Targeting of REST with rationally-designed small molecule compounds exhibits synergetic therapeutic potential in human glioblastoma cells.

BACKGROUND: Glioblastoma (GBM) is an aggressive brain cancer associated with poor prognosis, intrinsic heterogeneity, plasticity, and therapy resistance. In some GBMs, cell proliferation is fueled by a transcriptional regulator, repressor element-1 silencing transcription factor (REST). RESULTS: Using CRISPR/Cas9, we identified GBM cell lines dependent on REST activity. We developed new small molecule inhibitory compounds targeting small C-terminal domain phosphatase 1 (SCP1) to reduce REST protein level and transcriptional activity in glioblastoma cells. Top leads of the series like GR-28 exhibit potent cytotoxicity, reduce REST protein level, and suppress its transcriptional activity. Upon the loss of REST protein, GBM cells can potentially compensate by rewiring fatty acid metabolism, enabling continued proliferation. Combining REST inhibition with the blockade of this compensatory adaptation using long-chain acyl-CoA synthetase inhibitor Triacsin C demonstrated substantial synergetic potential without inducing hepatotoxicity. CONCLUSIONS: Our results highlight the efficacy and selectivity of targeting REST alone or in combination as a therapeutic strategy to combat high-REST GBM.

Tracking Inhibition of Human Small C-Terminal Domain Phosphatase 1 Using 193 nm Ultraviolet Photodissociation Mass Spectrometry.

Working in tandem with kinases via a dynamic interplay of phosphorylation and dephosphorylation of proteins, phosphatases regulate many cellular processes and thus represent compelling therapeutic targets. Here we leverage ultraviolet photodissociation to shed light on the binding characteristics of two covalent phosphatase inhibitors, T65 and rabeprazole, and their respective interactions with the human small C-terminal domain phosphatase 1 (SCP1) and its single-point mutant C181A, in which a nonreactive alanine replaces one key reactive cysteine. Top-down MS/MS analysis is used to localize the binding of T65 and rabeprazole on the two proteins and estimate the relative reactivities of each cysteine residue.

Extraction of Tooth Cusps based on DBSCAN Density Clustering and Neighborhood Search Algorithm.

A good tooth cusp extraction is helpful in evaluating the effect of cosmetic dental work in virtual tooth surgery. We propose a new tooth cusp extraction, which integrates the DBSCAN (density-based spatial clustering of applications with noise) clustering algorithm with the neighborhood search algorithm to extract tooth cusp from a three-dimensional cloud-point tooth model. This method used the point cloud height and curvature to screen out the dented point set. Then we employ the DBSCAN clustering algorithm to segment different feature regions of the tooth surface and generate the candidate point set. Finally, the candidate point set was accurately located at the tooth apex through the neighborhood search algorithm and the traversal search method of non-maximum suppression. The experimental results show that the proposed method is superior to the traditional watershed algorithm-based methods by calculating the recall rate and accuracy rate, and also has higher extraction speed and extraction precision than manual extraction methods.

Targeted Covalent Inhibition of Small CTD Phosphatase 1 to Promote the Degradation of the REST Transcription Factor in Human Cells.

The repressor element-1 silencing transcription factor (REST) represses neuronal gene expression, whose dysregulation is implicated in brain tumors and neurological diseases. A high level of REST protein drives the tumor growth in some glioblastoma cells. While transcription factors like REST are challenging targets for small-molecule inhibitors, the inactivation of a regulatory protein, small CTD phosphatase 1 (SCP1), promotes REST degradation and reduces transcriptional activity. This study rationally designed a series of α,ß-unsaturated sulfones to serve as potent and selective covalent inhibitors against SCP1. The compounds inactivate SCP1 via covalent modification of Cys181 located at the active site entrance. Cellular studies showed that the inhibitors inactivate SCP1 in a time- and dose-dependent manner with an EC50 ∼1.5 µM, reducing REST protein levels and activating specific REST-suppressed genes. These compounds represent a promising line of small-molecule inhibitors as a novel lead for glioblastoma whose growth is driven by REST transcription activity.

Bacterial magnetic particles-polyethylenimine vectors deliver target genes into multiple cell types with a high efficiency and low toxicity.

Bacterial magnetic particles (BMPs) are biosynthesized magnetic nano-scale materials with excellent dispersibility and biomembrane enclosure properties. In this study, we demonstrate that BMPs augment the ability of polyethylenimine (PEI) to deliver target DNA into difficult-to-transfect primary porcine liver cells, with transfection efficiency reaching over 30%. Compared with standard lipofection and polyfection, BMP-PEI gene vectors significantly enhanced the transfection efficiencies for the primary porcine liver cells and C2C12 mouse myoblast cell lines. To better understand the mechanism of magnetofection using BMP-PEI/DNA vectors, transmission electron microscopy (TEM) images of transfected Cos-7, HeLa, and HEP-G2 cells were observed. We found that the BMP-PEI/DNA complexes were trafficked into the cytoplasm and nucleus by way of vesicular transport and endocytosis. Our study builds support for the versatile BMP-PEI vector transfection system, which might be exploited to transfect a wide range of cell types or even to reach specific targets in the treatment of disease. KEY POINTS: • We constructed a BMP-PEI gene delivery vector by combining BMPs and PEI. • The vector significantly enhanced transfection efficiencies in eukaryotic cell lines. • The transfection mechanism of this vector was explained in our study.

Mapping RNAPII CTD Phosphorylation Reveals That the Identity and Modification of Seventh Heptad Residues Direct Tyr1 Phosphorylation.

The C-terminal domain (CTD) of the largest subunit in eukaryotic RNA polymerase II has a repetitive heptad sequence of Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 which is responsible for recruiting transcriptional regulatory factors. The seventh heptad residues in mammals are less conserved and subject to various post-translational modifications, but the consequences of such variations are not well understood. In this study, we use ultraviolet photodissociation mass spectrometry, kinetic assays, and structural analyses to dissect how different residues or modifications at the seventh heptad position alter Tyr1 phosphorylation. We found that negatively charged residues in this position promote phosphorylation of adjacent Tyr1 sites, whereas positively charged residues discriminate against it. Modifications that alter the charges on seventh heptad residues such as arginine citrullination negate such distinctions. Such specificity can be explained by conserved, positively charged pockets near the active sites of ABL1 and its homologues. Our results reveal a novel mechanism for variations or modifications in the seventh heptad position directing subsequent phosphorylation of other CTD sites, which can contribute to the formation of various modification combinations that likely impact transcriptional regulation.

Betaine Ameliorates Experimental Autoimmune Encephalomyelitis by Inhibiting Dendritic Cell-Derived IL-6 Production and Th17 Differentiation.

IL-17-secreting T cells (Th17 cells) play a pathogenic role in multiple autoimmune diseases, including multiple sclerosis (MS), and dendritic cell (DC)-derived cytokines play pivotal roles in promoting the differentiation of naive CD4+ T cells into Th cell subsets (Th1 and Th17). Therefore, small molecules blocking the key cytokines produced by DCs will be beneficial in MS. In this article, we report that betaine treatment ameliorates MS pathogenesis by inhibiting DC-derived IL-6 production and Th17 differentiation. Using experimental autoimmune encephalomyelitis, a widely used mouse model of MS, we found that, compared with the vehicle-treated group, betaine-treated mice exhibited less severe experimental autoimmune encephalomyelitis symptoms, including lower clinical scores, reduced leukocyte infiltration, and less extensive demyelination in the CNS. Moreover, a significantly lower percentage of Th17 cells, one of the major pathogenic effector cells in MS progression, was observed in the peripheral immune system and in the CNS. Interestingly, in the in vitro Th17-differentiation assay, no significant change in Th17 cells was observed between the vehicle- and betaine-treated groups, whereas in the in vitro DC culture experiment, betaine treatment significantly decreased DC-derived IL-6 production. In the DC-T cell coculture experiment, a significantly decreased Th17 differentiation was observed upon betaine treatment. All of these data demonstrated that betaine inhibited Th17 differentiation indirectly by reducing IL-6 production by DCs. In brief, our findings demonstrated the pivotal roles of betaine in modulating MS pathogenesis and suggested that it may serve as a potential novel drug candidate for the treatment of MS.

[Clinical study on pulmonary circulation changes in patients with acute respiratory distress syndrome induced by lung contusion].

OBJECTIVE: To analyze the variation and significance of pulmonary circulation in patients with acute respiratory distress syndrome (ARDS) induced by lung contusion by means of Swan-Ganz catheter and the pulse index continuous cardiac output (PiCCO) monitoring. METHODS: A prospective, randomized, non-blinded clinical trial was conducted. All patients admitted hospital from August 2009 to August 2011 met the inclusion criteria, were divided into the group with ARDS induced by lung contusion (contusion group, n = 18) and the group without lung contusion and ARDS (control group, n = 22). The measured parameters included pulmonary artery systolic pressure (PAS), pulmonary artery diastolic pressure (PAD), pulmonary artery wedge pressure (PAWP), pulmonary artery diastolic-pulmonary wedge pressure gradient (PAD-PAWP), and extravascular lung water index (ELWI) of each group at 0 hour after placing the catheter and at 12, 24, 36, 48, 60, 72 hours after contusion. The differences of all the parameters were compared within a group and among the different groups. RESULTS: In the survivors with ARDS induced by lung contusion, PAS, PAD and PAD-PAWP were significantly higher than those in the control group and then gradually declined. PAS and PAD returned to the level of control group at 60 hours after contusion, and the PAD-PAWP restored to the level of control group at 48 hours after contusion. For the patients in the control group, there were no significant differences in PAS and PAD from the 0 hour after placing catheter to 72 hours after contusion, but PAD-PAWP increased at 72 hours after contusion compared with at 48 hours. Compared with the control group, the ELWI in contusion group increased significantly, and peaked at 12 hours after contusion and then gradually declined, and restored to the level of control group at 60 hours after contusion. For the patients in the control group, ELWI were lower at 60 hours and 72 hours after contusion than at 48 hours. Compared with control group, PAWP in contusion group decreased at 0 hour, and returned to the level of control group at 48 hours after contusion. For the patients in the control group, there were no significant differences in PAWP from the 0 hour after placing the catheter to 72 hours after contusion. The positive correlation were found between ELWI and PAS, PAD, PAD-PAWP from 0 hour after placing the catheter to 48 hours after contusion in contusion group (r value, 0.554, 0.498, 0.629, respectively, all P < 0.01). CONCLUSION: Among the patients with ARDS induced by lung contusion, it appears that changes in PAS, PAD and PAD-PAWP, as well as ELWI play important roles in assessing fluid status, guiding mechanical ventilation and severity.