Regulation of PDGFR-ß gene expression by targeting the G-vacancy bearing G-quadruplex in promoter.

G-quadruplex is an essential element in gene transcription that serves as a promising drug target. Guanine-vacancy-bearing G-quadruplex (GVBQ) is a newly identified G-quadruplex that has distinct structural features from the canonical G-quadruplex. Potential GVBQ-forming motifs are widely distributed in gene promoter regions. However, whether GVBQ can form in genomic DNA and be an effective target for manipulating gene expression is unknown. Using photo-crosslinking, dimethyl sulfate footprinting, exonuclease digestion and in vitro transcription, we demonstrated the formation of a GVBQ in the G-rich nuclease hypersensitivity element within the human PDGFR-ß gene promoter region in both single-stranded and double-stranded DNA. The formation of GVBQ in dsDNA could be induced by negative supercoiling created by downstream transcription. We also found that the PDGFR-ß GVBQ was specifically recognized and stabilized by a new synthetic porphyrin guanine conjugate (mPG). Targeting the PDGFR-ß GVBQ in human cancer cells using the mPG could specifically alter PDGFR-ß gene expression. Our work illustrates that targeting GVBQ with mPG in human cells can regulate the expression level of a specific gene, thus indicating a novel strategy for drug development.

LMP1-mediated glycolysis induces myeloid-derived suppressor cell expansion in nasopharyngeal carcinoma.

Myeloid-derived suppressor cells (MDSCs) are expanded in tumor microenvironments, including that of Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC). The link between MDSC expansion and EBV infection in NPC is unclear. Here, we show that EBV latent membrane protein 1 (LMP1) promotes MDSC expansion in the tumor microenvironment by promoting extra-mitochondrial glycolysis in malignant cells, which is a scenario for immune escape initially suggested by the frequent, concomitant detection of abundant LMP1, glucose transporter 1 (GLUT1) and CD33+ MDSCs in tumor sections. The full process has been reconstituted in vitro. LMP1 promotes the expression of multiple glycolytic genes, including GLUT1. This metabolic reprogramming results in increased expression of the Nod-like receptor family protein 3 (NLRP3) inflammasome, COX-2 and P-p65 and, consequently, increased production of IL-1ß, IL-6 and GM-CSF. Finally, these changes in the environment of malignant cells result in enhanced NPC-derived MDSC induction. One key step is the physical interaction of LMP1 with GLUT1 to stabilize the GLUT1 protein by blocking its K48-ubiquitination and p62-dependent autolysosomal degradation. This work indicates that LMP1-mediated glycolysis regulates IL-1ß, IL-6 and GM-CSF production through the NLRP3 inflammasome, COX-2 and P-p65 signaling pathways to enhance tumor-associated MDSC expansion, which leads to tumor immunosuppression in NPC.

Tumour YAP1 and PTEN expression correlates with tumour-associated myeloid suppressor cell expansion and reduced survival in colorectal cancer.

The expansion of myeloid-derived suppressor cells (MDSCs) correlates with tumorigenesis in colorectal cancer (CRC). Here, we found a significant association between CD33+ MDSC number and Yes-associated protein 1 (YAP1) and phosphatase and tensin homologue (PTEN) levels in CRC patients (P < 0·05). Moreover, the CD33+ MDSCs, YAP1 and PTEN were identified as predictors for the prognosis of CRC patients (P < 0·05). Notably, CD33+ MDSCs were an independent survival predictor for CRC patients through a Cox model analysis. In vitro data determined that the expression levels of YAP1 and PTEN in CRC-derived cell lines were associated with CRC-derived MDSC induction, and the blockade of YAP1 and PTEN decreased CRC-derived MDSC induction. A mechanistic analysis revealed that YAP1 promoted CRC-derived MDSC induction by suppressing PTEN expression to up-regulate COX-2, P-AKT and P-p65 in CRC-derived cells, leading to secretion of the cytokine granulocyte-macrophage colony-stimulating factor. Our findings establish a novel mechanism of pro-tumorigenic MDSC induction mediated by ectopic YAP1 and PTEN expression in CRC.

Exosomal miR-24-3p impedes T-cell function by targeting FGF11 and serves as a potential prognostic biomarker for nasopharyngeal carcinoma.

Recent studies have shown that extracellular microRNAs are not only potential biomarkers but are also involved in cell interactions to regulate the intercommunication between cancer cells and their microenvironments in various types of malignancies. In this study, we isolated exosomes from nasopharyngeal carcinoma (NPC) cell lines and patient sera (T-EXOs), or control NP69 cells and healthy donor sera (HD-EXOs). We found that miR-24-3p was markedly enriched in T-EXOs as compared with HD-EXOs; the serum exosomal miR-24-3p level was correlated with worse disease-free survival of patients (p < 0.05). Knockdown of exosomal miR-24-3p (miR-24-3p-sponge-T-EXOs) by a sponge RNA targeting miR-24-3p restored the T-EXO-mediated (control-sponge-T-EXO) inhibition of T-cell proliferation and Th1 and Th17 differentiation, and the induction of regulatory T cells (Tregs). Mechanistic analyses revealed that administration of exosomal miR-24-3p increased P-ERK, P-STAT1 and P-STAT3 expression while decreasing P-STAT5 expression during T-cell proliferation and differentiation. Moreover, by in vivo and in vitro assessments, we found FGF11 to be a direct target of miR-24-3p. However, both miR-24-3p-sponge-T-EXOs and T-EXOs (control-sponge-T-EXOs) impeded proliferation and Th1 and Th17 differentiation, but induced Treg differentiation, of lenti-shFGF11-transfected T cells. The levels of phosphorylated ERK and STAT proteins were different in lenti-ScshRNA-transfected T cells and lenti-shFGF11-transfected T cells following administration of miR-24-3p-sponge-T-EXO. Interestingly, tumour FGF11 expression was positively correlated with the number of CD4+ and CD8+ T cells in vivo, and predicted favourable patient DFS (p < 0.05). Additionally, hypoxia increased cellular and exosomal miR-24-3p levels and enhanced the inhibitory effect of T-EXO on T-cell proliferation and differentiation. Collectively, our findings suggest that exosomal miR-24-3p is involved in tumour pathogenesis by mediating T-cell suppression via repression of FGF11, and may serve as a potential prognostic biomarker in NPC. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Solvent-assisted electrospray ionization for direct analysis of various compounds (complex) from low/nonpolar solvents and eluents.

Electrospray ionization (ESI) is a powerful ionization technique with a wide range of applications. However, the analytes in low/nonpolar solvents cannot be analyzed directly in electrospray ionization-mass spectrometry (ESI-MS), because low/nonpolar solvents are incompatible with ESI, because of their low conductivity. To circumvent this problem, we introduce an electrospray-based ionization method termed solvent-assisted electrospray ionization (SAESI). With the help of electrospray solvents at the tip of the spray needle, compounds in "non-electrospray ionization-friendly" solvents can be ionized directly using solvent-assisted electrospray ionization-mass spectrometry (SAESI-MS). The key features that the assistant solvent can be chosen flexibly and makes little interference to samples lead to better ionization performance in detection of organic reaction intermediates and real-time analysis of polymers and chiral drugs separated by gel permeation chromatography (GPC) and normal phase liquid chromatography (NPLC). Furthermore, it can achieve online hydrogen/deuterium (H/D) exchange reaction and even mitigate the signal suppression caused by strong acid modifiers in liquid chromatography. In addition, all parts of this device are commercially available and it only requires two parameters to be optimized, which makes SAESI easy to handle.

Single-Cell RNA Sequencing Analysis of Steroidogenesis and Spermatogenesis Impairment in the Testis of db/db Mice.

Objective: The mechanism of steroidogenesis and spermatogenesis impairment in men with type 2 diabetes remains unclear. We aimed to explore the local changes of steroidogenesis and spermatogenesis in the testis of db/db mice. Research Design and Methods. We performed single-cell RNA sequencing analysis in the testis of db/db and C57BL/6J mice. The differentially expressed genes were then confirmed by real-time PCR. The histopathological characteristics of testis in db/db mice and C57BL/6J control were also performed. Results: The 20-week-old db/db mice had significantly higher blood glucose and body weight (both p < 0.001). The serum testosterone levels (4.4 ± 0.8 vs. 9.8 ± 0.7 ng/ml, p=0.001) and weight of the testis (0.16 ± 0.01 vs. 0.24 ± 0.01 g, p < 0.001) were significantly lower in db/db mice than that in C57BL/6J controls. db/db mice had a lower cross-sectional area of seminiferous tubules and thickness of the cell layer (both p < 0.05). The numbers of Sertoli cells and Leydig cells decreased in db/db mice (both p < 0.01). Single-cell RNA sequencing analysis showed that compared with the control group, the percentage of spermatogonia was significantly higher in the db/db mouse (p < 0.001), while the proportions of spermatocytes, round and elongating spermatids, and sperms were all lower in the db/db mouse (p all < 0.001). The most differentially expressed genes were found in round spermatids (n = 86), which were not found in spermatogonia, spermatocyte, and sperm. Igfbp5 was the most significantly decreased gene in Leydig cells of the db/db mouse, while the expression of Cd74, H2-Aa, and H2-Eb1 was elevated. Ccl7 and Ptgds were the most significantly increased and decreased genes in Sertoli cells of the db/db mouse. Conclusions: The present study indicates spermiogenesis and steroidogenesis defects in db/db mice. The mechanism of steroidogenesis impairment in the testis of db/db mice deserves further investigation.

Septin 9 expression regulates 'don't eat me' signals and identifies an immune-epithelial class of intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) is a highly heterogeneous and aggressive liver cancer with limited therapeutic options. Precise classification and immunotherapy are perspectives to improve the treatments. We reported the role of septin 9 in apico-basal polarity and epithelial-to-mesenchymal transition (EMT). Here, we aim to elucidate its role in iCCA. We analyzed single-cell transcriptomes from human iCCA tumor cells based on phenotype and cell state. Knockdown of the septin 9 gene (SEPT9) was done using small interfering RNA (siRNA); interferon-γ (IFN-γ) stimulation was performed using different CCA cells; gene expressions were analyzed by reverse transcription and real-time PCR analysis (RT-qPCR); and immunofluorescence, immunoblotting, and flow cytometry were performed to assess the expression of proteins. The differential distributions of SEPT9 and vimentin (VIM) gene expressions allowed us to define specific cellular trajectories of malignant cells and thus identified distinct clusters of iCCA cells. One cluster was enriched in VIM and extracellular-matrix (ECM) remodeling molecules, and another had high expression of SEPT9 and genes from the 'don't eat me' signal involved in immune escape. This antagonism between SEPT9 and VIM was confirmed by in vitro experiments. Notably, SEPT9 and 'don't eat me' gene expressions were inversely correlated to those of vimentin and the EMT markers. SEPT9 expression was upregulated by IFN-γ and SEPT9 knockdown decreased expression of 'don't eat me' signal genes and increased expression of mesenchymal markers. Cancer Cell Line Encyclopedia (CCLE) transcriptome database analyses confirmed that iCCA cells enriched in septin 9 exhibit epithelial-like features. This study revealed septin 9 as a cytoskeleton element of iCCA epithelial-like cells and a regulator of the immune system response. It also brings new insights into the enigmatic relationship between EMT and immune response. Notably, we decoded a potential mechanism that could sensitize patients to immunotherapies.

Low Total Testosterone Levels in Men with Newly Diagnosed Early-Onset Type 2 Diabetes: A Cross-Sectional Study in China.

Objective: There is a bidirectional interaction between circulating testosterone and blood glucose levels. We aim to investigate the testosterone levels in men with early-onset type 2 diabetes (T2DM). Methods: A total of 153 drug naive men with T2DM were enrolled in the study. Early- (n = 63) and late-onset (n = 90) T2DM was classified according to age 40 years old. Clinical characteristics and plasma for biochemical criterions were collected. Gonadal hormones were measured using chemiluminescent immunometric assay. The concentrations of 3ß- and 17ß-HSD were determined using ELISA. Results: Compared with men with late-onset T2DM, those with early-onset T2DM had lower serum total testosterone (TT), sex hormone-binding globulin (SHBG), and FSH, but higher dehydroepiandrosterone sulfate (DHEA-S) level (p < 0.05). The mediating effect analysis showed that the decreased TT levels in patients with early-onset T2DM were associated with the higher HbA1c, BMI, and triglyceride in these patients (both p < 0.05). The early-onset of T2DM directly correlated with increased DHEA-S (both p < 0.01). The 3ß-HSD concentration in the early-onset T2DM group was lower than that in the late-onset T2DM group (11.07 ± 3.05 vs. 12.40 ± 2.72 pg/mL, p = 0.048) and was positively correlated with fasting C-peptide, while negatively correlated with HbA1c and fasting glucagon (p all < 0.05). Conclusions: Patients with early-onset T2DM showed inhibition of conversion from DHEA to testosterone, which may attribute to the low level of 3ß-HSD and high blood glucose in these patients.

Septin 9 and phosphoinositides regulate lysosome localization and their association with lipid droplets.

The accumulation of lipid droplets (LDs) in the liver is a hallmark of steatosis, which is often associated with lysosomal dysfunction. Nevertheless, the underlying mechanisms remain unclear. Here, using Huh7 cells loaded with oleate as a model to study LD metabolism, we show that cellular content and distribution of LDs are correlated with those of the lysosome and regulated by oleate and septin 9. High expression of septin 9 promotes perinuclear clustering of lysosomes which co-localized with Golgi and not with their surrounding LDs. On the other hand, knockdown of septin 9 disperses the two organelles which colocalize at the cell periphery. The Rab7 is present around these peripheral LDs. PtdIns5P which binds septin 9 and MTMR3 which converts PtdIns(3,5)P2 into PtdIns(5) recapitulates the effects of septin 9. By contrast, PtdIns(3,5)P2 promotes LD/lysosome co-localization. Overall, our data reveal a phosphoinositide/septin 9-dependent mechanism that regulates LD behavior through the control of their association with lysosomes.

Short-time intensive insulin therapy upregulates 3 beta- and 17 beta-hydroxysteroid dehydrogenase levels in men with newly diagnosed T2DM.

Objective: Our previous study has found that short-term intensive insulin therapy in patients with newly diagnosed type 2 diabetes mellitus (T2DM) increased serum testosterone levels, but the underlying mechanisms remain unclear. Design and methods: In this self-controlled study, 43 men with newly diagnosed drug naïve T2DM, aged 18-60 years, with HbA1c >9.0% were treated with continuous subcutaneous insulin infusion (CSII) to normalize blood glucose within one week. Venous blood specimens were collected for measuring of serum total testosterone, dehydroepiandrosterone sulfate (DHEA-S), 3ß- and 17ß-hydroxysteroid dehydrogenase (3ß- and 17ß-HSD) concentrations before and after insulin therapy. Results: Testosterone increased from 13.0 (11.3, 14.6) nmol/L to 15.7 (13.9, 17.5) nmol/L after intensive insulin therapy (p<0.001), while the levels of DHEA-S decreased significantly after treatment (from 6.5 (5.7, 7.3) µmol/L to 6.0 (5.3, 6.7) µmol/L, p=0.001). The ratio of testosterone/DHEA-S increased significantly (2.4 (2.0, 2.8) vs. 3.1 (2.6, 3.7) nmol/µmol, p<0.001). After blood glucose normalization with the short-term CSII therapy, 3ß-HSD increased from 11.0 (9.5, 12.5) pg/mL to 14.6 (13.5, 15.7) pg/mL, p=0.001, and 17ß-HSD increased from 20.7 (16.3, 25.2) pg/mL to 28.2 (23.8, 32.5) pg/mL, p=0.009. Conclusions: Blood glucose normalization via short-term intensive insulin therapy increases plasma total testosterone levels in men with newly diagnosed type 2 diabetes, associated with a decreased level of DHEA-S, probably because of the enhanced conversion from DHEA to testosterone catalyzed by 3ß-HSD and 17ß-HSD.