STAT1 transcriptionally regulates the expression of S1PR1 by binding its promoter region.

Sphingosine 1-phosphate receptor 1 (S1PR1) plays a pivotal role in mediating trafficking and migration of immune cells. Previous reports also identify S1PR1 as an important susceptibility gene of asthma and other autoimmune disorders. However, little has been known about the regulatory mechanism of S1PR1 expression. Thus we systematically investigated the transcriptional regulation of S1PR1 in this study. Promoter activity of S1PR1 gene was carefully screened using series of pGL3-Basic reporter vectors, containing full length (range from transcription start site to upstream -1 kb region) or several truncated fragments of S1PR1 promoter. We identified an area (from -29 to -12 bp) of the S1PR1 promoter as the minimal promoter region. Bioinformatics prediction results showed that several transcription factors were recruited to these sites. EMSA and ChIP assays demonstrated the transcriptional factor STAT1 could bind to the region. We also found that the level of S1PR1 level was significantly reduced when STAT1 was knocked-down. Consistent with the reduction of S1PR1 caused by depletion of STAT1, overexpression of STAT1 resulted in up-regulation of S1PR1. In addition, both mRNA and protein levels of S1PR1 were increased when STAT1 was activated by IFN-γ, and decreased when STAT1 was inhibited by fludarabine. Besides, the levels of STAT1 and S1PR1 expression were positively correlated in peripheral blood leukocytes derived from 41 healthy individuals. Our study showed that transcription factor STAT1 could bind to upstream region of -29 bp to -12 bp of the S1PR1 promoter and stimulate the expression of S1PR1.

Soluble Urokinase Receptor and Acute Kidney Injury.

BACKGROUND: Acute kidney injury is common, with a major effect on morbidity and health care utilization. Soluble urokinase plasminogen activator receptor (suPAR) is a signaling glycoprotein thought to be involved in the pathogenesis of kidney disease. We investigated whether a high level of suPAR predisposed patients to acute kidney injury in multiple clinical contexts, and we used experimental models to identify mechanisms by which suPAR acts and to assess it as a therapeutic target. METHODS: We measured plasma levels of suPAR preprocedurally in patients who underwent coronary angiography and patients who underwent cardiac surgery and at the time of admission to the intensive care unit in critically ill patients. We assessed the risk of acute kidney injury at 7 days as the primary outcome and acute kidney injury or death at 90 days as a secondary outcome, according to quartile of suPAR level. In experimental studies, we used a monoclonal antibody to urokinase plasminogen activator receptor (uPAR) as a therapeutic strategy to attenuate acute kidney injury in transgenic mice receiving contrast material. We also assessed cellular bioenergetics and generation of reactive oxygen species in human kidney proximal tubular (HK-2) cells that were exposed to recombinant suPAR. RESULTS: The suPAR level was assessed in 3827 patients who were undergoing coronary angiography, 250 who were undergoing cardiac surgery, and 692 who were critically ill. Acute kidney injury developed in 318 patients (8%) who had undergone coronary angiography. The highest suPAR quartile (vs. the lowest) had an adjusted odds ratio of 2.66 (95% confidence interval [CI], 1.77 to 3.99) for acute kidney injury and 2.29 (95% CI, 1.71 to 3.06) for acute kidney injury or death at 90 days. Findings were similar in the surgical and critically ill cohorts. The suPAR-overexpressing mice that were given contrast material had greater functional and histologic evidence of acute kidney injury than wild-type mice. The suPAR-treated HK-2 cells showed heightened energetic demand and mitochondrial superoxide generation. Pretreatment with a uPAR monoclonal antibody attenuated kidney injury in suPAR-overexpressing mice and normalized bioenergetic changes in HK-2 cells. CONCLUSIONS: High suPAR levels were associated with acute kidney injury in various clinical and experimental contexts. (Funded by the National Institutes of Health and others.).

CTLA4 blockade promotes vessel normalization in breast tumors via the accumulation of eosinophils.

Immune checkpoint blockade (ICB) has shown long-term survival benefits, but only in a small fraction of cancer patients. Recent studies suggest that improved vessel perfusion by ICB positively correlates with its therapeutic outcomes. However, the underlying mechanism of such a process remains unclear. Here, we show that anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) treatment-induced tumor vessel normalization was accompanied by an increased infiltration of eosinophils into breast tumors. Eosinophil accumulation was positively correlated with the responsiveness of a breast tumor to anti-CTLA4 therapy. Depletion of eosinophils subsequently negated vessel normalization, reduced antitumor immunity and attenuated tumor growth inhibition by anti-CTLA4 therapy. Moreover, intratumoral accumulation of eosinophils relied on T lymphocytes and interferon γ production. Together, these results suggest that eosinophils partially mediate the antitumor effects of CTLA4 blockade through vascular remodeling. Our findings uncover an unidentified role of eosinophils in anti-CTLA4 therapy, providing a potential new target to improve ICB therapy and to predict its efficacy.

METTL3 Promotes the Proliferation and Mobility of Gastric Cancer Cells.

Methyltransferase-like 3 (METTL3) was originally known to be responsible for N6-methyladenosine (m6A) modification of mRNA. Recent studies have found that METTL3 plays important roles in a variety of tumors by regulating the translation of oncogenes. However, the functional and regulating mechanisms of METTL3 in human gastric cancer have not yet been understood. Here we knocked down METTL3 in human gastric cancer cell lines, AGS and MKN45, by using shRNA transfection. RT-qPCR assay and western blotting verified the effectiveness of RNA interference on mRNA and protein levels, respectively. Then we found that METTL3 knockdown inhibited cell proliferation, migration and invasion in AGS and MKN45 cells. Moreover, METTL3 knockdown decreased Bcl2 and increased Bax and active Caspase-3 in gastric cancer cells, which suggested the apoptotic pathway was activated. Mechanistic investigation suggested that METTL3 led to inactivation of the AKT signaling pathway in human gastric cancer cells, including decreased phosphorylation levels of AKT and expression of down-stream effectors p70S6K and Cyclin D1. In conclusion, our study reveals that down-regulation of METTL3 inhibits the proliferation and mobility of human gastric cancer cells and leads to inactivation of the AKT signaling pathway, suggesting that METTL3 may be a potential target for the treatment of human gastric cancer.

Prominin-like, a homolog of mammalian CD133, suppresses di lp6 and TOR signaling to maintain body size and weight in Drosophila.

CD133 (AC133/prominin-1) has been identified as a stem cell marker and a putative cancer stem cell marker in many solid tumors. Its biologic function and molecular mechanisms remain largely elusive. Here, we show that a fly mutant for prominin-like, a homolog of mammalian CD133, shows a larger body size and excess weight accompanied with higher fat deposits as compared with the wild type. The expression levels of prominin-like are mediated by ecdysone signaling where its protein levels increase dramatically in the fat body during metamorphosis. Prominin-like mutants exhibit higher Drosophila insulin-like peptide 6 (di lp6) levels during nonfeeding stages and increased Akt/ Drosophila target of rapamycin (dTOR) signaling. On an amino acid-restricted diet, prominin-like mutants exhibit a significantly larger body size than the wild type does, similar to that which occurs upon the activation of the dTOR pathway in the fat body. Our data suggest that prominin-like functions by suppressing TOR and dilp6 signaling to control body size and weight. The identification of the physiologic function of prominin-like in Drosophila may provide valuable insight into the understanding of the metabolic function of CD133 in mammals.-Zheng, H., Zhang, Y., Chen, Y., Guo, P., Wang, X., Yuan, X., Ge, W., Yang, R., Yan, Q., Yang, X., Xi, Y. Prominin-like, a homolog of mammalian CD133, suppresses di lp6 and TOR signaling to maintain body size and weight in Drosophila.

Fetal Renal Echogenicity Associated with Maternal Focal Segmental Glomerulosclerosis: The Effect of Transplacental Transmission of Permeability Factor suPAR.

We report a case of a pregnant woman with nephrotic syndrome due to biopsy-proven focal segmental glomerulosclerosis (FSGS) whose fetus developed echogenic kidneys and severe oligohydramnios by 27 weeks of gestation. Maternal treatment with prednisone resulted in normalization of the amniotic fluid indices and resolution of fetal renal echogenicity. The newborn was noted to have transient renal dysfunction and proteinuria, resolving by 6 weeks postpartum. The transplacental passage of permeability factors is postulated to have caused both the fetal and newborn renal presentation, with significantly elevated levels of soluble urokinase-type plasminogen activator receptor (suPAR) noted in the cord blood. This case documents the transplacental maternal-fetal transmission of suPAR, demonstrating the potential for maternal-fetal transmission of deleterious, disease-causing entities, and adds to the differential diagnosis of fetal echogenic kidneys. Further, this is the first documentation of a fetal response to maternal systemic therapy.

Effect of perioperative infusion of Dexmedetomidine combined with Sufentanil on quality of postoperative analgesia in patients undergoing laparoscopic nephrectomy: a CONSORT-prospective, randomized, controlled trial.

BACKGROUND: Postoperative pain is one of the most common symptoms after surgery, which brings physical discomfort to patients. In addition, it may cause a series of complications, and even affect the long-term quality of life. The purpose of this prospective, randomized, double-blinded, controlled trial is to investigate the efficacy and safety of dexmedetomidine combined with sufentanil to attenuate postoperative pain in patients after laparoscopic nephrectomy. METHODS: Ninety patients undergoing laparoscopic nephrectomy were randomized into three groups: the control (sufentanil 0.02 µg/kg/h, Group C), sufentanil plus low dose of dexmedetomidine (0.02 µg/kg/h each, Group D1), and sufentanil plus high dose of dexmedetomidine (0.04 µg/kg/h, Group D2). The patient-controlled analgesia was programmed to deliver a bolus dose of 0.5 ml, followed by an infusion of 2 ml/h and a lockout time of 10 min. The primary goal was to calculate the cumulative amount of self-administered sufentanil; the secondary goals were to estimate pain intensity using the numerical rating scale (NRS), level of sedation, the first bowel movement, concerning adverse effects as well as duration of postoperative hospital stay. RESULTS: The total consumption of sufentanil in group D1 and D2 were significantly lower than in group C during the first 8 h after surgery (P < 0.05), whereas there were no statistically significant differences (P > 0.05) between group D1 and D2. Compared with group C, the NRS scores at rest during first 8 h after surgery were significantly lower in group D1 (P < 0.05). The NRS scores, neither at rest nor with movement, show statistically significant differences between group D1 and D2 at each time point following surgery (P > 0.05). The time to first flatus was shorter in group D1 compared with the control group (P < 0.05). In addition, compared with group C, group D1 and D2 had a shorter time for first defecation (P < 0.05). CONCLUSIONS: Dexmedetomidine combined with sufentanil showed better postoperative analgesia without adverse effects, as well as facilitated bowel movements for patients undergoing laparoscopic nephrectomy. TRIAL REGISTRATION: We registered this study in a Chinese Clinical Trial Registry (ChiCTR) centre on Dec 23 2015 and received the registration number: ChiCTR-IPR-15007628 .

Lentinan inhibits tumor angiogenesis via interferon γ and in a T cell independent manner.

BACKGROUND: Antiangiogenic agents are commonly used in lung and colon cancer treatments, however, rapid development of drug resistance limits their efficacy. METHODS: Lentinan (LNT) is a biologically active compound extracted from Lentinus edodes. The effects of LNT on tumor angiogenesis were evaluated by immunohistochemistry in murine LAP0297 lung and CT26 colorectal tumor models. The impacts of LNT on immune cells and gene expression in tumor tissues were determined by flow cytometry, qPCR, and ELISA. Nude mice and IFNγ blockade were used to investigate the mechanism of LNT affecting on tumor angiogenesis. The data sets were compared using two-tailed student's t tests or ANOVA. RESULTS: We found that LNT inhibited tumor angiogenesis and the growth of lung and colon cancers. LNT treatments elevated the expression of angiostatic factors such as IFNγ and also increased tumor infiltration of IFNγ-expressing T cells and myeloid cells. Interestingly, IFNγ blockade, but not T cell deficiency, reversed the effects of LNT treatments on tumor blood vessels. Moreover, long-lasting LNT administration persistently suppressed tumor angiogenesis and inhibited tumor growth. CONCLUSIONS: LNT inhibits tumor angiogenesis by increasing IFNγ production and in a T cell-independent manner. Our findings suggest that LNT could be developed as a new antiangiogenic agent for long-term treatment of lung and colon cancers.

Radiomic analysis of pulmonary ground-glass opacity nodules for distinction of preinvasive lesions, invasive pulmonary adenocarcinoma and minimally invasive adenocarcinoma based on quantitative texture analysis of CT.

OBJECTIVE: To identify the differences among preinvasive lesions, minimally invasive adenocarcinomas (MIAs) and invasive pulmonary adenocarcinomas (IPAs) based on radiomic feature analysis with computed tomography (CT). METHODS: A total of 109 patients with ground-glass opacity lesions (GGOs) in the lungs determined by CT examinations were enrolled, all of whom had received a pathologic diagnosis. After the manual delineation and segmentation of the GGOs as regions of interest (ROIs), the patients were subdivided into three groups based on pathologic analyses: the preinvasive lesions (including atypical adenomatous hyperplasia and adenocarcinoma in situ) subgroup, the MIA subgroup and the IPA subgroup. Next, we obtained the texture features of the GGOs. The data analysis was aimed at finding both the differences between each pair of the groups and predictors to distinguish any two pathologic subtypes using logistic regression. Finally, a receiver operating characteristic (ROC) curve was applied to accurately evaluate the performances of the regression models. RESULTS: We found that the voxel count feature (P<0.001) could be used as a predictor for distinguishing IPAs from preinvasive lesions. However, the surface area feature (P=0.040) and the extruded surface area feature (P=0.013) could be predictors of IPAs compared with MIAs. In addition, the correlation feature (P=0.046) could distinguish preinvasive lesions from MIAs better. CONCLUSIONS: Preinvasive lesions, MIAs and IPAs can be discriminated based on texture features within CT images, although the three diseases could all appear as GGOs on CT images. The diagnoses of these three diseases are very important for clinical surgery.

Increased vessel perfusion predicts the efficacy of immune checkpoint blockade.

Immune checkpoint blockade (ICB) has demonstrated curative potential in several types of cancer, but only for a small number of patients. Thus, the identification of reliable and noninvasive biomarkers for predicting ICB responsiveness is an urgent unmet need. Here, we show that ICB increased tumor vessel perfusion in treatment-sensitive EO771 and MMTV-PyVT breast tumor as well as CT26 and MCA38 colon tumor models, but not in treatment-resistant MCaP0008 and 4T1 breast tumor models. In the sensitive tumor models, the ability of anti-cytotoxic T lymphocyte-associated protein 4 or anti-programmed cell death 1 therapy to increase vessel perfusion strongly correlated with its antitumor efficacy. Moreover, globally enhanced tumor vessel perfusion could be detected by Doppler ultrasonography before changes in tumor size, which predicted final therapeutic efficacy with more than 90% sensitivity and specificity. Mechanistically, CD8+ T cell depletion, IFN-γ neutralization, or implantation of tumors in IFN-γ receptor knockout mice abrogated the vessel perfusion enhancement and antitumor effects of ICB. These results demonstrated that ICB increased vessel perfusion by promoting CD8+ T cell accumulation and IFN-γ production, indicating that increased vessel perfusion reflects the successful activation of antitumor T cell immunity by ICB. Our findings suggest that vessel perfusion can be used as a novel noninvasive indicator for predicting ICB responsiveness.

Intrastriatal transplantation of stem cells from human exfoliated deciduous teeth reduces motor defects in Parkinsonian rats.

BACKGROUND: This study explored the neural differentiation and therapeutic effects of stem cells from human exfoliated deciduous teeth (SHED) in a rat model of Parkinson's disease (PD). METHODS: The SHED were isolated from fresh dental pulp and were induced to differentiate to neurons and dopamine neurons by inhibiting similar mothers against dpp (SMAD) signaling with Noggin and increase conversion of dopamine neurons from SHED with CHIR99021, Sonic Hedgehog (SHH) and FGF8 in vitro. The neural-primed SHED were transplanted to the striatum of 6-hydroxydopamine (6-OHDA)-induced PD rats to evaluate their neural differentiation and functions in vivo. RESULTS: These SHED were efficiently differentiated to neurons (62.7%) and dopamine neurons (42.3%) through a newly developed method. After transplantation, the neural-induced SHED significantly improved recovery of the motor deficits of the PD rats. The grafted SHED were differentiated into neurons (61%), including dopamine neurons (22.3%), and integrated into the host rat brain by forming synaptic connections. Patch clamp analysis showed that neurons derived from grafted SHED have the same membrane potential profile as dopamine neurons, indicating these cells are dopamine neuron-like cells. The potential molecular mechanism of SHED transplantation in alleviating motor deficits of the rats is likely to be mediated by neuronal replacement and immune-modulation as we detected the transplanted dopamine neurons and released immune cytokines from SHED. CONCLUSION: Using neural-primed SHED to treat PD showed significant restorations of motor deficits in 6-OHDA-induced rats. These observations provide further evidence that SHED can be used for cell-based therapy of PD.

Mitochondrial Dysfunctions Contribute to Hypertrophic Cardiomyopathy in Patient iPSC-Derived Cardiomyocytes with MT-RNR2 Mutation.

Hypertrophic cardiomyopathy (HCM) is the most common cause of sudden cardiac death in young individuals. A potential role of mtDNA mutations in HCM is known. However, the underlying molecular mechanisms linking mtDNA mutations to HCM remain poorly understood due to lack of cell and animal models. Here, we generated induced pluripotent stem cell-derived cardiomyocytes (HCM-iPSC-CMs) from human patients in a maternally inherited HCM family who carry the m.2336T>C mutation in the mitochondrial 16S rRNA gene (MT-RNR2). The results showed that the m.2336T>C mutation resulted in mitochondrial dysfunctions and ultrastructure defects by decreasing the stability of 16S rRNA, which led to reduced levels of mitochondrial proteins. The ATP/ADP ratio and mitochondrial membrane potential were also reduced, thereby elevating the intracellular Ca2+ concentration, which was associated with numerous HCM-specific electrophysiological abnormalities. Our findings therefore provide an innovative insight into the pathogenesis of maternally inherited HCM.

Premature remodeling of fat body and fat mobilization triggered by platelet-derived growth factor/VEGF receptor in Drosophila.

In Drosophila, fat-body remodeling accompanied with fat mobilization is an ecdysone-induced dynamic process that only occurs during metamorphosis. Here, we show that the activated Drosophila platelet-derived growth factor/VEGF receptor (PVR) is sufficient to induce shape changes in the fat body, from thin layers of tightly conjugated polygonal cells to clusters of disaggregated round-shaped cells. These morphologic changes are reminiscent of those seen during early pupation upon initiation of fat-body remodeling. Activation of PVR also triggers an early onset of lipolysis and mobilization of internal storage, as revealed by the appearance of small lipid droplets and up-regulated lipolysis-related genes. We found that PVR displays a dynamic expression pattern in the fat body and peaks at the larval-prepupal transition under the control of ecdysone signaling. Removal of PVR, although it does not prevent ecdysone-induced fat-body remodeling, causes ecdysone signaling to be up-regulated. Our data reveal that PVR is active in a dual-secured mechanism that involves an ecdysone-induced fat-body remodeling pathway and a reinforced PVR pathway for effective lipid mobilization. Ectopic expression of activated c-kit-the mouse homolog of PVR in the Drosophila fat body-also results in a similar phenotype. This may suggest a novel function of c-kit as it relates to lipid metabolism in mammals.-Zheng, H., Wang, X., Guo, P., Ge, W., Yan, Q., Gao, W., Xi, Y., Yang, X. Premature remodeling of fat body and fat mobilization triggered by platelet-derived growth factor/VEGF receptor in Drosophila.