Automatic Electrochemiluminescence Method for the Detection of Cancerous Exosomes Incorporating Specific Aptamer-Magnetic Beads and Signal Nanoprobes.

Exosomes, as an emerging biomarker, have exhibited remarkable promise in early cancer diagnosis. Here, a highly sensitive, selective, and automatic electrochemiluminescence (ECL) method for the detection of cancerous exosomes was developed. Specific aptamer-(EK)4 peptide-tagged magnetic beads (MBs-(EK)4-aptamer) were designed as a magnetic capture probe in which the (EK)4 peptide was used to reduce the steric binding hindrance of cancerous exosomes with a specific aptamer. One new universal ECL signal nanoprobe (CD9 Ab-PEG@SiO2ϵRu(bpy)32+) was designed and synthesized by using microporous SiO2 nanoparticles as the carrier for loading ECL reagent Ru(bpy)32+, polyethylene glycol (PEG) layer, and anticluster of differentiation 9 antibody (CD9 Ab). A "sandwich" biocomplex was formed on the surface of the magnetic capture probe after mixing the capture probe, target exosomes, and ECL signal nanoprobe, and then it was introduced into an automated ECL analyzer for rapid and automatic ECL measurement. It was found that the designed signal nanoprobe shows a 270-fold improvement in the signal-to-noise ratio than that of the ruthenium complex-labeled CD9 antibody signal probe. The relative ECL intensity was proportional to MCF-7 exosomes as a model in the range of 102 to 104 particle/µL, with a detection limit of 11 particle/µL. Furthermore, the ECL method was employed to discriminate cancerous exosomes based on fingerprint responses using the designed multiple magnetic capture probes and the universal ECL signal nanoprobe. This work demonstrates that the utilization of a designed automated ECL tactic using the MBs-(EK)4-aptamer capture probe and the CD9 Ab-PEG@SiO2ϵRu(bpy)32+ signal nanoprobe will provide a unique and robust method for the detection and discrimination of cancerous exosomes.

Small but powerful: RALF peptides in plant adaptive and developmental responses.

Plants live in a highly dynamic environment and require to rapidly respond to a plethora of environmental stimuli, so that to maintain their optimal growth and development. A small plant peptide, rapid alkalization factor (RALF), can rapidly increase the pH value of the extracellular matrix in plant cells. RALFs always function with its corresponding receptors. Mechanistically, effective amount of RALF is induced and released at the critical period of plant growth and development or under different external environmental factors. Recent studies also highlighted the role of RALF peptides as important regulators in plant intercellular communications, as well as their operation in signal perception and as ligands for different receptor kinases on the surface of the plasma membrane, to integrate various environmental cues. In this context, understanding the fine-print of above processes may be essential to solve the problems of crop adaptation to various harsh environments under current climate trends scenarios, by genetic means. This paper summarizes the current knowledge about the structure and diversity of RALF peptides and their roles in plant development and response to stresses, highlighting unanswered questions and problems to be solved.

[miR-185-5p alleviates the inflammatory response of acute gouty arthritis by inhibiting of IL-1ß].

Objective To investigate the relationship between interleukin-1ß (IL-1ß) and miR-185-5p in the process of joint injury in acute gouty arthritis (AGA). Methods The serum miR-185-5p levels of 89 AGA patients and 91 healthy volunteers were detected by real-time quantitative PCR. The correlation between miR-185-5p expression level and VAS score or IL-1ß expression level was evaluated by Pearson correlation coefficient method. Receiver operating characteristic (ROC) curve was used to evaluate the diagnostic value of miR-185-5p in AGA. THP-1 cells were induced by sodium urate (MSU) to construct an in vitro acute gouty inflammatory cell model. After the expression level of miR-185-5p in THP-1 cells was upregulated or downregulated by transfection of miR-185-5p mimics or inhibitors in vitro, inflammatory cytokines of THP-1 cells, such as IL-1ß, IL-8 and tumor necrosis factor α (TNF-α), were detected by ELISA. The luciferase reporter gene assay was used to determine the interaction between miR-185-5p and the 3'-UTR of IL-1ß. Results Compared with the healthy control group, the expression level of serum miR-185-5p in AGA patients was significantly reduced. The level of serum miR-185-5p was negatively correlated with VAS score and IL-1ß expression level. The area under the curve (AUC) was 0.905, the sensitivity was 80.17% and the specificity was 83.52%. Down-regulation of miR-185-5p significantly promoted the expression of IL-1ß, IL-8 and tumor necrosis factor (TNF-α), while overexpression of miR-185-5p showed the opposite results. Luciferase reporter gene assay showed that IL-1ß was the target gene of miR-185-5p, and miR-185-5p negatively regulated the expression of IL-1ß. Conclusion miR-185-5p alleviates the inflammatory response in AGA by inhibiting IL-1ß.

ST3GAL3 Promotes the Inflammatory Response of Fibroblast-Like Synoviocytes in Rheumatoid Arthritis by Activating the TLR9/MyD88 Pathway.

This study is aimed at investigating the role of ß-galactoside-α2,3-sialyltransferase III (ST3GAL3) in fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA), as well as its potential mechanism of action. The Gene Expression Omnibus (GEO) database and gene set enrichment analysis (GSEA) were used to analyse the expression of ST3GAL3 and the enrichment signalling pathways associated with ST3GAL3 in RA. The effects of ST3GAL3 on tumour necrosis factor- (TNF-) α and interleukin- (IL-) 1ß-treated MH7A cells were determined using methyl thiazolyl tetrazolium (MTT), transwell, and enzyme-linked immunosorbent assays (ELISA). The expression of proliferation-associated proteins and Toll-like receptor (TLR) pathway-enriched proteins was analysed using western blotting. As a main result, ST3GAL3 was screened as an overlapping upregulated gene from GSE101193 and GSE94519 datasets. ST3GAL3 expression in MH7A cells significantly increased with increasing treatment time with TNF-α or IL-1ß. TLR9/myeloid differentiation primary response protein 88 (MyD88) is a downstream activation pathway of ST3GAL3. ST3GAL3 overexpression promoted MH7A cell proliferation and migration. Additionally, ST3GAL3 overexpression upregulated the expression of proliferation-associated proteins (cyclinD, cyclinE, and proliferating cell nuclear antigen) and TLR pathway enrichment factors (TLR9 and MyD88) and increased the production of matrix metallopeptidase (MMP) 1, MMP3, interleukin- (IL-) 6, and IL-8, whereas si-ST3GAL3 had the opposite effect. The addition of TLR9 agonists (CpG 2216 and CpG 2006) reversed the effects of si-ST3GAL3 on MH7A cell proliferation, migration, and inflammation. TLR9-specific siRNA reversed the effects of ST3GAL3 overexpression on MH7A cell proliferation, migration, and inflammation. In conclusion, ST3GAL3 is likely involved in RA pathogenesis by activating the TLR9/MyD88 pathway.

Treatment with Humanized Selective CD19CAR-T Cells Shows Efficacy in Highly Treated B-ALL Patients Who Have Relapsed after Receiving Murine-Based CD19CAR-T Therapies.

PURPOSE: CD19 chimeric antigen receptor (CAR)-T therapy has shown impactful results in treatment of B-cell malignancies. However, immune recognition of the murine scFv may render subsequent infusion(s) ineffective. Also, nonselective expansion of both CAR-transduced and nontransduced T cells during the production stage affects the yield and purity of final products. Here, we aim to develop a humanized selective (hs) CD19 CAR to solve the above problems.Experimental Design: A CD19 hsCAR was designed, which incorporated a short selective domain between the humanized heavy chain and light chain. The CAR was examined for its property, and then trialed in 5 highly treated B-ALL patients. RESULTS: hsCAR possessed around 6-fold higher affinity to CD19 versus murine CAR (mCAR). Incubation with selective domain-specific mAbs (SmAb) selectively expanded CAR-transduced T cells, and led to a higher proportion of central memory T cells in the final products. SmAb-stimulated CD19 hsCAR-T cells exhibited superior antitumor cytotoxic functions in vitro and in vivo. Autologous (n = 2) and allogeneic donor (n = 3, with hematopoietic stem cell transplantation) hsCAR-T cells were infused into 5 patients who had relapsed after receiving mCAR-T treatments. Two patients received mCAR-T treatments twice previously but the second treatments were ineffective. In contrast, subsequent hsCAR-T treatments proved effective in all 5 patients and achieved complete molecular remission in four, including one with extramedullary disease with central nervous system involvement. CONCLUSIONS: hsCD19 CAR-T treatment shows efficacy in highly treated B-ALL patients who have relapsed after receiving CD19 mCAR-T therapies.

Long noncoding RNA profiling revealed differentially expressed lncRNAs associated with disease activity in PBMCs from patients with rheumatoid arthritis.

Long noncoding RNAs (lncRNAs) have recently emerged as important biological regulators, and the aberrant expression of lncRNAs has been reported in numerous diseases. However, the expression of lncRNAs in peripheral blood mononuclear cells (PBMCs) in rheumatoid arthritis (RA) has not been well documented. We applied a microarray analysis to profile the lncRNA and mRNA expression in 3 pairs of samples. Each sample was mixed with equivalent PBMCs from 9 female RA patients and 9 corresponding healthy controls, and the data were validated via qPCR using another cohort that comprised 36 RA patients and 24 healthy controls. A bioinformatic analysis was performed to investigate the potential functions of differentially expressed genes. Overall, 2,099 lncRNAs and 2,307 mRNAs were differentially expressed between the RA patients and healthy controls. The bioinformatic analysis indicated that the differentially expressed lncRNAs regulated the abnormally expressed mRNAs, which were involved in the pathogenesis of RA through several different pathways. The qPCR results showed that the expression levels of ENST00000456270 and NR_002838 were significantly increased in the RA patients, whereas the expression levels of NR_026812 and uc001zwf.1 were significantly decreased. Furthermore, the expression level of ENST00000456270 was strongly associated with the serum levels of IL-6 and TNF-a and the Simplified Disease Activity Index (SDAI) of the RA patients. Our data provided comprehensive evidence regarding the differential expression of lncRNAs in PBMCs of RA patients, which shed light on the understanding of the molecular mechanisms of lncRNAs in the pathogenesis of RA.

Elevated expression of microRNA-873 facilitates Th17 differentiation by targeting forkhead box O1 (Foxo1) in the pathogenesis of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease, and its pathogenesis remains mostly unknown. MicroRNAs (miRs) has drawn much attention as a crucial regulator of autoimmune diseases. In this study, we demonstrated that miR-873 expression was significantly up-regulated in patients with SLE, and its expression was positively associated with the disease severity. CD4+ T cells, especially the Th17 subset, were found to be the major source of miR-873 expression. Using gain- and loss-of-function approaches, we further showed that miR-873 could facilitate the differentiation of CD4+ T cells into Th17 lineage. Moreover, forkhead box O1 (Foxo1), one member of the Foxo family, was identified as a novel target gene of miR-873, and Foxo1 has been known as an inhibitor of Th17 cell differentiation. Foxo1 was observed to be markedly decreased in PBMC of patients with SLE. Notably, in vivo lentivirus-mediated inhibition of miR-873 significantly alleviated the disease severity of spontaneous SLE in MRL/lpr mice, with down-regulated levels of autoantibodies, proteinuria, and IL-17A. Our data reveal a novel mechanism in which the elevated miR-873 in PBMC of SLE promotes Th17 cell differentiation through down-regulation of Foxo1. In vivo blockade of miR-873 may serve as a novel therapeutic approach in the treatment of SLE.

Functionally defined therapeutic targets in diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG) is a fatal childhood cancer. We performed a chemical screen in patient-derived DIPG cultures along with RNA-seq analyses and integrated computational modeling to identify potentially effective therapeutic strategies. The multi-histone deacetylase inhibitor panobinostat demonstrated therapeutic efficacy both in vitro and in DIPG orthotopic xenograft models. Combination testing of panobinostat and the histone demethylase inhibitor GSK-J4 revealed that the two had synergistic effects. Together, these data suggest a promising therapeutic strategy for DIPG.

Numblike and Numb differentially affect p53 and Sonic Hedgehog signaling.

Numb serves as a key regulator of Notch and Sonic Hedgehog signaling and also modulates p53 protein levels. Numblike is a highly conserved homolog to mammalian Numb, but considerably less is known about its function. To address the role of Numblike, we have generated a mouse embryonic stem (ES) cell line, Nbl(TetOn), in which expression of Numblike can be induced and analyzed the effect of activation of Numblike. Induction of Numblike, similar to Numb, reduced the amount of Notch receptor, whereas Numblike differed from Numb with regard to p53 and Shh signaling. In contrast to Numb, Numblike did not elevate the level of p53 protein and Numblike potentiated, rather than reduced, the immediate downstream response of Shh signaling. In keeping with a role for Numblike in potentiating Shh signaling, Shh and Numblike synergistically increased the proportion of ES cells expressing pluripotency markers. In conclusion, the data demonstrate that Numb and Numblike have evolved to acquire at least partially distinct functions.

Notch signaling induces SKP2 expression and promotes reduction of p27Kip1 in T-cell acute lymphoblastic leukemia cell lines.

In T-cell acute lymphoblastic leukemia (T-ALL) NOTCH 1 receptors are frequently mutated. This leads to aberrantly high Notch signaling, but how this translates into deregulated cell cycle control and the transformed cell type is poorly understood. In this report, we analyze downstream responses resulting from the high level of NOTCH 1 signaling in T-ALL. Notch activity, measured immediately downstream of the NOTCH 1 receptor, is high, but expression of the canonical downstream Notch response genes HES 1 and HEY 2 is low both in primary cells from T-ALL patients and in T-ALL cell lines. This suggests that other immediate Notch downstream genes are activated, and we found that Notch signaling controls the levels of expression of the E3 ubiquitin ligase SKP2 and its target protein p27Kip1. We show that in T-ALL cell lines, recruitment of NOTCH 1 intracellular domain (ICD) to the SKP2 promoter was accompanied by high SKP2 and low p27Kip1 protein levels. In contrast, pharmacologically blocking Notch signaling reversed this situation and led to loss of NOTCH 1 ICD occupancy of the SKP2 promoter, decreased SKP2 and increased p27Kip1 expression. T-ALL cells show a rapid G1-S cell cycle transition, while blocked Notch signaling resulted in G0/G1 cell cycle arrest, also observed by transfection of p27Kip1 or, to a smaller extent, a dominant negative SKP2 allele. Collectively, our data suggest that the aberrantly high Notch signaling in T-ALL maintains SKP2 at a high level and reduces p27Kip1, leading to more rapid cell cycle progression.

High levels of Notch signaling down-regulate Numb and Numblike.

Inhibition of Notch signaling by Numb is critical for many cell fate decisions. In this study, we demonstrate a more complex relationship between Notch and the two vertebrate Numb homologues Numb and Numblike. Although Numb and Numblike at low levels of Notch signaling negatively regulated Notch, high levels of Notch signaling conversely led to a reduction of Numb and Numblike protein levels in cultured cells and in the developing chick central nervous system. The Notch intracellular domain but not the canonical Notch downstream proteins Hes 1 and Hey 1 caused a reduction of Numb and Numblike. The Notch-mediated reduction of Numblike required the PEST domain in the Numblike protein and was blocked by the proteasome inhibitor MG132. Collectively, these observations reveal a reciprocal negative regulation between Notch and Numb/Numblike, which may be of relevance for stabilizing asymmetric cell fate switches and for tumor development.

Aromatase-deficient mice spontaneously develop a lymphoproliferative autoimmune disease resembling Sjogren's syndrome.

Sjögren's syndrome (SS) is an incurable, autoimmune exocrinopathy that predominantly affects females and whose pathogenesis remains unknown. Like rheumatoid arthritis, its severity increases after menopause, and estrogen deficiency has been implicated. We have reported that estrogen receptor-alpha and -beta-knockout mice develop autoimmune nephritis and myeloid leukemia, respectively, but neither develops SS. One model of estrogen deficiency in rodents is the aromatase-knockout (ArKO) mouse. In these animals, there is elevated B lymphopoiesis in bone marrow. We now report that ArKO mice develop severe autoimmune exocrinopathy resembling SS. By 1 year of age, there is B cell hyperplasia in the bone marrow, spleen, and blood of ArKO mice and spontaneous autoimmune manifestations such as proteinuria and severe leukocyte infiltration in the salivary glands and kidney. Also, as is typically found in human SS, there were proteolytic fragments of alpha-fodrin in the salivary glands and anti-alpha-fodrin antibodies in the serum of both female and male ArKO mice. When mice were raised on a phytoestrogen-free diet, there was a mild but significant incidence of infiltration of B lymphocytes in WT mice and severe destructive autoimmune lesions in ArKO mice. In age-matched WT mice fed a diet containing normal levels of phytoestrogen, there were no autoimmune lesions. These results reveal that estrogen deficiency results in a lymphoproliferative autoimmune disease resembling SS and suggest that estrogen might have clinical value in the prevention or treatment of this disease.