The extracellular domain of epithelial cell adhesion molecule (EpCAM) enhances multipotency of mesenchymal stem cells through EGFR-LIN28-LET7 signaling.

Mesenchymal stem cells (MSCs) are widely considered to be an attractive cell source for regenerative therapies, but maintaining multipotency and self-renewal in cultured MSCs is especially challenging. Hence, the development and mechanistic description of strategies that help promote multipotency in MSCs will be vital to future clinical use. Here, using an array of techniques and approaches, including cell biology, RT-quantitative PCR, immunoblotting, immunofluorescence, flow cytometry, and ChIP assays, we show that the extracellular domain of epithelial cell adhesion molecule (EpCAM) (EpEX) significantly increases the levels of pluripotency factors through a signaling cascade that includes epidermal growth factor receptor (EGFR), signal transducer and activator of transcription 3 (STAT3), and Lin-28 homolog A (LIN28) and enhances the proliferation of human bone marrow MSCs. Moreover, we found that EpEX-induced LIN28 expression reduces the expression of the microRNA LET7 and up-regulates that of the transcription factor high-mobility group AT-hook 2 (HMGA2), which activates the transcription of pluripotency factors. Surprisingly, we found that EpEX treatment also enhances osteogenesis of MSCs under differentiation conditions, as evidenced by increases in osteogenic markers, including Runt-related transcription factor 2 (RUNX2). Taken together, our results indicate that EpEX stimulates EGFR signaling and thereby context-dependently controls MSC states and activities, promoting cell proliferation and multipotency under maintenance conditions and osteogenesis under differentiation conditions.

Down-regulation of ATF1 leads to early neuroectoderm differentiation of human embryonic stem cells by increasing the expression level of SOX2.

We reveal by high-throughput screening that activating transcription factor 1 (ATF1) is a novel pluripotent regulator in human embryonic stem cells (hESCs). The knockdown of ATF1 expression significantly up-regulated neuroectoderm (NE) genes but not mesoderm, endoderm, and trophectoderm genes. Of note, down-regulation or knockout of ATF1 with short hairpin RNA (shRNA), small interfering RNA (siRNA), or clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) was sufficient to up-regulate sex-determining region Y-box (SOX)2 and paired box 6 (PAX6) expression under the undifferentiated or differentiated conditions, whereas overexpression of ATF1 suppressed NE differentiation. Endogenous ATF1 was spontaneously down-regulated after d 1-3 of neural induction. By double-knockdown experiments, up-regulation of SOX2 was critical for the increase of PAX6 and SOX1 expression in shRNA targeting Atf1 hESCs. Using the luciferase reporter assay, we identified ATF1 as a negative transcriptional regulator of Sox2 gene expression. A novel function of ATF1 was discovered, and these findings contribute to a broader understanding of the very first steps in regulating NE differentiation in hESCs.-Yang, S.-C., Liu, J.-J., Wang, C.-K., Lin, Y.-T., Tsai, S.-Y., Chen, W.-J., Huang, W.-K., Tu, P.-W. A., Lin, Y.-C., Chang, C.-F., Cheng, C.-L., Lin, H., Lai, C.-Y., Lin, C.-Y., Lee, Y.-H., Chiu, Y.-C., Hsu, C.-C., Hsu, S.-C., Hsiao, M., Schuyler, S. C., Lu, F. L., Lu, J. Down-regulation of ATF1 leads to early neuroectoderm differentiation of human embryonic stem cells by increasing the expression level of SOX2.

Understanding Breast Implant Illness, Before and After Explantation: A Patient-Reported Outcomes Study.

BACKGROUND: Breast implant illness (BII) after aesthetic breast augmentation remains a poorly defined syndrome encompassing a wide spectrum of symptoms. While previously published series have observed overall symptomatic improvement after breast implant removal, there is a lack of studies evaluating changes in specific symptoms over time. The purpose of this study was to gain an understanding of symptoms associated with BII, and to evaluate how these symptoms change after removal of breast implants and total capsulectomy (explantation). We hypothesized that patients presenting with BII would experience both immediate and sustained improvement in constitutional symptoms after explantation. METHODS: A retrospective study of all patients who underwent explantation by a single surgeon over 2 years was conducted. Repeated-measures analysis of variance accounting for dependency was used to compare symptoms before and after surgery. Multivariate analyses and linear regression models were used to examine the impact of patient- and implant-related factors on changes in symptoms. RESULTS: Seven hundred fifty patients met inclusion criteria. Mean preoperative survey score (26.19 ± 11.24) was significantly different from mean postoperative survey score at less than 30 days (9.49 ± 7.56) and greater than 30 days (9.46 ± 7.82, P < 0.001). Patients with a BMI greater than 30 or those with clinically detectable contracture on examination showed greater improvement on their survey scores (P = 0.039, 0.034, respectively). CONCLUSIONS: Although BII encompasses a large range of symptoms, subjects in this study demonstrated significant and sustained improvement in 11 common symptom domains. This improvement was demonstrable within the first 30 days postoperatively and was maintained beyond 30 days. The study demonstrated a strong association of explantation and specific symptom improvement within the patient population studied. Future investigation will further elucidate possible biologic phenomena to better characterize the pathophysiology and mechanism of BII.

Aesthetic assessment after root coverage of multiple adjacent recessions with coronally advanced flap with adjunctive collagen matrix or connective tissue graft: Randomized clinical trial.

BACKGROUND: Few studies have looked at professional assessment or patient perception of aesthetics after root coverage procedures. The addition of connective tissue grafts (CTG) seems to improve aesthetic outcomes. The objective of this a priori analysis was to compare aesthetics after addition of CTG or a collagen matrix (CMX) to coronally advanced flap (CAF). METHODS: Two independent, trained and calibrated assessors analysed baseline and 6-month post-operative Images from 183 subjects with 475 recessions from a previously reported multicentre multinational randomized clinical trial. The root coverage aesthetic score (RES) was assessed in its five constituent components after assessing the suitability of images blindly with regard to treatment assignment and centre. Data were analysed at the tooth and subject level. RESULTS: One hundred and fifty-five subjects (81 CTG) and 393 teeth (207 CTG) were included in the analysis. CTG control subjects had higher total RES scores (mean adjusted difference of 1.3 ± 0.8 RES units, p = 0.002). Analyses of RES subcomponents showed that the CTG group had higher scores in terms of gingival margin position but that better marginal tissue contour (OR 3.0, 95% CI 1.2-7.7) and soft tissue texture (OR 3.3, 95% CI 1.9-5.8) was observed for the CMX group. No significant differences were observed for mucogingival alignment and gingival colour. CONCLUSION: Better overall RES scores were observed for the CTG group. Better marginal tissue texture and marginal contour were observed in the CMX group. More research and development is needed to optimize materials to be used in conjunction with CAF to improve root coverage without negatively affecting tissue texture and marginal contour.

PARP1 controls KLF4-mediated telomerase expression in stem cells and cancer cells.

Telomerase is highly expressed in cancer and embryonic stem cells (ESCs) and implicated in controlling genome integrity, cancer formation and stemness. Previous studies identified that Krüppel-like transcription factor 4 (KLF4) activates telomerase reverse transcriptase (TERT) expression and contributes to the maintenance of self-renewal in ESCs. However, little is known about how KLF4 regulates TERT expression. Here, we discover poly(ADP-ribose) polymerase 1 (PARP1) as a novel KLF4-interacting partner. Knockdown of PARP1 reduces TERT expression and telomerase activity not only in cancer cells, but also in human and mouse ESCs. Recruitment of KLF4 to TERT promoter is reduced in PARP1-suppressed cells. The poly(ADP-ribose) polymerase activity is dispensable, while the oligo(ADP-ribose) polymerase activity is required for the PARP1- and KLF4-mediated TERT activation. Repression of Parp1 in mouse ESCs decreases expression of pluripotent markers and induces differentiation. These results suggest that PARP1 recruits KLF4 to activate telomerase expression and stem cell pluripotency, indicating a positive regulatory role of the PARP1-KLF4 complex in telomerase expression in cancer and stem cells.

Critical involvement of atypical chemokine receptor CXCR7 in allergic airway inflammation.

Trafficking and recruitment of immune cells to the site of inflammation with spatial and temporal synchronization is crucial for the development of allergic airway inflammation. Particularly, chemokines are known to be key players in these processes. Previous studies revealed that the CXCL12/CXCR4 axis plays an important role in regulating allergic airway inflammation. However, the role of CXCR7, a recently discovered second receptor for CXCL12, in regulating airway inflammation has not been explored. Initially, CXCR7 was considered as a decoy receptor; however, numerous subsequent studies revealed that engagement of CXCR7 triggered its own signalling or modulated CXCR4-mediated signalling. In the present study, we detected the expression of CXCR7 in airway epithelial cells. Use of a lentiviral delivery system to knock down the expression of CXCR7 in the lung of sensitized mice abrogated the cardinal features of asthma, indicating that CXCR7 plays a role in regulating allergic airway inflammation. The activation of mitogen-activated protein kinase and Akt signalling in response to CXCL12 in the mouse epithelial cell line MLE-12 was reduced when CXCR7 expression was knocked down. However, either knockdown or overexpression of CXCR7 in MLE-12 did not affect CXCL12-mediated calcium influx, indicating that CXCR7 does not modulate CXCR4-mediated signalling, and that it functions as a signalling receptor rather than a decoy receptor. Finally, we found that the expression of chemokine CCL2 is regulated by CXCR7/CXCL12-mediated signalling through ß-arrestin in airway epithelial cells. Hence, regulating the expression of CCL2 in airway epithelial cells may be one mechanism by which CXCR7 participates in regulating allergic airway inflammation.

Regulation of EBV LMP1-triggered EphA4 downregulation in EBV-associated B lymphoma and its impact on patients' survival.

Epstein-Barr virus (EBV), an oncogenic human virus, is associated with several lymphoproliferative disorders, including Burkitt lymphoma, Hodgkin disease, diffuse large B-cell lymphoma (DLBCL), and posttransplant lymphoproliferative disorder (PTLD). In vitro, EBV transforms primary B cells into lymphoblastoid cell lines (LCLs). Recently, several studies have shown that receptor tyrosine kinases (RTKs) play important roles in EBV-associated neoplasia. However, details of the involvement of RTKs in EBV-regulated B-cell neoplasia and malignancies remain largely unclear. Here, we found that erythropoietin-producing hepatocellular receptor A4 (EphA4), which belongs to the largest RTK Eph family, was downregulated in primary B cells post-EBV infection at the transcriptional and translational levels. Overexpression and knockdown experiments confirmed that EBV-encoded latent membrane protein 1 (LMP1) was responsible for this EphA4 suppression. Mechanistically, LMP1 triggered the extracellular signal-regulated kinase (ERK) pathway and promoted Sp1 to suppress EphA4 promoter activity. Functionally, overexpression of EphA4 prevented LCLs from proliferation. Pathologically, the expression of EphA4 was detected in EBV(-) tonsils but not in EBV(+) PTLD. In addition, an inverse correlation of EphA4 expression and EBV presence was verified by immunochemical staining of EBV(+) and EBV(-) DLBCL, suggesting EBV infection was associated with reduced EphA4 expression. Analysis of a public data set showed that lower EphA4 expression was correlated with a poor survival rate of DLBCL patients. Our findings provide a novel mechanism by which EphA4 can be regulated by an oncogenic LMP1 protein and explore its possible function in B cells. The results provide new insights into the role of EphA4 in EBV(+) PTLD and DLBCL.

RBMY, a novel inhibitor of glycogen synthase kinase 3ß, increases tumor stemness and predicts poor prognosis of hepatocellular carcinoma.

UNLABELLED: Male predominance of hepatocellular carcinoma (HCC) occurs particularly among young children aged 6-9 years, indicative of a possible role of the Y chromosome-encoded oncogene in addition to an androgenic effect. The discovery of oncogenic activation of RBMY (RNA-binding motif on Y chromosome), which is absent in normal hepatocytes but present in male HCC tissues, sheds light on this issue. Herein, we report on a critical hepatocarcinogenic role of RBMY and its ontogenic origin. During liver development, the Ser/Thr phosphorylated RBMY is expressed in the cytoplasm of human and rodent fetal livers. It is then silenced in mature hepatocytes and restricted to scarce expression in the bile ductular cells. Upon hepatocarcinogenesis, a noteworthy increase of cytoplasmic and nuclear RBMY is observed in HCC tissues; however, only the former is expressed dominantly in hepatic cancer stem cells and correlates significantly to a poor prognosis and decreased survival rate in HCC patients. Cytoplasmic expression of RBMY, which is mediated by binding to nuclear exporter chromosome region maintenance 1 and further enriched upon Wnt-3a stimulation, confers upon tumor cells the traits of cancer stem cell by augmenting self-renewal, chemoresistance, cell-cycle progression, proliferation, and xenograft tumor growth. This is achieved mechanistically through increasing Ser9 phosphorylation-inactivation of glycogen synthase kinase 3ß by RBMY, thereby impeding the glycogen synthase kinase 3ß-dependent degradation of ß-catenin and eventually inducing the nuclear entry of ß-catenin for the transcription of downstream oncogenes. CONCLUSION: RBMY is a novel oncofetal protein that plays a key role in attenuating glycogen synthase kinase 3ß activity, leading to aberrant activation of Wnt/ß-catenin signaling, which facilitates malignant hepatic stemness; because of its absence from normal human tissues except the testis, RBMY represents a feasible therapeutic target for the selective eradication of HCC cells in male patients.

Mesenchymal stem cells tune the development of monocyte-derived dendritic cells toward a myeloid-derived suppressive phenotype through growth-regulated oncogene chemokines.

Mesenchymal stem/stromal cells (MSCs) are promising potential candidates for the treatment of immunological diseases because of their immunosuppressive functions. However, the molecular mechanisms that mediate MSCs' immunosuppressive activity remain elusive. In this article, we report for the first time, to our knowledge, that secreted growth-regulated oncogene (GRO) chemokines, specifically GRO-γ, in human MSC-conditioned media have an effect on the differentiation and the function of human monocyte-derived dendritic cells. The monocyte-derived dendritic cells were driven toward a myeloid-derived suppressor cell (MDSC)-like phenotype by the GRO chemokines. GRO-γ-treated MDSCs had a tolerogenic phenotype that was characterized by an increase in the secretion of IL-10 and IL-4, and a reduction in the production of IL-12 and IFN-γ. We have also shown that the mRNA expression levels of the arginase-1 and inducible NO synthase genes, which characterize MDSCs, were upregulated by GRO-γ-primed mouse bone marrow cells. In addition, the ability of GRO-γ-treated bone marrow-derived dendritic cells to stimulate the OVA-specific CD8(+) T (OT-1) cell proliferation and the cytokine production of IFN-γ and TNF-α were significantly decreased in vivo. Our findings allow a greater understanding of how MDSCs can be generated and offer new perspectives to exploit the potential of MDSCs for alternative approaches to treat chronic inflammation and autoimmunity, as well as for the prevention of transplant rejection.

Squalene synthase induces tumor necrosis factor receptor 1 enrichment in lipid rafts to promote lung cancer metastasis.

RATIONALE: Metabolic alterations contribute to cancer development and progression. However, the molecular mechanisms relating metabolism to cancer metastasis remain largely unknown. OBJECTIVES: To identify a key metabolic enzyme that is aberrantly overexpressed in invasive lung cancer cells and to investigate its functional role and prognostic value in lung cancer. METHODS: The differential expression of metabolic enzymes in noninvasive CL1-0 cells and invasive CL1-5 cells was analyzed by a gene expression microarray. The expression of target genes in clinical specimens from patients with lung cancer was examined by immunohistochemistry. Pharmacologic and gene knockdown/overexpression approaches were used to investigate the function of the target gene during invasion and metastasis in vitro and in vivo. The association between the target gene expression and clinicopathologic parameters was further analyzed. Bioinformatic analyses were used to discover the signaling pathways involved in target gene-regulated invasion and migration. MEASUREMENTS AND MAIN RESULTS: Squalene synthase (SQS) was up-regulated in CL1-5 cells and in the tumor regions of the lung cancer specimens. Loss of function or knockdown of SQS significantly inhibited invasion/migration and metastasis in cell and animal models and vice versa. High expression of SQS was significantly associated with poor prognosis among patients with lung cancer. Mechanistically, SQS contributed to a lipid-raft-localized enrichment of tumor necrosis factor receptor 1 in a cholesterol-dependent manner, which resulted in the enhancement of nuclear factor-κB activation leading to matrix metallopeptidase 1 up-regulation. CONCLUSIONS: Up-regulation of SQS promotes metastasis of lung cancer by enhancing tumor necrosis factor-α receptor 1 and nuclear factor-κB activation and matrix metallopeptidase 1 expression. Targeting SQS may have considerable potential as a novel therapeutic strategy to treat metastatic lung cancer.

The pathological effects of CCR2+ inflammatory monocytes are amplified by an IFNAR1-triggered chemokine feedback loop in highly pathogenic influenza infection.

BACKGROUND: Highly pathogenic influenza viruses cause high levels of morbidity, including excessive infiltration of leukocytes into the lungs, high viral loads and a cytokine storm. However, the details of how these pathological features unfold in severe influenza infections remain unclear. Accumulation of Gr1 + CD11b + myeloid cells has been observed in highly pathogenic influenza infections but it is not clear how and why they accumulate in the severely inflamed lung. In this study, we selected this cell population as a target to investigate the extreme inflammatory response during severe influenza infection. RESULTS: We established H1N1 IAV-infected mouse models using three viruses of varying pathogenicity and noted the accumulation of a defined Gr1 + CD11b + myeloid population correlating with the pathogenicity. Herein, we reported that CCR2+ inflammatory monocytes are the major cell compartments in this population. Of note, impaired clearance of the high pathogenicity virus prolonged IFN expression, leading to CCR2+ inflammatory monocytes amplifying their own recruitment via an interferon-α/ß receptor 1 (IFNAR1)-triggered chemokine loop. Blockage of IFNAR1-triggered signaling or inhibition of viral replication by Oseltamivir significantly suppresses the expression of CCR2 ligands and reduced the influx of CCR2+ inflammatory monocytes. Furthermore, trafficking of CCR2+ inflammatory monocytes from the bone marrow to the lung was evidenced by a CCR2-dependent chemotaxis. Importantly, leukocyte infiltration, cytokine storm and expression of iNOS were significantly reduced in CCR2-/- mice lacking infiltrating CCR2+ inflammatory monocytes, enhancing the survival of the infected mice. CONCLUSIONS: Our results indicated that uncontrolled viral replication leads to excessive production of inflammatory innate immune responses by accumulating CCR2+ inflammatory monocytes, which contribute to the fatal outcomes of high pathogenicity virus infections.

Hybrid Closed Loop in Adults With Type 1 Diabetes and Severely Impaired Hypoglycemia Awareness.

BACKGROUND: Benefits of hybrid closed-loop (HCL) systems in a high-risk group with type 1 diabetes and impaired awareness of hypoglycemia (IAH) have not been well-explored. METHODS: Adults with Edmonton HYPO scores ≥1047 were randomized to 26-weeks HCL (MiniMed™ 670G) vs standard therapy (multiple daily injections or insulin pump) without continuous glucose monitoring (CGM) (control). Primary outcome was percentage CGM time-in-range (TIR; 70-180 mg/dL) at 23 to 26 weeks post-randomization. Major secondary endpoints included magnitude of change in counter-regulatory hormones and autonomic symptom responses to hypoglycemia at 26-weeks post-randomization. A post hoc analysis evaluated glycemia risk index (GRI) comparing HCL with control groups at 26 weeks post-randomization. RESULTS: Nine participants (median [interquartile range (IQR)] age 51 [41, 59] years; 44% male; enrolment HYPO score 1183 [1058, 1308]; Clarke score 6 [6, 6]; n = 5 [HCL]; n = 4 [control]) completed the study. Time-in-range was higher using HCL vs control (70% [68, 74%] vs 48% [44, 50%], P = .014). Time <70 mg/dL did not differ (HCL 3.8% [2.7, 3.9] vs control 6.5% [4.3, 8.6], P = .14) although hypoglycemia episode duration was shorter (30 vs 50 minutes, P < .001) with HCL. Glycemia risk index was lower with HCL vs control (38.1 [30.0, 39.2] vs 70.8 [58.5, 72.4], P = .014). Following 6 months of HCL use, greater dopamine (24.0 [12.3, 27.6] vs -18.5 [-36.5, -4.8], P = .014), and growth hormone (6.3 [4.6, 16.8] vs 0.5 [-0.8, 3.0], P = .050) responses to hypoglycemia were observed. CONCLUSIONS: Six months of HCL use in high-risk adults with severe IAH increased glucose TIR and improved GRI without increased hypoglycemia, and partially restored counter-regulatory responses. CLINICAL TRIAL REGISTRATION: ACTRN12617000520336.

Advances in Automated Insulin Delivery with the Medtronic 780G: The Australian Experience.

Aim: To assess the real-world performance of MiniMed™ 780G for Australians with type 1 diabetes (T1D) following advanced hybrid closed loop (AHCL) activation and to evaluate the effect of changing from MiniMed 670/770G to 780G. Methods: We analyzed deidentified Carelink™ continuous glucose monitoring (CGM) data from Australian users from January 2020 to December 2022, including the proportion attaining three major consensus targets: Glucose management indicator (GMI <7.0%), time in range (TIR 70-180 mg/dL >70%), and time below range (TBR 70 mg/dL <4%). Results: Comparing 670/770G users (n = 5676) for mean ± standard deviation 364 ± 244 days with 780G users (n = 3566) for 146 ± 145 days, the latter achieved a higher TIR (72.6% ± 10.6% vs. 67.3% ± 11.4%; P < 0.001), lower time above range (TAR) (25.5% ± 10.9% vs. 30.6% ± 11.7%; P < 0.001), and lower GMI (6.9% ± 0.4% vs. 7.2% ± 0.4%; P < 0.001) without compromising TBR (1.9% ± 1.8% vs. 2.0% ± 1.8%; P = 0.0015). Of 1051 670/770G users transitioning to 780G, TIR increased (70.0% ± 10.7% to 74.0% ± 10.2%; P < 0.001), TAR decreased (28.1% ± 10.9% to 24.0% ± 10.7%; P < 0.001), and TBR was unchanged. The percentage of users attaining all three CGM targets was higher in 780G users (50.1% vs. 29.5%; P < 0.001). CGM metrics were stable at 12 months post-transition. Conclusion: Real-world data from Australia shows that a higher proportion of MiniMed 780G users meet clinical targets for CGM consensus metrics compared to MiniMed 670/770G users and glucose control was sustained over 12 months.

Combining an Electrochemical Continuous Glucose Sensor With an Insulin Delivery Cannula: A Feasibility Study.

BACKGROUND: Combining a continuous glucose monitor with an insulin delivery cannula (CGM-IS) could benefit clinical outcomes. We evaluated the feasibility of a single-needle insertion electrochemical investigational CGM-IS (Pacific Diabetes Technologies, Portland, Oregon) in type 1 diabetes adults. METHODS: Following 48 hours run-in using a Medtronic 780G in manual mode with a commercial insulin set, 12 participants commenced insulin delivery using the CGM-IS. A standardized test meal was eaten on the mornings of days 1 and 4. Venous samples were collected every 10 minutes one hour prior to and 15 minutes post-meal for four hours. CGM-IS glucose measurements were post-processed with a single capillary blood calibration during warm-up and benchmarked against YSI. A Dexcom G6 sensor was worn post-consent to study end. RESULTS: Mean absolute relative difference (MARD) for the CGM-IS glucose measurements was 9.2% (484 paired data points). Consensus error grid revealed 88.6% within zone A and 100% in A + B. Mean (SD) % bias was -3.5 (11.7) %. There were 35 paired YSI readings <100 mg/dL cutoff and 449 ≥100 mg/dL with 81.4% within ±15 mg/dL or ±15%, and 89.9% within ±20 mg/dL or ±20%. Two cannula occlusions required discontinuation of insulin delivery: one at 70 hours post insertion and another during the day 4 meal test. Mean (SD) Dexcom glucose measurements during run-in and between meal tests was respectively 161.3 ± 27.3 mg/dL versus 158.0 ± 25.6 mg/dL; P = .39 and corresponding mean total daily insulin delivered by the pump was 58.0 ± 25.4 Units versus 57.1 ± 28.8 Units; P = .47. CONCLUSIONS: Insulin delivery and glucose sensing with the investigational CGM-IS was feasible. Longer duration studies are needed.

Involvement of recepteur d'origine nantais receptor tyrosine kinase in Epstein-Barr virus-associated nasopharyngeal carcinoma and its metastasis.

Nasopharyngeal carcinoma (NPC) is characteristic for its strong association with Epstein-Barr virus (EBV) and high metastatic rate. Recently, overexpressed recepteur d'origine nantais (RON) (MST1R), receptor tyrosine kinase has been reported in human cancers and tumor metastasis. Therefore, the role of RON in EBV-associated NPC and its metastasis was investigated. Here we show that RON was found in NPC but not in control tissues. A significant correlation of latent membrane protein 1 (LMP1) and RON expression was found in NPC (Pearson's χ(2) test; P = 0.0023). At the molecular level, LMP1 stimulates nuclear factor-κB binding to the RON promoter through its carboxyl-terminal activation region 1 to induce expression of RON. Knockdown of RON in cells expressing LMP1 significantly reverses LMP1-induced epithelial-mesenchymal transition and suppresses LMP1-induced cell migration and invasion. These results suggest an important role of RON in the tumorigenesis and metastasis of NPC and RON may be a novel therapeutic target for EBV-associated NPC.

Requirement for LMP1-induced RON receptor tyrosine kinase in Epstein-Barr virus-mediated B-cell proliferation.

EBV, an oncogenic human herpesvirus, can transform primary B lymphocytes into immortalized lymphoblastoid cell lines (LCLs) through multiple regulatory mechanisms. However, the involvement of protein tyrosine kinases in the infinite proliferation of B cells is not clear. In this study, we performed kinase display assays to investigate this subject and identified a specific cellular target, Recepteur d'Origine Nantais (RON) tyrosine kinase, expressed in LCLs but not in primary B cells. Furthermore, we found that latent membrane protein 1 (LMP1), an important EBV oncogenic protein, enhanced RON expression through its C-terminal activation region-1 (CTAR1) by promoting NF-κB binding to the RON promoter. RON knockdown decreased the proliferation of LCLs, and transfection with RON compensated for the growth inhibition caused by knockdown of LMP1. Immunohistochemical analysis revealed a correlation between LMP1 and RON expression in biopsies from posttransplantation lymphoproliferative disorder (PTLD), suggesting that LMP1-induced RON expression not only is essential for the growth of LCLs but also may contribute to the pathogenesis of EBV-associated PTLD. Our study is the first to reveal the impact of RON on the proliferation of transformed B cells and to suggest that RON may be a novel therapeutic target for EBV-associated lymphoproliferative diseases.

Sonic hedgehog antagonists induce cell death in acute myeloid leukemia cells with the presence of lipopolysaccharides, tumor necrosis factor-α, or interferons.

Due to the development of drug resistance, the outcome for the majority of patients with acute myeloid leukemia (acute myelogenous leukemia; AML) remains poor. To prevent drug resistance and increase the therapeutic efficacy of treating AML, the development of new combinatory drug therapies is necessary. Sonic hedgehog (Shh) is expressed in AML biopsies and is essential for the drug resistance of cancer stem cells of AML. AML patients are frequently infected by bacteria and exposed to lipopolysaccharide (LPS). LPS itself, its derivatives, and its downstream effectors, such as tumor necrosis factor-α (TNF-α) and interferons (IFNs), have been shown to provoke anti-tumor effects. The application of a Shh inhibitor against AML cells in the presence of LPS/TNF-α/IFNs has not been investigated. We found that the Shh inhibitor cyclopamine in combination with LPS treatment synergistically induced massive cell apoptosis in THP-1 and U937 cells. The cytotoxic effects of this combined drug treatment were confirmed in 5 additional AML cell lines, in primary AML cells, and in an AML mouse model. Replacing cyclopamine with another Shh inhibitor, Sant-1, had the same effect. LPS could be substituted by TNF-α or IFNs to induce AML cell death in combination with cyclopamine. Our results suggest a potential strategy for the development of new therapies employing Shh antagonists in the presence of LPS/TNF-α/IFNs for the treatment of AML patients.

A shRNA functional screen reveals Nme6 and Nme7 are crucial for embryonic stem cell renewal.

In contrast to the somatic cells, embryonic stem cells (ESCs) are characterized by its immortalization ability, pluripotency, and oncogenicity. Revealing the underlying mechanism of ESC characteristics is important for the application of ESCs in clinical medicine. We performed systematic functional screen in mouse ESCs with 4,801 shRNAs that target 929 kinases and phosphatases. One hundred and thirty-two candidate genes that regulate both ESC expansion and stem cell marker expression were identified. Twenty-seven out of the 132 genes were regarded as most important since knockdown of each gene induces morphological changes from undifferentiated to differentiated state. Among the 27 genes, we chose nonmetastatic cell 6 (Nme6, also named as Nm23-H6) and nonmetastatic cell 7 (Nme7, also designated as Nm23-H7) to study first. Nme6 and Nme7 both belong to the members of nucleoside diphosphate kinase family. We demonstrate that Nme6 and Nme7 are important for the regulation of Oct4, Nanog, Klf4, c-Myc, telomerase, Dnmt3B, Sox2, and ERas expression. Either knockdown of Nme6 or Nme7 reduces the formation of embryoid body (EB) and teratoma. The overexpression of either Nme6 or Nme7 can rescue the stem cell marker expression and the EB formation in the absence of leukemia inhibiting factor. This implies the importance of Nme6 and Nme7 in ESC renewal. This finding not only pinpoints Nme6 or Nme7 can regulate several critical regulators in ESC renewal but also increases our understanding of the ESC renewal and oncogenesis.

Challenges of Glycemic Control in People With Diabetes and Advanced Kidney Disease and the Potential of Automated Insulin Delivery.

Diabetes is the leading cause of chronic kidney disease (CKD) and end-stage kidney disease in the world. It is known that maintaining optimal glycemic control can slow the progression of CKD. However, the failing kidney impacts glucose and insulin metabolism and contributes to increased glucose variability. Conventional methods of insulin delivery are not well equipped to adapt to this increased glycemic lability. Automated insulin delivery (AID) has been established as an effective treatment in patients with type 1 diabetes mellitus, and there is emerging evidence for their use in type 2 diabetes mellitus. However, few studies have examined their role in diabetes with concurrent advanced CKD. We discuss the potential benefits and challenges of AID use in patients with diabetes and advanced CKD, including those on dialysis.

Development of a Chemical Cocktail That Rescues Mouse Brain Demyelination in a Cuprizone-Induced Model.

Oligodendrocytes are glial cells located in the central nervous system (CNS) that play essential roles in the transmission of nerve signals and in the neuroprotection of myelinated neurons. The dysfunction or loss of oligodendrocytes leads to demyelinating diseases such as multiple sclerosis (MS). To treat demyelinating diseases, the development of a therapy that promotes remyelination is required. In the present study, we established an in vitro method to convert human fibroblasts into induced oligodendrocyte-like cells (iOLCs) in 3 days. The induced cells displayed morphologies and molecular signatures similar to oligodendrocytes after treatment with valproic acid and exposure to the small molecules Y27632, SU9516, and forskolin (FSK). To pursue the development of a cell-free remyelination therapy in vivo, we used a cuprizone-induced demyelinated mouse model. The small molecules (Y27632, SU9516, and FSK) were directly injected into the demyelinated corpus callosum of the mouse brain. This combination of small molecules rescued the demyelination phenotype within two weeks as observed by light and electron microscopy. These results provide a foundation for exploring the development of a treatment for demyelinating diseases via regenerative medicine.