Reliability of Droplet Digital PCR Alone and in Combination with Interleukin-6 and Procalcitonin for Prognosis of Bloodstream Infection.

Purpose: Bloodstream infection(BSI) is linked with high mortality, underscoring the significance of prompt etiological diagnosis for timely and precise treatment. This study aims to investigate the diagnostic value of droplet digital polymerase chain reaction(ddPCR) in combination with conventional inflammatory markers [interleukin-6(IL-6) and procalcitonin(PCT)] concerning disease progression and treatment prognosis in BSI patients. Furthermore, the study aims to explore a more efficient clinical application strategy. Patients and Methods: This prospective case seried study centers on 176 patients suspected of or confirmed with BSI. Blood samples were collected to extract nucleic acids for identifying pathogens (bacteria, fungi, and viruses) and determining copy loads via ddPCR. Results: The sensitivity of ddPCR was markedly higher compared to the culture method (74.71% vs 31.03%). A positive correlation existed between bacterial load and levels of inflammatory markers [IL-6 (P=0.0182), PCT (P=0.0029), and CRP (P=0.0005)]. In suspected BSI cases, the combination of ddPCR and inflammatory markers could predict sepsis risk [ROC: Area under the curve(AUC)=0.6071, P=0.0383]. Within confirmed BSI patients, the ddPCR bacterial load of those with SOFA<7 was lower than that of the SOFA≥7 (P=0.0334). ddPCR (OR: 1.789, P=0.035) monitoring combined with PCT (OR: 1.787, P=0.035) holded predictive value for SOFA progression (AUC=0.7913, P=0.0003). Similarly, BSI survivors displayed a lower burden than non-survivors (P=0.0170). Additionally, ddPCR combinated with IL-6 provided a more accurate and expedited insight into clinical outcomes prediction for BSI confirmed patients (AUC=0.7352, P=0.0030). Serial monitoring of bacterial load by ddPCR effectively mirrored the clinical course of BSI in patients. Notably, patients with positive ddPCR virus infection exhibited significantly reduced lymphocyte counts (P=0.0003). Conclusion: In a clinical context, qualitative ddPCR results and quantitative continuous monitoring can more precisely assess sepsis progression and treatment prognosis in BSI patients. Furthermore, ddPCR results offer quicker and more accurate reference points for clinical antibacterial and antiviral interventions.

Correction: MiR-101 reverses the hypomethylation of the LMO3 promoter in glioma cells.

[This corrects the article DOI: 10.18632/oncotarget.3181.].

Palladium/Copper Cocatalyzed C-H Activation and C-C Bond Regioselective Cleavage Reaction for the Synthesis of Fused Chromeno Quinolines.

A method for the synthesis of fused chromeno quinolines by the palladium/copper cocatalyzed C-H bond activation and C-C bond cleavage reaction has been developed. A variety of fused chromeno quinoline derivatives could be synthesized by coumarin derivatives and anilines through the C-C bond cleavage/C-H functionalization/C-C bond formation process. The MTT assay was used to evaluate the chromeno quinolines against the human cervix tumor cells (HeLa), 3c and 9c without coordination metals showed good inhibition effect.

Bonded- and discreted-Lindqvist hexatungstate-based copper hybrids as heterogeneous catalysts for the one-pot synthesis of 2-phenylquinoxalines via 2-haloanilines with vinyl azides or 3-phenyl-2H-azirines.

One bonded- and one discreted-Lindqvist hexatungstate-based copper hybrids (Cu-POMs) ([Cu2(O)OH(phen)2]2[W6O19]·6H2O (1) and [Cu2(phen)4Cl] [HW6O19]·2H2O (2) (phen = 1,10-phenanthroline)) were controllably synthesized and routinely characterized. Cu-POMs 1-2 consisted of identical [W6O19] unit and similar copper-phen complexes, the two units are bonded via four Cu-O chemical bonds in compound 1; however, compound 2 is discreted and stabilized by intermolecular electrostatic interactions. Importantly, these Cu-POMs catalysts were first applied in the novel reaction for the preparation of 2-phenylquinoxalines via the one-pot coupling and oxidation reactions of 2-haloanilines with vinyl azides or 3-phenyl-2H-azirines under mild conditions, and Cu-POMs 1 showed higher catalytic performance in good yields (79-84%). The reactions exhibit some functional group tolerance and allow for the preparation of a number of 2-phenylquinoxalines.

The EGFR-ZNF263 signaling axis silences SIX3 in glioblastoma epigenetically.

The homeotic protein SIX3 is a transcription factor vital for neurogenesis and has a bivalent promoter. We previously showed that SIX3 can be transcriptionally silenced by DNA hypermethylation, functions as a tumor suppressor gene, and inhibits human glioblastoma transcriptionally. Here, we show that the activation of epidermal growth factor (EGFR) induces DNA methylation of SIX3 promoter through the MAPK pathway. ERK, when activated, binds with ZNF263, consequently abrogating the ubiquitination of ZNF263 and leading to its stabilization. ZNF263 binds to the core promoter region of SIX3 and recruits the KAP1/HATS/DNMT corepressor complex to induce transcriptional silencing of SIX3 through H3K27me3 and methylation of SIX3 promoter. Activation of the EGFR-ZNF263 signaling axis in phenotypically normal astrocytes or glioblastoma cells triggers or enhances tumorigenic activities, while elevated expression of the EGFR-ZNF263 signaling components in glioblastoma tissues is associated with poor prognosis of the patients. Together, our findings demonstrate that epigenetic silencing of SIX3 is controlled by a sophisticated and highly ordered oncogenic signaling pathway and therefore provide new insights into initiation and progression of glioblastoma.

Knockdown of LncRNA RHPN1-AS1 Inhibits Cell Migration, Invasion and Proliferation in Head and Neck Squamous Cell Carcinoma.

Previous studies have revealed that long non-coding RNAs (lncRNAs) are involved in head and neck squamous cell carcinoma (HNSCC) progression. However, the detailed roles of lncRNA RHPN1-AS1 remain to be elucidated. In this study, by analyzing online RNA-Seq data, we found that RHPN1-AS1 was upregulated in HNSCC tissues and that its expression level was associated with neoplasm histologic grade. High expression of RHPN1-AS1 was also confirmed in HNSCC tissues. Knockdown of RHPN1-AS1 inhibited tumor cell migration, invasion and proliferation in HNSCC. Furthermore, inhibition of RHPN1-AS1 suppressed the expression of epithelial-mesenchymal transition (EMT)-related genes (ß-Catenin, Claudin-1 and Vimentin) in HNSCC cells. Collectively, our results suggest that RHPN1-AS1, acting as an oncogene, may be a potential diagnostic and therapeutic target in HNSCC.

Retraction Note: Upregulation of the long non-coding RNA AFAP1-AS1 affects the proliferation, invasion and survival of tongue squamous cell carcinoma via the Wnt/ß-catenin signaling pathway.

The authors have retracted this article [1] because there are errors in Figures 4a and 4b.

Cellular prion protein is involved in decidualization of mouse uterus.

Prion protein (PrP) is encoded by a single copy gene Prnp in many cell and tissue types. PrP is very famous for its infectious conformers (PrPSC) resulting in transmissible spongiform encephalopathies. At present, physiological functions of its cellular isoform (PrPC) remain ambiguous. Although PrPC expression has been found in uterus, whether it functions in maternal-fetal dialogue during early pregnant is unknown. In this study, we examined PrPC mRNA and protein in the uterus of peri-implantation mice, and found that they were expressed with a spatiotemporal dynamic pattern. Interestingly, PrPC was significantly increased in the decidual zones around the implanting embryos at the implantation window stage. To further demonstrate that PrPC is involved in the decidualization of mouse uterus during embryo implantation, we constructed the artificial decidualization models and the delayed implantation models. Once the pseudopregnant mice were artificially induced to decidualization, the PrPC expression then increased significantly in the decidua zone. And also, if the delayed implantation embryos were allowed to implant, PrPC protein was also simultaneously improved in stromal cells surrounding the implanting embryos. Moreover, PrPC expression can be inhibited by progesterone but upregulated by estrogen in mouse uterus. These results suggest that PrPC may play an important role in embryo implantation and decidualization.

Upregulation of the long non-coding RNA AFAP1-AS1 affects the proliferation, invasion and survival of tongue squamous cell carcinoma via the Wnt/ß-catenin signaling pathway.

BACKGROUND: Long non-coding RNA (lncRNA) actin filament associated protein 1 antisense RNA1 (AFAP1-AS1) is oriented in an antisense direction to the protein-coding gene AFAP1 in the opposite strand. Previous studies showed that lncRNA AFAP1-AS1 was upregulated and acted as an oncogene in a variety of tumors. However, the expression and biological functions of lncRNA AFAP1-AS1 in tongue squamous cell carcinoma (TSCC) are still unknown. METHODS: The expression level of AFAP1-AS1 was measured in 103 pairs of human TSCC tissues and corresponding adjacent normal tongue mucous tissues. The correlation between AFAP1-AS1 and the clinicopathological features was evaluated using the chi-square test. The effects of AFAP1-AS1 on TSCC cells were determined via a CCK-8 assay, clone formation assay, flow cytometry, wound healing assay and transwell assay. Furthermore, the effect of AFAP1-AS1 knockdown on the activation of the Wnt/ß-catenin signaling pathway was investigated. Finally, CAL-27 cells with AFAP1-AS1 knockdown were subcutaneously injected into nude mice to evaluate the effect of AFAP1-AS1 on tumor growth in vivo. RESULTS: In this study, we found that lncRNA AFAP1-AS1 was increased in TSCC tissues and that patients with high AFAP1-AS1 expression had a shorter overall survival. Short hairpin RNA (shRNA)-mediated AFAP1-AS1 knockdown significantly decreased the proliferation of TSCC cells. Furthermore, AFAP1-AS1 silencing partly inhibited cell migration and invasion. Inhibition of AFAP1-AS1 decreased the activity of the Wnt/ß-catenin pathway and suppressed the expression of EMT-related genes (SLUG, SNAIL1, VIM, CADN, ZEB1, ZEB2, SMAD2 and TWIST1) in TSCC cells. In addition, CAL-27 cells with AFAP1-AS1 knockdown were injected into nude mice to investigate the effect of AFAP1-AS1 on tumorigenesis in vivo. Downregulation of AFAP1-AS1 suppressed tumor growth and inhibited the expression of EMT-related genes (SLUG, SNIAL1, VIM, ZEB1, NANOG, SMAD2, NESTIN and SOX2) in vivo. CONCLUSIONS: Taken together, our findings present a road map for targeting the newly identified lncRNA AFAP1-AS1 to suppress TSCC progression, and these results elucidate a novel potential therapeutic strategy for TSCC.

SIX3, a tumor suppressor, inhibits astrocytoma tumorigenesis by transcriptional repression of AURKA/B.

BACKGROUND: SIX homeobox 3 (SIX3) is a member of the sine oculis homeobox transcription factor family. It plays a vital role in the nervous system development. Our previous study showed that the SIX3 gene is hypermethylated, and its expression is decreased in astrocytoma, but the role of SIX3 remains unknown. METHODS: Chromatin-immunoprecipitation (ChIP) and luciferase reporter assay were used to confirm the binding of SIX3 to the promoter regions of aurora kinase A (AURKA) and aurora kinase B (AURKB). Confocal imaging and co-immunoprecipitation (Co-IP) were used to detect the interaction between AURKA and AURKB. Flow cytometry was performed to assess the effect of SIX3 on cell cycle distribution. Colony formation, EdU incorporation, transwell, and intracranial xenograft assays were performed to demonstrate the effect of SIX3 on the malignant phenotype of astrocytoma cells. RESULTS: SIX3 is identified as a novel negative transcriptional regulator of AURKA and AURKB, and it decreases the expression of AURKA and AURKB in a dose-dependent manner in astrocytoma cells. Importantly, interactions between AURKA and AURKB stabilize and protect AURKA/B from degradation, and overexpression of SIX3 does not affect these interactions; SIX3 also acts as a tumor suppressor, and it increases p53 activity and expression at the post-translational level by the negative regulation of AURKA or AURKB, reduces the events of numerical centrosomal aberrations and misaligned chromosomes, and significantly inhibits the proliferation, invasion, and tumorigenesis of astrocytoma in vitro and in vivo. Moreover, experiments using primary cultured astrocytoma cells indicate that astrocytoma patients with a low expression of SIX3 and mutant p53 are more sensitive to treatment with aurora kinase inhibitors. CONCLUSION: SIX3 is a novel negative transcriptional regulator and acts as a tumor suppressor that directly represses the transcription of AURKA and AURKB in astrocytoma. For the first time, the functional interaction of AURKA and AURKB has been found, which aids in the protection of their stability, and partially explains their constant high expression and activity in cancers. SIX3 is a potential biomarker that could be used to predict the response of astrocytoma patients to aurora kinase inhibitors.

Positive association between serum apolipoprotein M levels and hepatitis B virus DNA load in HBeAg-negative chronic hepatitis B.

BACKGROUND: Hepatitis virus B (HBV) has infected millions of people worldwide. Notably, such infections can be associated with hepatic complications. Levels of apolipoprotein M (apoM), a component of high-density lipoprotein (HDL), are known to be significantly elevated in patients with chronic hepatitis B (CHB). The aim of this study was to investigate the relationship between HBV DNA load in serum and serum apoM levels in patients with CHB. METHODS: A total of 73 HBeAg-negative CHB patients, 50 HBeAg-positive CHB patients, and 79 non-CHB controls were included in the study cohort. The age and body mass index (BMI) of the study participants were matched. Serum levels of apoM and the HBV antigens HBsAg and HBeAg were measured by enzyme-linked immunosorbent assay (ELISA) analysis. Serum levels of alanine aminotransferase (ALT), aspartate transaminase (AST), cholesterol, and triglycerides (TG) were assessed using an automatic biochemical analyzer. Serum HBV DNA levels were quantified by real-time PCR analysis. Data were analyzed by Spearman's rank correlation coefficient, Pearson correlation coefficient, and multivariate linear regression model (continuous variables), or Student's t-test (mean differences). RESULTS: Both the HBeAg-negative CHB and HBeAg-positive CHB patient groups exhibited elevated serum levels of apoM. Moreover, serum apoM levels were positively correlated with serum HBV DNA levels in HBeAg-negative CHB patients (r = 0.394, p < 0.001). Conversely, there was no significant relationship between apoM and HBV DNA levels in the HBeAg-positive CHB group (r = 0.197, p = 0.170). The median log copies/mL value for HBV DNA (4.00) was considered the cutoff point for the HBeAg-negative CHB group. Notably, a significant number of patients with HBV DNA levels above the cutoff point also had higher serum apoM levels (63.38 ± 29.84 vs. 41.41 ± 21.84; p = 0.001). CONCLUSIONS: Our findings reveal that the correlation between serum apoM levels and viral loads may depend on HBeAg status, as serum apoM levels were positively correlated with HBV DNA levels in HBeAg-negative CHB patients. These results suggest that HBeAg may play a role in apoM-related lipid metabolism and anti-inflammatory functions in hepatitis B patients. Thus, our findings may facilitate the clinical management of HBV infection.

The D Domain of LRRC4 anchors ERK1/2 in the cytoplasm and competitively inhibits MEK/ERK activation in glioma cells.

BACKGROUND: As a well-characterized key player in various signal transduction networks, extracellular-signal-regulated kinase (ERK1/2) has been widely implicated in the development of many malignancies. We previously found that Leucine-rich repeat containing 4 (LRRC4) was a tumor suppressor and a negative regulator of the ERK/MAPK pathway in glioma tumorigenesis. However, the precise molecular role of LRRC4 in ERK signal transmission is unclear. METHODS: The interaction between LRRC4 and ERK1/2 was assessed by co-immunoprecipitation and GST pull-down assays in vivo and in vitro. We also investigated the interaction of LRRC4 and ERK1/2 and the role of the D domain in ERK activation in glioma cells. RESULTS: Here, we showed that LRRC4 and ERK1/2 interact via the D domain and CD domain, respectively. Following EGF stimuli, the D domain of LRRC4 anchors ERK1/2 in the cytoplasm and abrogates ERK1/2 activation and nuclear translocation. In glioblastoma cells, ectopic LRRC4 expression competitively inhibited the interaction of endogenous mitogen-activated protein kinase (MEK) and ERK1/2. Mutation of the D domain decreased the LRRC4-mediated inhibition of MAPK signaling and its anti-proliferation and anti-invasion roles. CONCLUSIONS: Our results demonstrated that the D domain of LRRC4 anchors ERK1/2 in the cytoplasm and competitively inhibits MEK/ERK activation in glioma cells. These findings identify a new mechanism underlying glioblastoma progression and suggest a novel therapeutic strategy by restoring the activity of LRRC4 to decrease MAPK cascade activation.

MicroRNA-155 in exosomes secreted from helicobacter pylori infection macrophages immunomodulates inflammatory response.

Exosomes containing microRNA-155 act as molecule carriers during immune cell-cell communication and play an important role in the inflammatory response of H. pylori infection macrophages. Previous reports have found that miR-155 was over-expressed in H. pylori infection macrophages, but the significance of which is still unknown. In this study, we analyzed the impact of miR-155 loaded in exosomes derived from macrophages to the inflammatory response of H. pylori infection macrophages and possible mechanisms. We found that miR-155 promoted the expression of inflammatory cytokines including TNF-a, IL-6, IL-23, but also increased the expression of CD40, CD63, CD81, and MCH-I. Meanwhile, inflammatory signal pathways proteins, such as MyD88, NF-κB in H. pylori infection macrophages were down-regulated due to the over-expression of miR-155. Experiments in vitro or in vivo revealed that miR-155 promoted macrophages to inhibit or kill H. pylori by regulating the inflammatory response of cells to prevent the gastritis caused by H. pylori infection. These findings contribute to the understanding of miR-155 contained in exosomes in inflammatory responses of H. pylori infection macrophages.

Up-Regulation of microRNA-183 Promotes Cell Proliferation and Invasion in Glioma By Directly Targeting NEFL.

Glioblastoma multiforme (GBM) is the most common and lethal type of primary malignant brain tumor. In recent years, increasing reports suggest that discovery of microRNAs (miRNAs) might provide a novel therapeutical target for human cancers, including GBM. The expression and roles of microRNA-183 (miR-183) has been explored in several types of human cancers, including in GBM, and plays important roles in tumor initiation and progression. However, its biological functions in GBM remain largely unknown. In this study, we demonstrated that miR-183 was significantly up-regulated in astrocytoma tissues and glioblastoma cell lines. Introduction of miR-183 mimics into U251 cells could promoted, while its antisense oligos inhibited cell proliferation and invasion. Moreover, we identified neurofilament light polypeptide (NEFL) as a novel target gene of miR-183. The expression levels of NEFL are inversely correlated with that of miR-183 in human astrocytoma clinical specimens. In addition, NEFL-siRNA could significantly attenuate the inhibitory effects of knockdown miR-183 on the proliferation and invasion of U251 cells via mTOR signaling pathway. Overall, This study revealed that miR-183 promotes glioma cell proliferation by targeting NEFL, and also demonstrated that miR-183 could be a potential target for GBM treatment.

Circulating MicroRNA-21 Is a Potential Diagnostic Biomarker in Gastric Cancer.

MicroRNA-21 was upexpressed in gastric cancer (GC) indicating that it is a potential diagnostic biomarker for GC. In this study, 50 GC patients and 50 healthy controls were recruited. miR-21 levels in serum and peripheral blood mononuclear cells (PBMCs) were quantified using quantitative real-time PCR. CA199, and CEA were measured using electrochemiluminescence assay. The sensitivity and specificity of circulating miR-21, CA199 and CEA in GC diagnosis, the correlation of circulating miR-21 to clinicopathological features, and the diagnostic value of miR-21 in different GC stages were determined. The levels of miR-21 in both serum and PBMCs increased significantly in GC patients comparing to healthy controls; however, no correlation was observed between circulating miR-21 level and clinicopathological features. The sensitivity and specificity of miR-21 in serum and PBMCs, and CA199 and CEA in GC diagnosis were 88.4%, 79.6%, 81.3%, 73.4%, 60.5%, 55.9%, and 68.6%, 59.3%, respectively. The positive prediction rates of circulating miR-21 in GC stages I to IV were all around 90%, while those of CA199 and CEA were around or less than 50%. Our data suggest circulating miR-21 (both in serum and in PBMCs) can serve as a good biomarker for GC and could be used in diagnosis of early (stage I) and late GC (stage IV).

Macrophage-secreted Exosomes Delivering miRNA-21 Inhibitor can Regulate BGC-823 Cell Proliferation.

Exosomes, membranous nanovesicles, naturally carry bio-macromolecules or miRNA and play impoetant roles in tumor pathogenesis. Here, we showed that macrophages cell-derived exosomes can function as vehicles to deliver exogenous miR-21 inhibitor into BGC-823 gastric cancer cells. Exosomes loaded with miR-21 inhibitor significantly increased miR-21 levels in BGC-823, but miR-21 inhibitor loaded in exosomes exerted an opposite effect. miRNA transfected with exosomes had less cellular toxicity to host cells compared to conventional transfection methods. The miR-21 inhibitor loaded exosomes promoted the migration ability and reduced apoptosis of BGC-823 gastric cancer cells. These observations indicate that miR-21 acts as a tumor promoter by targeting the PDCD4 gene and preventing apoptosis of gastric cancer cells through inhibition of PDCD4 expression. Furthermore, exosome -mediated miR-21 inhibitor delivery resulted in functionally more efficient inhibition and less cellular toxicity compared to conventional transfection methods. Similar approaches could be useful in modification of target biomolecules in vitro and in vivo. These findings contribute to our understanding of the functions of miR-21 and exosomes as a carrier for therapy of gastric cancer.

NGL-2 Is a New Partner of PAR Complex in Axon Differentiation.

Neuronal polarization is pivotal for neural network formation during brain development. Axon differentiation is a hallmark of initial neuronal polarization. Here, we report that the leucine-rich repeat-containing protein netrin-G ligand-2 (NGL-2) as a polarity regulator that localizes asymmetrically in rat hippocampal neurons and is required for differentiation of the future axon. NGL-2 was associated with PAR complex, and this interaction resulted in local stabilization of axonal microtubules. Further study showed that the C terminal of NGL-2 binds to the PDZ domain of PAR6, and NGL-2 interacts with PAR3 and atypical PKCζ (aPKCζ), with PAR6 acting as a bridge or modifier. Then, NGL-2 regulates the local stabilization of microtubules and promotes axon differentiation by the aPKCζ/microtubule affinity-regulating kinase 2 pathway. These findings reveal the critical role of NGL-2 in regulating axon differentiation in rat hippocampal neurons and reveal a novel partner of the PAR complex.

The fibroblast growth factor-2 arrests Mycobacterium avium sp. paratuberculosis growth and immunomodulates host response in macrophages.

Mycobacterium tuberculosisis (M. tb) epidemic is one of the most severe health problem worldwide, while mechanisms underlying its pathogenesis and host immune responses remain unclear. Mycobacterium avium (M. avium), a mycobacterial species related to M. tb, shares similarities with M. tb in many ways. In this study, using M. avium infection of macrophages as a model, we systematically studied the effect of fibroblast growth factor-2 (FGF-2) on M. avium infection of macrophages. Our results showed that M. avium infection could increase FGF-2 expression on both mRNA and protein levels. M. avium infection elevated TNF-α and IFN-γ production while the addition of FGF-2 could further increase TNF-α but not IFN-γ level. M. avium infection could increase the expression of oxygen/nitrogen metabolism proteins iNOS and SOD-1, and FGF-2 had additive effect on the expression of these two proteins. M. avium infection had inhibitive effect on actin expression while FGF-2 could partly counteract such inhibition. Moreover, FGF-2 could inhibit M. avium proliferation in macrophages. Our results together indicate that macrophage-secreted FGF-2 upon M. avium infection could suppress M. avium proliferation through various ways including cytokine production, enhancement of phagocytosis as well as oxygen/nitrogen metabolism.