), as was the 3-year rate of OS (93.8% vs. 81.8% and 61.6%, P<0.001). CD8+ TIL combined with PD-L1 expression showed better predicting accuracy for RFS than CD8+ TIL alone., Conclusions: The density of CD8+ TIL combined with PD-L1 expression in liver metastasis was a predictor of RFS for patients with SCLM undergoing R0 resection, and therefore can be used for guiding the postoperative treatment of these patients., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/atm-20-2762a). All authors report grants from National Natural Science Foundation of China, grants from Natural Science Foundation of Guangdong Province, China, during the conduct of the study., (2020 Annals of Translational Medicine. All rights reserved.)

Purpose: This study aimed to explore the role of the protein kinase A (PKA) pathway in proliferative vitreoretinopathy (PVR) and the effect of the PKA inhibitor H89 on experimental PVR., Methods: Epiretinal membranes (ERMs) were acquired from PVR patients and analyzed by frozen-section immunofluorescence. An in vivo model was developed by intravitreal injecting rat eyes with ARPE-19 cells and platelet-rich plasma, and changes in eye structures and vision function were observed. An in vitro epithelial-mesenchymal transition (EMT) cell model was established by stimulating ARPE-19 cells with transforming growth factor (TGF)-β. Alterations in EMT-related genes and cell function were detected. Mechanistically, PKA activation and activity were explored to assess the relationship between TGF-β1 stimulation and the PKA pathway. The effect of H89 on the TGF-β-Smad2/3 pathway was detected. RNA sequencing was used to analyze gene expression profile changes after H89 treatment., Results: PKA was activated in human PVR membranes. In vivo, H89 treatment protected against structural changes in the retina and prevented decreases in electroretinogram b-wave amplitudes. In vitro, H89 treatment inhibited EMT-related gene alterations and partially reversed the functions of the cells. TGF-β-induced PKA activation was blocked by H89 pretreatment. H89 did not affect the phosphorylation or nuclear translocation of regulatory Smad2/3 but increased the expression of inhibitory Smad6., Conclusions: PKA pathway activation is involved in PVR pathogenesis, and the PKA inhibitor H89 can effectively inhibit PVR, both in vivo and in vitro. Furthermore, the protective effect of H89 is related to an increase in inhibitory Smad6.

Adipose-derived stem cells (ASCs) have shown a strong protective effect on retinal degenerative diseases (RDD) after being transplanted into the subretinal space in an animal model. Recently, several clinical trials have been conducted to treat RDD with intravitreal transplantation of stem cells, including ASCs. However, the outcomes of the clinical trials were not satisfactory. To investigate if the transplantation site alters the outcome of stem cell-based therapy for RDD, we isolated rat ASCs (rASCs) and labeled them with green fluorescent protein. Autologous rASCs were grafted into the vitreous chamber or subretinal space in a rat RDD model induced by sodium iodate (SI). The electric response was recorded by ERG. The anatomic structure of the retina was observed in cryosections of rat eyes at posttransplantation weeks 1, 2, and 4. Neural retina apoptosis and epiretinal membrane- (ERM-) like structure formation were investigated by immunostaining. The intravitreal transplantation of rASCs resulted in an extinguished electric response, although the rosette formation and apoptosis of neural retina were reduced. However, the rASCs that grafted in the subretinal space protected the retina from the damage caused by SI, including a partial recovering of the electric response and a reduction in rosette formation. Intravitreally grafted rASCs formed a membrane, resulting in retina folding at the injection site. Müller cells, retinal pigment epithelial cells, and microglial cells migrated from the retina to the rASC-formed membrane and subsequently formed an ERM-like structure. Furthermore, vitreous fluid promoted rASC migration, and rASC-conditioned medium enhanced Müller cell migration as indicated by in vitro studies. These data suggested that the vitreous chamber is not a good transplantation site for ASC-based therapy for RDD and that a deliberate decision should be made before transplantation of stem cells into the vitreous chamber to treat RDD in clinical trials., Competing Interests: All the authors indicate no potential conflict of interest., (Copyright © 2020 Chengyu Hu et al.)

Background: The aim of this study was to assess trophoblast antigen protein 2 (TROP2) expression in liver oligometastases and its prognostic value for colorectal liver oligometastasis (CLO) patients undergoing liver resection., Methods: We retrospectively selected 129 consecutive CLO patients who underwent curative liver resection between June 1999 and December 2016. Immunohistochemistry (IHC) was performed to detect TROP2 expression in paraffin-embedded specimens. Recurrence-free survival (RFS) and overall survival (OS) were analyzed using the Kaplan-Meier method and log-rank test, and independent prognostic factors were identified with Cox regression modeling., Results: TROP2 was expressed in 72.9% (94/129) of liver oligometastatic tissues. TROP2 expression in primary tumors and liver oligometastases was significantly positively correlated ( r  = 0.758, p  < 0.001). Survival analysis indicated that CLO patients with high TROP2 expression had worse 3-year RFS (44.2% versus 66.4%, p  = 0.007) and 3-year OS rates (70.3% versus 85.4%, p  = 0.035) than did those with low TROP2 expression. Multivariate analysis indicated that high TROP2 expression was independently associated with poor RFS [hazard ratio (HR) = 2.017; 95% confidence interval (CI) 1.198-3.396; p  = 0.023] and OS (HR = 2.090; 95% CI 1.037-4.214; p  = 0.039). Gene expression profile analysis indicated that high TROP2 expression was associated with TNFα signaling via NF-κB, the inflammatory response and epithelial-mesenchymal transition (EMT)., Conclusions: TROP2 overexpression was associated with an unfavorable oncologic prognosis in patients with CLO undergoing liver resection. Detecting TROP2 expression may be valuable for guiding postoperative treatment among CLO patients., Competing Interests: Conflict of interest statement: The authors declare that there is no conflict of interest., (© The Author(s), 2019.)

Background: It was reported that tumor-expressed dickkopf-related (DKK) proteins affect micro-environment. However, the influence of DKK1 on colorectal cancer (CRC) liver oligometastases (CRCLOM) remains unclear., Methods: CRC cases after resection of liver oligometastases were enrolled in Sun Yat-Sen University Cancer Center with intact clinical data. Serum DKK1 was detected by ELISA assay. Immunofluorescent staining examination for CD3 and CD8 in slices were also conducted., Results: Among 65 patients included, the recurrence-free survival (RFS) and overall survival (OS) were significantly better in the low serum DKK1 group (RFS: P = 0.021; OS: P = 0.043). DKK1 was overexpressed in stage IV CRC patients in TCGA data. The number of CD8+ tumor-infiltrating lymphocytes (TILs) in invasive margin of CRC liver oligometastases was significantly higher in low serum DKK1 group (P = 0.042)., Conclusion: Elevated serum DKK1 level was associated with poorer RFS and OS, and less CD8+ TILs in invasive margin in CRC liver oligometastases. DKK1 might serve as a supplementalprognostic factor for clinical risk score and a potential target for immunotherapy.

: Background: A comprehensive investigation into immune cell infiltration provides more accurate and reliable prognostic information for patients with colorectal liver oligometastases (CLO) after liver metastasectomy., Methods: Simultaneous detection of the immune constituents CD3 + , CD8 + , Foxp3 + T, and α-SMA + cells in the liver oligometastasis of 133 patients was conducted using a four-colour immunohistochemical multiplex technique. Immune cells were quantified, and tumour-infiltrating lymphocyte (TIL) ratios were subsequently calculated. Correlation analysis was performed using Pearson's correlation. Recurrence-free survival (RFS) and overall survival (OS) for TIL ratios were analysed using the Kaplan-Meier method and Cox regression models., Results: Significantly fewer CD3 + , CD8 + , and Foxp3 + T cells were observed in the intratumoural region than in the peritumoural region of liver metastases. CD3 + , CD8 + , Foxp3 + T, and α-SMA + cells showed significantly positive correlations with each other both in the intratumoural and peritumoural regions of liver metastases. Only the CD8/CD3 TIL ratio demonstrated a positive correlation between intratumoural and peritumoural regions of liver metastases (r = 0.541, p < 0.001). Patients with high intratumoural CD8/CD3 ratios had significantly longer 3-year RFS (59.0% vs. 47.4%, p = 0.035) and 3-year OS rates (83.3% vs. 65.8%, p = 0.007) than those with low intratumoural CD8/CD3 ratios. Multivariate analyses revealed that the intratumoural CD8/CD3 ratio was independently associated with RFS (HR = 0.593; 95% CI = 0.357-0.985; p = 0.043) and OS (HR = 0.391; 95% CI = 0.193-0.794; p = 0.009)., Conclusion: These findings offer a better understanding of the prognostic value of immune cell infiltration on liver oligometastasis from colorectal cancer., Competing Interests: The authors declare that they have no conflicts of interest.

Background: We launched a screening program for colorectal cancer (CRC) in Yuexiu District, Guangzhou, China, in 2014. Here we aimed to report the early results of the program and evaluate the benefits of a screening questionnaire., Methods: Residents aged between 50 and 74 were eligible for the screening. A questionnaire and two consecutive fecal immunological tests (FITs) were used as primary screening methods. Subjects who were positive for any of the two tests were referred for further examination with colonoscopy. Neoplasms were removed either colonoscopically or by colectomy. Atypical adenoma and CRC were defined as advanced neoplasms., Results: A total of 6,971 residents in Dadong Street, Yuexiu District were screened with a questionnaire, and among them, 5,343 underwent at least one FIT. Four thousand and two hundred eleven (60.4%) were female, and 2,760 (39.6%) were male, with a median age of 62.0 years. Questionnaire and FITs identified 1,219 candidates for further examination with colonoscopy, among whom only 647 (53.1%) comply. As of this writing, 623 colonoscopy results were obtained, among which 270 (43.3%) had positive findings. The adenoma detection rate (ADR) was 43.3% (270/623). The ADR was 43.3% (270/623). Of the 270 patients, 10 (3.07%) had CRC, 81 (30.0%) had advanced adenoma, 178 had low-grade adenoma or other benign polyps, one had carcinoid. Except for three advanced CRC, all neoplasms detected were benign or in an early stage., Conclusions: Our screening program help identified patients with colonic neoplasms at an early stage, precluding them from developing into the malignant disease. The addition of the questionnaire significantly increased the sensitivity of primary screening, while also decreasing the specificity. Long-term results should evaluate the social and economic benefits of this program., Competing Interests: Conflicts of Interest: The authors have no conflicts of interest to declare., (2019 Annals of Translational Medicine. All rights reserved.)

Background: Follistatin-like protein 1 (FSTL1) has been demonstrated to play a controversial role in cancer. In this study, we aimed to investigate the expression of FSTL1 and its characteristics in patients with colorectal cancer (CRC)., Methods: Gene expression microarray assays in 30 CRC patients and a real-time quantitative polymerase chain reaction (RT-qPCR) of 22 patients were performed to compare the mRNA level of FSTL1 in tumor lesions and paired normal tissues. Also, 332 consecutive patients with pathologically confirmed CRC were selected to detect FSTL1 expression by using immunohistochemistry (IHC). Enzyme-linked immunosorbent assay (ELISA) was also applied to determine the serum level of FSTL1 in 60 CRC patients, as well as 34 healthy donors., Results: Gene expression microarray assays and RT-qPCR in CRC tissues, as well as ELISA in the serum all, revealed that the expression level of FSTL1 was higher in cancer tissue of CRC patients compared with paired normal tissue or healthy donors. The IHC results suggested that FSTL1 was also higher in tumor tissues than in its normal counterparts, however interestingly, a narrow scan focusing on the stromal region indicated that FSTL1 was significantly higher in normal tissues than in cancerous tissues. Besides, higher FSTL1 expression in cancer tissue, as well as lower FSTL1 expression in cancer stroma, both correlated with a worse prognosis, and the latter was an independent prognostic factor., Conclusions: Our results provide novel insight into the role of FSTL1 in CRC, and it might be an essential factor in CRC development., Competing Interests: Conflicts of Interest: The authors have no conflicts of interest to declare., (2019 Annals of Translational Medicine. All rights reserved.)

MicroRNAs (miRNAs) have been shown to play critical roles in the pathogenesis and progression of degenerative retinal diseases like age-related macular degeneration (AMD). In this study, we first demonstrated that miR-24 plays an important role in maintaining retinal structure and visual function of rats by targeting chitinase-3-like protein 1 (CHI3L1). In the retinal pigment epithelial (RPE) cells of Royal College of Surgeons (RCS) rats, an animal model of genetic retinal degeneration (RD), miR-24 was found lower and CHI3L1 level was higher in comparison with those in Sprague-Dawley (SD) rats. Other changes in the eyes of RCS rats include activated AKT/mTOR and ERK pathways and abnormal autophagy in the RPE cells. Such roles of miR-24 and CHI3L1 were further confirmed in RCS rats by subretinal injection of agomiR-24, which decreased CHI3L1 level and preserved retinal structure and function. Upstream, NF-κB was identified as the regulator of miR-24 in the RPE cells of these rats. On the other hand, in SD rats, intraocular treatment of antagomiR-24 induced pathological changes similar to those in RCS rats. The results revealed the protective roles for miR-24 to RPE cells and a mechanism for RD in RCS rats was proposed: extracellular stress stimuli first activate the NF-κB signaling pathway, which lowers miR-24 expression so that CHI3L1 increased. CHI3L1 sequentially results in aberrant autophagy and RPE dysfunction by activating AKT/mTOR and ERK pathways. Taken together, although the possibility, that the therapeutic effects in RCS rats are caused by other transcriptional changes regulated by miR-24, cannot be excluded, these findings indicate that miR-24 protects rat retina by targeting CHI3L1. Thus, miR-24 and CHI3L1 might be the targets for developing more effective therapy for degenerative retinal diseases like AMD., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objective: As a member of the N-myc downregulated gene family, N-Myc downstream-regulated gene 2 (NDRG2) contributes to tumorigenesis of various types of cancer. The expression status of NDRG2 in colorectal cancer (CRC) and its prognostic value remain to be elucidated. The goal of this study was to determine the expression pattern of NDRG2 in human CRC and its association of NDRG2 expression with prognosis. Methods: Immunohistochemistry was used to determine the level of NDRG2 expressions in 316 CRC tissues. The medical records of consecutive CRC patients undergoing primary tumor resection from September 2000 to February 2015 were retrospectively selected. Then, we compared to specific clinicopathological features in patients with different level of NDRG2 expressions. The correlation of NDRG2 expression with 3-year survival rate was assessed by Kaplan-Meier method and Cox regression modeling. Results: NDRG2 was expressed in 94.6% (299/316) of CRC tissues. The median IHC score of NDRG2 expression was significantly lower in tumor tissues compared with that of tumor-adjacent normal tissues [4.50(range 0.00-12.00) vs. 10.00 (range 0.00-12.00), P < 0.001].Survival analysis indicated that patients with low NDRG2 expression had poorer 3-year OS than those with high NDRG2 expression (59.9% vs. 76.6%, P = 0.017). Low NDRG2 expression also presented a significantly poorer 3-year OS rate in patient with stage IV disease (29.4% vs. 56.5%, P = 0.002), liver metastasis(32.2% vs. 54.7%, P = 0.005) and those receiving liver resection(56.5% vs. 71.9% , P = 0.012). Multivariate analysis indicated that high NDRG2 expression was independently associated with poor OS (hazard ratio [HR]: 1.499; 95% confidence interval [CI]: 1.037-2.165; P = 0.031). Conclusions: Low expression of NDRG2 was associated with unfavorable prognosis in CRC patients with primary tumor resection. Detection of NDRG2 expression might be useful for providing valuable information of individualized therapy for CRC patients., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Background: Millions of people are affected with retinal diseases that eventually cause blindness, and retinal progenitor cell (RPC) transplantation is a promising therapeutic avenue. However, RPC expansion and the underlying regulation mechanisms remain elusive., Methods: Adult mouse neural RPCs (mNRPCs) were isolated and amplified with the combination of basic fibroblast growth factor (bFGF) and glycogen synthase kinase 3 (GSK3) inhibitor CHIR99021. The progenitor characteristics were evaluated with RT-PCR, immunocytochemistry (ICC), western blot, flow cytometry, and transcriptome analysis prior to transplantation. By treating cells with or without bFGF and CHIR99021 at different time points, the mechanism for mNRPCs' self-renewal was investigated by transcriptome analysis and western blot assay., Results: mNRPCs were self-renewing in the presence of bFGF and CHIR99021 and showed prominent RPC characteristics. bFGF was essential in promoting cell cycle by facilitating G1/S and G2/M transitions. bFGF combined with CHIR99021 activated the non-canonical Wnt5A/Ca 2+ pathway and form a calcium homeostasis. In addition, the self-renewing mNRPCs could differentiate into rod photoreceptor-like cells and retinal pigment epithelium (RPE)-like cells by in vitro induction. When green fluorescent protein (GFP)-labeled cells were transplanted into the subretinal space (SRS) of Pde6b (rd1) mice (also known as RD1 mice, or rodless mice), the cells survived for more than 12 weeks and migrated into the retina. Parts of the recipient retina showed positive expression of photoreceptor marker rhodopsin. Transplanted cells can migrate into the retina, mainly into the inner cell layer (INL) and ganglion cell layer (GCL). Some cells can differentiate into astrocytes and amacrine cells. Cultured mNRPCs did not form tumors after transplanted into NOD/SCID mice for 6 months., Conclusions: Present study developed an approach to maintain long-term self-renewal of RPCs from adult retinal tissues and revealed that activation of the non-canonical Wnt5A/Ca 2+ pathway may participate in regulating RPC self-renewal in vitro. This study presents a very promising platform to expand RPCs for future therapeutic application.

The pathological change of retinal pigment epithelial (RPE) cells is one of the main reasons for the development of age-related macular degeneration (AMD). Thus, cultured RPE cells are a proper cell model for studying the etiology of AMD in vitro. However, such cultured RPE cells easily undergo epithelial-mesenchymal transition (EMT) that results in changes of cellular morphology and functions of the cells. To restore and maintain the mesenchymal-epithelial transition (MET) of the cultured RPE cells, we cultivated dedifferentiated porcine RPE (pRPE) cells and compared their behaviors in four conditions: 1) in cell culture dishes with DMEM/F12 containing FBS (CC dish-FBS), 2) in petri dishes with DMEM/F12 containing FBS (Petri dish-FBS), 3) in cell culture dishes with DMEM/F12 containing N2 and B27 supplements (CC dish-N2B27), and 4) in petri dishes with DMEM/F12 containing N2 and B27 (Petri dish-N2B27). In addition to observing the cell morphology and behavior, RPE specific markers, as well as EMT-related genes and proteins, were examined by immunostaining, quantitative real-time PCR and Western blotting. The results showed that dedifferentiated pRPE cells maintained EMT in CC dish-FBS, Petri dish-FBS and CC dish-N2B27 groups, whereas MET was induced when the dedifferentiated pRPE cells were cultured in Petri dish-N2B27. Such induced pRPE cells showed polygonal morphology with increased expression of RPE-specific markers and decreased EMT-associated markers. Similar results were observed in induced pluripotent stem cell-derived RPE cells. Furthermore, during the re-differentiation of those dedifferentiated pRPE cells, Petri dish-N2B27 reduced the activity of RhoA and induced F-actin rearrangement, which promoted the nuclear exclusion of transcriptional co-activator with PDZ-binding motif (TAZ) and TAZ target molecule zinc finger E-box binding protein (ZEB1), both of which are EMT inducing factors. This study provides a simple and reliable method to reverse dedifferentiated phenotype of pRPE cells into epithelialized phenotype, which is more appropriate for studying AMD in vitro, and suggests that MET of other cell types might be induced by a similar approach., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Since early diagnosis is very important for treating CRC, we decided to detect peripheral serum canopy fibroblast growth factor signaling regulator 2 (CNPY2) isoform 2 to verify its diagnostic value for CRC patients. Serum samples were collected from 430 CRC patients and 201 healthy controls. Enzyme-linked immunosorbent assay (ELISA) detection kits for CNPY2 isoform 2 were generated and then applied to measure serum CNPY2 isoform 2 concentrations. Serum carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) were also measured. The median serum CNPY2 isoform 2 concentrations in all CRC patients were significantly higher than those in the healthy control group (all P<0.001). Those with stage I CRC presented the highest area under the receiver operating characteristic curve (AUC) for CNPY2 isoform 2 [0.707, 95% confidence interval (CI): 0.649-0.765, P<0.001]. The diagnostic efficiency of the combination of CNPY2 isoform 2, CEA and CA19-9 was significantly higher than that of each biomarker detected separately (all P<0.0167). Serum CNPY2 isoform 2 may be a valuable biomarker for the early detection of CRC and presents an improvement in the diagnostic efficiency by combination of CEA and CA19-9.

Background: The poor clinical prognosis of Stage IIIB colon cancer patients is due in part to the current lack of an effective diagnostic method being available and highlights a need for the identification of novel biomarkers like microRNA (miRNA)., Patients and Methods: We used microarray analysis to compare the miRNA expression profiles of eight Stage IIIB colon cancer patients with worse clinical outcome (those who developed liver metastases between 8 and 18 months after surgery) against eight 'cured' Stage IIIB colon cancer patients (those who remained disease free following surgery during the same monitoring period). In addition, quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed examining miRNAs in tumor tissue of 98 patients with Stage IIIB colon cancer., Results: We found, miRNA-192-3p and miRNA-192-5p were down regulated in the patients with worsening disease compared to the control 'cured' Stage IIIB colon cancer patients (P = 0.026 and P = 0.042, respectively). Patients with higher expression of miRNA-192-5p had higher 5-year disease-free survival (DFS) (84.21%) and overall survival (OS) (89.47%) than those with lower targeted miRNA expression DFS (38.8%; hazard ratio (HR): 3.74, 95% confidence interval (CI): 1.52-9.23, P = 0.042) and OS (48.57%; HR: 5.01, 95% CI: 1.75-14.38, P = 0.033). In contrast, patients with higher expression of miRNA-192-3p did not appear to statistically impact the survival of patients in this setting (DFS 73.33% vs 64.7%, HR: 0.68, 95% CI: 0.31-1.52, P = 0.35; OS 76.67% vs 66.17%, HR: 0.62, 95% CI: 0.27-1.45, P = 0.27)., Conclusions: The decreased expression of miRNA-192-5p found for patients with relapsing disease might represent a highly predictive marker to use for the prognosis of Stage IIIB colon cancer patients.

Background: Animal models play critical roles in studies of the etiology and therapy of retinal degeneration (RD)., Objective: To establish an RD model without severe systemic side effects in monkeys., Methods: Cynomolgus monkeys and Sprague-Dawley rats were treated with intravenous and intravitreal sodium iodate (SI). Electroretinographic (ERG) recording, fluorescein fundus angiography (FFA), optical coherence tomography (OCT) and a retinal morphology examination were conducted to evaluate retinal function and structure. ARPE-19 cells were treated with SI to assess cell viability and morphology. Glutathione (GSH) was administered to SI-treated cultured cells and rats for mechanistic studies., Results: Intravenous SI failed to induce RD in monkeys due to its lethal toxicity and the spontaneous recovery of visual function. However, intravitreal SI injection induced very rapid and severe retinal damage in both monkeys and rats. Different doses of SI were tested in both rats and monkeys, and the SI dose appropriate for the model was calculated. GSH partially rescued oxidative damage to SI-treated retinas. A combination of the appropriate dose of intravitreal SI and intravenous GSH generated moderate subacute RD., Conclusions: An RD model was established in cynomolgus monkeys by intravitreal SI injection. The key advantages of this model are that lethal SI side effects can be avoided and that the structural and functional changes are similar to those in patients with RD, although the development of RD in the model is too rapid and more severe. An appropriate dose of SI plus systemic GSH generates delayed and moderate RD; this prolonged therapeutic window allows the development of new therapies, such as gene or stem cell-based therapy, for RD., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Colorectal cancer (CRC) is a leading cause of cancer-related mortality. Recently, we identified a novel biomarker, canopy fibroblast growth factor signaling regulator 2 (CNPY2) isoform2, and subsequently investigated its expression and prognostic value in CRC patients. We initially generated CNPY2 isoform2 monoclonal antibodies and examined CNPY2 isoform2 expression in CRC cell lines and tissues using quantitative real-time polymerase chain reaction, western blot and immunohistochemistry analyses. We found that CNPY2 isoform2 expression significantly increased in tumor cell lines and tissues compared with that in normal colon epithelial cells and tumor-adjacent normal tissues. Survival analysis indicated that patients with low CNPY2 isoform2 expression had poorer 5-year overall survival (OS) in both the training cohort (41.7% vs. 77.7%, P = 0.007) and validation cohort (47.1% vs. 78.8%, P = 0.002). In multivariable analysis, CNPY2 isoform2 was identified as a predictor of 5-year OS in both the training cohort [hazard ratio (HR) = 5.001; 95% confidence interval (CI) 2.156-11.598, P < 0.001) and validation cohort (HR= 2.443; 95% CI 1.197- 4.983, P = 0.014). In conclusion, CNPY2 isoform2 represents as a novel and valuable prognostic indicator for CRC patients, while the oncologic function of CNPY2 requires further study.