Directionally non-rotating electric field therapy delivered through implanted electrodes as a glioblastoma treatment platform: A proof-of-principle study.

Background: While directionally rotating tumor-treating fields (TTF) therapy has garnered considerable clinical interest in recent years, there has been comparatively less focus on directionally non-rotating electric field therapy (dnEFT). Methods: We explored dnEFT generated through customized electrodes as a glioblastoma therapy in in vitro and in vivo preclinical models. The effects of dnEFT on tumor apoptosis and microglia/macrophages in the tumor microenvironment were tested using flow-cytometric and qPCR assays. Results: In vitro, dnEFT generated using a clinical-grade spinal cord stimulator showed antineoplastic activity against independent glioblastoma cell lines. In support of the results obtained using the clinical-grade electrode, dnEFT delivered through a customized, 2-electrode array induced glioblastoma apoptosis. To characterize this effect in vivo, a custom-designed 4-electrode array was fabricated such that tumor cells can be implanted into murine cerebrum through a center channel equidistant from the electrodes. After implantation with this array and luciferase-expressing murine GL261 glioblastoma cells, mice were randomized to dnEFT or placebo. Relative to placebo-treated mice, dnEFT reduced tumor growth (measured by bioluminescence) and prolonged survival (median survival gain of 6.5 days). Analysis of brain sections following dnEFT showed a notable increase in the accumulation of peritumoral macrophage/microglia with increased expression of M1 genes (IFNγ, TNFα, and IL-6) and decreased expression of M2 genes (CD206, Arg, and IL-10) relative to placebo-treated tumors. Conclusions: Our results suggest therapeutic potential in glioblastoma for dnEFT delivered through implanted electrodes, supporting the development of a proof-of-principle clinical trial using commercially available deep brain stimulator electrodes.

Machine learning on multiple epigenetic features reveals H3K27Ac as a driver of gene expression prediction across patients with glioblastoma.

Cancer cells show remarkable plasticity and can switch lineages in response to the tumor microenvironment. Cellular plasticity drives invasiveness and metastasis and helps cancer cells to evade therapy by developing resistance to radiation and cytotoxic chemotherapy. Increased understanding of cell fate determination through epigenetic reprogramming is critical to discover how cancer cells achieve transcriptomic and phenotypic plasticity. Glioblastoma is a perfect example of cancer evolution where cells retain an inherent level of plasticity through activation or maintenance of progenitor developmental programs. However, the principles governing epigenetic drivers of cellular plasticity in glioblastoma remain poorly understood. Here, using machine learning (ML) we employ cross-patient prediction of transcript expression using a combination of epigenetic features (ATAC-seq, CTCF ChIP-seq, RNAPII ChIP-seq, H3K27Ac ChIP-seq, and RNA-seq) of glioblastoma stem cells (GSCs). We investigate different ML and deep learning (DL) models for this task and build our final pipeline using XGBoost. The model trained on one patient generalizes to another one suggesting that the epigenetic signals governing gene transcription are consistent across patients even if GSCs can be very different. We demonstrate that H3K27Ac is the epigenetic feature providing the most significant contribution to cross-patient prediction of gene expression. In addition, using H3K27Ac signals from patients-derived GSCs, we can predict gene expression of human neural crest stem cells suggesting a shared developmental epigenetic trajectory between subpopulations of these malignant and benign stem cells. Our cross-patient ML/DL models determine weighted patterns of influence of epigenetic marks on gene expression across patients with glioblastoma and between GSCs and neural crest stem cells. We propose that broader application of this analysis could reshape our view of glioblastoma tumor evolution and inform the design of new epigenetic targeting therapies.

Dopamine pre-treatment impairs the anti-cancer effect of integrated stress response- and TRAIL pathway-inducing ONC201, ONC206 and ONC212 imipridones in pancreatic, colorectal cancer but not DMG cells.

ONC201 (originally discovered as TRAIL-Inducing Compound #10 or TIC10) and analogue ONC206 have been found to induce an integrated stress response with suggested primary targets and mechanisms involving targeting mitochondrial protein ClpP and antagonism of dopamine receptors D2/3 (DRD2/3). We hypothesized that dopamine, the agonist of DRD2, may counteract ONC201 or ONC206 for DRD2/3 and impair the anti-cancer effect of ONC201 or ONC206, thus protect the tumor cells from the cytotoxic effect of ONC201 or ONC206. We therefore pre-treated cancer cells from different tissue origins including breast cancer, pancreatic cancer, colorectal cancer, and diffuse midline glioma (DMG) with dopamine, followed by treatment of ONC201, ONC206 or ONC212. We observed that 48 hours of pre-treatment with dopamine impaired the cell viability suppression effect of ONC201, ONC206 and ONC212 in pancreatic cancer cells and colorectal cancer cells. We pre-treated multiple cancer cell lines with dopamine for one week followed by ONC201, ONC206, or ONC212 treatment and performed colony assays. Pre-treatment with dopamine impaired the anti-cancer effect of ONC201 or ONC206 in pancreatic cancer and colorectal cancer. Impairment of ONC212 effect by pre-treatment with dopamine was also seen in colony assay for colorectal cancer, but not in pancreatic cancer cells by colony assay. No protection from killing by imipridones was observed with DRD2 agonist sumanirole in tumor cells, or with brain tumor cell lines pretreated with dopamine. Immunoblotting was conducted to investigate whether dopamine pre-treatment impacts signaling pathways reported to be affected by ONC201. The dopamine pre-treatment did not impact changes in ATF4, CHOP, DR5 and ClpX which were reported to be affected by ONC201. The mechanism of impairment of ONC201/206/212 effect caused by dopamine pre-treatment appears to involve upregulation of anti-apoptotic p-Bad, XIAP, FLIP and pAkt. Our results shed light on mechanisms of cancer cell protection by dopamine after imipridone treatment, heterogeneity among different tumor cell types, and suggest that effects of dopamine adaptation on tumor cells may impact on cell survival pathways in ways that may or may not depend on expression of dopamine receptors.

Chi3l1 Is a Modulator of Glioma Stem Cell States and a Therapeutic Target in Glioblastoma.

Chitinase 3-like 1 (Chi3l1) is a secreted protein that is highly expressed in glioblastoma. Here, we show that Chi3l1 alters the state of glioma stem cells (GSC) to support tumor growth. Exposure of patient-derived GSCs to Chi3l1 reduced the frequency of CD133+SOX2+ cells and increased the CD44+Chi3l1+ cells. Chi3l1 bound to CD44 and induced phosphorylation and nuclear translocation of ß-catenin, Akt, and STAT3. Single-cell RNA sequencing and RNA velocity following incubation of GSCs with Chi3l1 showed significant changes in GSC state dynamics driving GSCs towards a mesenchymal expression profile and reducing transition probabilities towards terminal cellular states. ATAC-seq revealed that Chi3l1 increases accessibility of promoters containing a Myc-associated zinc finger protein (MAZ) transcription factor footprint. Inhibition of MAZ downregulated a set of genes with high expression in cellular clusters that exhibit significant cell state transitions after treatment with Chi3l1, and MAZ deficiency rescued the Chi3L-induced increase of GSC self-renewal. Finally, targeting Chi3l1 in vivo with a blocking antibody inhibited tumor growth and increased the probability of survival. Overall, this work suggests that Chi3l1 interacts with CD44 on the surface of GSCs to induce Akt/ß-catenin signaling and MAZ transcriptional activity, which in turn upregulates CD44 expression in a pro-mesenchymal feed-forward loop. The role of Chi3l1 in regulating cellular plasticity confers a targetable vulnerability to glioblastoma. SIGNIFICANCE: Chi3l1 is a modulator of glioma stem cell states that can be targeted to promote differentiation and suppress growth of glioblastoma.

The germline factor DDX4 contributes to the chemoresistance of small cell lung cancer cells.

Human cancers often re-express germline factors, yet their mechanistic role in oncogenesis and cancer progression remains unknown. Here we demonstrate that DEAD-box helicase 4 (DDX4), a germline factor and RNA helicase conserved in all multicellular organisms, contributes to increased cell motility and cisplatin-mediated drug resistance in small cell lung cancer (SCLC) cells. Proteomic analysis suggests that DDX4 expression upregulates proteins related to DNA repair and immune/inflammatory response. Consistent with these trends in cell lines, DDX4 depletion compromised in vivo tumor development while its overexpression enhanced tumor growth even after cisplatin treatment in nude mice. Further, the relatively higher DDX4 expression in SCLC patients correlates with decreased survival and shows increased expression of immune/inflammatory response markers. Taken together, we propose that DDX4 increases SCLC cell survival, by increasing the DNA damage and immune response pathways, especially under challenging conditions such as cisplatin treatment.

Novel combination of imipridones and histone deacetylase inhibitors demonstrate cytotoxic effect through integrated stress response in pediatric solid tumors.

There is a demonstrated need for new chemotherapy options in pediatric oncology, as pediatric solid tumors continue to plateau at 60% with event-free survival. Imipridones, a novel class of small molecules, represent a potential new therapeutic option, with promising pre-clinical data and emerging clinical trial data in adult malignancies. ONC201, ONC206, and ONC212 are imipridones showing pro-apoptotic anti-cancer response. Using cell viability assays, and protein immunoblotting, we were able to demonstrate single-agent efficacy of all 3 imipridones inducing cell death in pediatric solid tumor cell lines, including osteosarcoma, malignant peripheral nerve sheath tumors, Ewing sarcoma (EWS), and neuroblastoma. ONC201 displayed IC50 values for non-H3K27M-mutated EWS cell lines ranging from 0.86 µM (SK-N-MC) to 2.76 µM (RD-ES), which were comparable to the range of IC50 values for H3K27M-mutated DIPG cells lines (range 1.06 to 1.56 µM). ONC212 demonstrated the highest potency in single-agent cell killing, followed by ONC206, and ONC201. Additionally, pediatric solid tumor cells were treated with single-agent therapy with histone deacetylase inhibitors (HDACi) vorinostat, entinostat, and panobinostat, showing cell killing with all 3 HDACi drugs, with panobinostat showing the greatest potency. We demonstrate that dual-agent therapy with combinations of imipridones and HDACi lead to synergistic cell killing and apoptosis in all pediatric solid tumor cell lines tested, with ONC212 and panobinostat combinations demonstrating maximal potency. The imipridones induced the integrated stress response with ATF4 and TRAIL receptor upregulation, as well as reduced expression of ClpX. Hyperacetylation of H3K27 was associated with synergistic killing of tumor cells following exposure to imipridone plus HDAC inhibitor therapies. Our results introduce a novel class of small molecules to treat pediatric solid tumors in a precision medicine framework. Use of impridones in pediatric oncology is novel and shows promising pre-clinical efficacy in pediatric solid tumors, including in combination with HDAC inhibitors.

Electric-field facilitated rapid and efficient dissociation of tissues Into viable single cells.

Single-Cell Analysis is a growing field that endeavors to obtain genetic profiles of individual cells. Disruption of cell-cell junctions and digestion of extracellular matrix in tissues requires tissue-specific mechanical and chemical dissociation protocols. Here, a new approach for dissociating tissues into constituent cells is described. Placing a tissue biopsy core within a liquid-filled cavity and applying an electric field between two parallel plate electrodes facilitates rapid dissociation of complex tissues into single cells. Different solution compositions, electric field strengths, and oscillation frequencies are investigated experimentally and with COMSOL Multiphysics. The method is compared with standard chemical and mechanical approaches for tissue dissociation. Treatment of tissue samples at 100 V/cm 1 kHz facilitated dissociation of 95 ± 4% of biopsy tissue sections in as little as 5 min, threefold faster than conventional chemical-mechanical techniques. The approach affords good dissociation of tissues into single cells while preserving cell viability, morphology, and cell cycle progression, suggesting utility for sample preparation of tissue specimens for direct Single-Cell Analysis.

Subventricular zone adult mouse neural stem cells require insulin receptor for self-renewal.

The insulin receptor (INSR) is an evolutionarily conserved signaling protein that regulates development and cellular metabolism. INSR signaling promotes neurogenesis in Drosophila; however, a specific role for the INSR in maintaining adult neural stem cells (NSCs) in mammals has not been investigated. We show that conditionally deleting the Insr gene in adult mouse NSCs reduces subventricular zone NSCs by ∼70% accompanied by a corresponding increase in progenitors. Insr deletion also produced hyposmia caused by aberrant olfactory bulb neurogenesis. Interestingly, hippocampal neurogenesis and hippocampal-dependent behaviors were unperturbed. Highly aggressive proneural and mesenchymal glioblastomas had high INSR/insulin-like growth factor (IGF) pathway gene expression, and isolated glioma stem cells had an aberrantly high ratio of INSR:IGF type 1 receptor. Moreover, INSR knockdown inhibited GBM tumorsphere growth. Altogether, these data demonstrate that the INSR is essential for a subset of normal NSCs, as well as for brain tumor stem cell self-renewal.

Potent preclinical sensitivity to imipridone-based combination therapies in oncohistone H3K27M-mutant diffuse intrinsic pontine glioma is associated with induction of the integrated stress response, TRAIL death receptor DR5, reduced ClpX and apoptosis.

The H3K27M oncohistone mutation, identified in approximately 80% of diffuse intrinsic pontine gliomas (DIPG), is a potential target for therapy. Imipridone ONC201/TIC10 (TRAIL-Inducing Compound #10) induces apoptosis of cancer cells, and has clinical efficacy against H3K27M-mutant DIPG. We demonstrate synergy between ONC201, ONC206 and ONC212, and targeted therapies with known preclinical activity against DIPG. We hypothesized that imipridone combinations with HDAC or proteasome inhibitors may be superior to single agent ONC201 treatment in H3K27M mutant DIPG. Six patient-derived DIPG cell lines (SU-DIPG-IV, SU-DIPG-13, SU-DIPG-25, SU-DIPG-27, SU-DIPG-29, SU-DIPG-36) were exposed to imipridones alone or combinations with histone de-acetylase inhibitors [HDACi], marizomib, etoposide, and temozolomide. Dose-dependent response to imipridones was observed in DIPG cells with half-maximal inhibitory concentration (IC50) of 1.46 µM, 0.11 µM, and 0.03 µM, for ONC201, ONC206, and ONC212, respectively. Upon treatment with the imipridones, DIPG cell lines engaged CLpP/CLPX, the integrated stress response with ATF4 activation, and TRAIL death receptor 5 (DR5) induction. Strong synergy was identified between ONC201 and HDACi panobinostat (combination index [CI] 0.01), romidepsin (CI 0.08) and proteasome inhibitor marizomib (CI 0.19). Synergy was demonstrated between ONC201 and etoposide (CI 0.54), although to a lesser degree than with panobinostat, romidepsin, and marizomib. ONC206 and ONC212 showed similar synergistic effects with panobinostat, romidepsin, and marizomib. Induction of apoptosis was demonstrated with imipridones and panobinostat or romidepsin combinations. Our results suggest increased sensitivity of H3K27M-mutant DIPG cell lines to second generation imipridone therapies, as compared to ONC201. Additionally, there is synergistic cell death with combination of imipridones and panobinostat, romidepsin, or marizomib, which may be further tested in vivo and in clinical trials.

EZH2i EPZ-6438 and HDACi vorinostat synergize with ONC201/TIC10 to activate integrated stress response, DR5, reduce H3K27 methylation, ClpX and promote apoptosis of multiple tumor types including DIPG.

ONC201/TIC10 activates TRAIL signaling through ATF4 and the integrated stress response (ISR). ONC201 demonstrated tumor regressions and disease stability in patients with histone H3K27M-mutated midline-glioma. H3K27M-mutation prevents H3K27-methylation on the mutated allele. EZH2 inhibitors (EZH2i) reduce H3K27 methylation and have anti-tumor effects. We hypothesized ONC201 sensitivity and tumor apoptosis may increase by reducing H3K27-methylation with EZH2i or HDACi as mimics of H3K27M-mutation. EZH2i EPZ-6438 (tazemetostat) or PF-06821497 and HDACi vorinostat were combined with ONC201 to treat multiple cancer cell lines and cell viability and histone modifications were analyzed. We observed synergistic effects towards cell viability in multiple cancers by EPZ-6438 or PF-06821497 plus ONC201 or triple therapy with vorinostat, EPZ-6438, and ONC201. EPZ-6438 and vorinostat synergized with ONC201 to enhance apoptosis. Activation of the ISR and TRAIL-DR5 were observed in cells treated with ONC201 -/+ epigenetic modulators. Knockdown of ATF4 reduced DR5 induction and apoptosis following EZH2i and ONC201 treatment of U251 glioma cells. mRNA expression of dopamine-receptors did not correlate with ONC201 sensitivity in the tumor cell lines tested (N = 12), including changes after epigenetic drugs. Dopamine did not rescue apoptosis by ONC201 in different tumor cell lines (N = 10) including 2 GBM, 3 DIPG and did not prevent DR5 activation or apoptosis. DRD2 agonist sumanirole did not protect brain tumor cells (N = 6 including 4 DIPG cell lines) from ONC201 reduction in viability. Although synergy was observed with ONC201 and vorinostat, there was no significant increase in H3K27 acetylation in cell lines including DIPG as compared to vorinostat alone, and in some cases the acetylation was less than vorinostat alone at 72 H. H3K27 methylation reduction correlated with synergy from combinations of either EPZ-6438 or vorinostat with ONC201 or triple combination. Our findings provide a rationale for combination of ONC201 and epigenetic modulators including triple therapy for in vivo and clinical testing in treatment of human malignancies including brain tumors and DIPG.

Advances in Lipid-Based Nanoparticles for Cancer Chemoimmunotherapy.

Nanomedicines have shown great potential in cancer therapy; in particular, the combination of chemotherapy and immunotherapy (namely chemoimmunotherapy) that is revolutionizing cancer treatment. Currently, most nanomedicines for chemoimmunotherapy are still in preclinical and clinical trials. Lipid-based nanoparticles, the most widely used nanomedicine platform in cancer therapy, is a promising delivery platform for chemoimmunotherapy. In this review, we introduce the commonly used immunotherapy agents and discuss the opportunities for chemoimmunotherapy mediated by lipid-based nanoparticles. We summarize the clinical trials involving lipid-based nanoparticles for chemoimmunotherapy. We also highlight different chemoimmunotherapy strategies based on lipid-based nanoparticles such as liposomes, nanodiscs, and lipid-based hybrid nanoparticles in preclinical research. Finally, we discuss the challenges that have hindered the clinical translation of lipid-based nanoparticles for chemoimmunotherapy, and their future perspectives.

miRNA-mediated loss of m6A increases nascent translation in glioblastoma.

Within the glioblastoma cellular niche, glioma stem cells (GSCs) can give rise to differentiated glioma cells (DGCs) and, when necessary, DGCs can reciprocally give rise to GSCs to maintain the cellular equilibrium necessary for optimal tumor growth. Here, using ribosome profiling, transcriptome and m6A RNA sequencing, we show that GSCs from patients with different subtypes of glioblastoma share a set of transcripts, which exhibit a pattern of m6A loss and increased protein translation during differentiation. The target sequences of a group of miRNAs overlap the canonical RRACH m6A motifs of these transcripts, many of which confer a survival advantage in glioblastoma. Ectopic expression of the RRACH-binding miR-145 induces loss of m6A, formation of FTO/AGO1/ILF3/miR-145 complexes on a clinically relevant tumor suppressor gene (CLIP3) and significant increase in its nascent translation. Inhibition of miR-145 maintains RRACH m6A levels of CLIP3 and inhibits its nascent translation. This study highlights a critical role of miRNAs in assembling complexes for m6A demethylation and induction of protein translation during GSC state transition.

Functions of histone modifications and histone modifiers in Schwann cells.

Schwann cells (SCs) are the main glial cells present in the peripheral nervous system (PNS). Their primary functions are to insulate peripheral axons to protect them from the environment and to enable fast conduction of electric signals along big caliber axons by enwrapping them in a thick myelin sheath rich in lipids. In addition, SCs have the peculiar ability to foster axonal regrowth after a lesion by demyelinating and converting into repair cells that secrete neurotrophic factors and guide axons back to their former target to finally remyelinate regenerated axons. The different steps of SC development and their role in the maintenance of PNS integrity and regeneration after lesion are controlled by various factors among which transcription factors and chromatin-remodeling enzymes hold major functions. In this review, we discussed how histone modifications and histone-modifying enzymes control SC development, maintenance of PNS integrity and response to injury. The functions of histone modifiers as part of chromatin-remodeling complexes are discussed in another review published in the same issue of Glia.

Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures.

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better understand the molecular mechanisms underlying glioma cell migration, an ability to study the interaction between hGCs and axons within the tumor microenvironment is essential. In order to model this cellular interaction, we developed a mixed culture system consisting of hGCs and dorsal root ganglia (DRG) axon-oligodendrocyte co-cultures. DRG cultures were selected because they can be isolated efficiently and can form the long, extensive projections which are ideal for migration studies of this nature. Purified rat oligodendrocytes were then added on purified rat DRG axons and induced to myelinate. After confirming the formation of compact myelin, hGCs were finally added to the co-culture and their interactions with DRG axons and oligodendrocytes was monitored in real-time using time-lapse microscopy. Under these conditions, hGCs form tumor-like aggregate structures that express GFAP and Ki67, migrate along both myelinated and non-myelinated axonal tracks and interact with these axons through the formation of pseudopodia. Our ex vivo co-culture system can be used to identify novel cellular and molecular mechanisms of hGC migration and could potentially be used for in vitro drug efficacy testing.

Determination of confocal profile and curved focal plane for OCT mapping of the attenuation coefficient.

The attenuation coefficient has proven to be a useful tool in numerous biological applications, but accurate calculation is dependent on the characterization of the confocal effect. This study presents a method to precisely determine the confocal effect and its focal plane within a sample by examining the ratio of two optical coherence tomography (OCT) images. The method can be employed to produce a single-value estimate, or a 2D map of the focal plane accounting for the curvature or tilt within the sample. Furthermore, this method is applicable to data obtained with both high numerical aperture (NA) and low-NA lenses, thereby furthering the applicability of the attenuation coefficient to high-NA OCT data. We test and validate this method using standard samples of Intralipid 20% and 5%, improving the accuracy to 99% from 65% compared to the traditional method and preliminarily show applicability to real biological data of glioblastoma acquired in vivo in a murine model.

Regulation of Peripheral Myelination through Transcriptional Buffering of Egr2 by an Antisense Long Non-coding RNA.

Precise regulation of Egr2 transcription is fundamentally important to the control of peripheral myelination. Here, we describe a long non-coding RNA antisense to the promoter of Egr2 (Egr2-AS-RNA). During peripheral nerve injury, the expression of Egr2-AS-RNA is increased and correlates with decreased Egr2 transcript and protein levels. Ectopic expression of Egr2-AS-RNA in dorsal root ganglion (DRG) cultures inhibits the expression of Egr2 mRNA and induces demyelination. In vivo inhibition of Egr2-AS-RNA using oligonucleotide GapMers released from a biodegradable hydrogel following sciatic nerve injury reverts the EGR2-mediated gene expression profile and significantly delays demyelination. Egr2-AS-RNA gradually recruits H3K27ME3, AGO1, AGO2, and EZH2 on the Egr2 promoter following sciatic nerve injury. Furthermore, expression of Egr2-AS-RNA is regulated through ERK1/2 signaling to YY1, while loss of Ser184 of YY1 regulates binding to Egr2-AS-RNA. In conclusion, we describe functional exploration of an antisense long non-coding RNA in peripheral nervous system (PNS) biology.

Nuc-ErbB3 regulates H3K27me3 levels and HMT activity to establish epigenetic repression during peripheral myelination.

Nuc-ErbB3 an alternative transcript from the ErbB3 locus binds to a specific DNA motif and associates with Schwann cell chromatin. Here we generated a nuc-ErbB3 knockin mouse that lacks nuc-ErbB3 expression in the nucleus without affecting the neuregulin-ErbB3 receptor signaling. Nuc-ErbB3 knockin mice exhibit hypermyelination and aberrant myelination at the paranodal region. This phenotype is attributed to de-repression of myelination associated gene transcription following loss of nuc-ErbB3 and histone H3K27me3 promoter occupancy. Nuc-ErbB3 knockin mice exhibit reduced association of H3K27me3 with myelination-associated gene promoters and increased RNA Pol-II rate of transcription of these genes. In addition, nuc-ErbB3 directly regulates levels of H3K27me3 in Schwann cells. Nuc-ErbB3 knockin mice exhibit significant decrease of histone H3K27me3 methyltransferase (HMT) activity and reduced levels of H3K27me3. Collectively, nuc-ErbB3 is a master transcriptional repressor, which regulates HMT activity to establish a repressive chromatin landscape on promoters of genes during peripheral myelination.

Sustained Local Release of Methylprednisolone From a Thiol-Acrylate Poly(Ethylene Glycol) Hydrogel for Treating Chronic Compressive Radicular Pain.

STUDY DESIGN: A preclinical animal model of chronic ligation of the sciatic nerve was used to compare the effectiveness of a slow-release hydrogel carrying methylprednisolone to methylprednisolone injection alone, which simulates the current standard of care for chronic compressive radiculopathy (CR). OBJECTIVE: To extend the short-term benefits of steroid injections by using a nonswelling, biodegradable hydrogel as carrier to locally release methylprednisolone in a regulated and sustained way at the site of nerve compression. SUMMARY OF BACKGROUND DATA: CR affects millions worldwide annually, and is a cause of costly disability with significant societal impact. Currently, a leading nonsurgical therapy involves epidural injection of steroids to temporarily alleviate the pain associated with CR. However, an effective way to extend the short-term effect of steroid treatment to address the chronic component of CR does not exist. METHODS: We induced chronic compression injury of the sciatic nerves of rats by permanent ligation. Forty-eight hours later we injected our methylprednisolone infused hydrogel and assessed the effectiveness of our treatment for 4 weeks. We quantified mechanical hyperalgesia using a Dynamic Plantar Aesthesiometer (Ugo Basile, Stoelting Co., IL, USA), whereas gait analysis was conducted using the Catwalk automated gait analysis platform (Noldus, Leesburg, VA, USA). Macrophage staining was performed with immunohistochemistry and quantification of monocyte chemoattractant protein-1 in sciatic nerve lysates was performed with multiplex immunoassay using a SECTOR Imager 2400A (Meso Scale Discovery, Rockville, MA, USA). RESULTS: We demonstrate that using the hydrogel to deliver methylprednisolone results in significant (P < 0.05) reduction of hyperalgesia and improvement in the gait pattern of animals with chronic lesions as compared with animals treated with steroid alone. In addition, animals treated with hydrogel plus steroid showed significant reduction in the number of infiltrating macrophages at the sciatic nerve and reduced expression of the neuroinflammatory chemokine monocyte chemoattractant protein-1 (P < 0.05). CONCLUSION: Use of hydrogels as carriers for sustained local release of steroids provides significantly better control of pain in an animal model of chronic CR. Our steroid-infused hydrogel could be an effective extender of the short-term benefits of epidural steroid injections for patients with chronic compression-induced radicular pain. LEVEL OF EVIDENCE: N/A.

Prediction of DNA binding motifs from 3D models of transcription factors; identifying TLX3 regulated genes.

Proper cell functioning depends on the precise spatio-temporal expression of its genetic material. Gene expression is controlled to a great extent by sequence-specific transcription factors (TFs). Our current knowledge on where and how TFs bind and associate to regulate gene expression is incomplete. A structure-based computational algorithm (TF2DNA) is developed to identify binding specificities of TFs. The method constructs homology models of TFs bound to DNA and assesses the relative binding affinity for all possible DNA sequences using a knowledge-based potential, after optimization in a molecular mechanics force field. TF2DNA predictions were benchmarked against experimentally determined binding motifs. Success rates range from 45% to 81% and primarily depend on the sequence identity of aligned target sequences and template structures, TF2DNA was used to predict 1321 motifs for 1825 putative human TF proteins, facilitating the reconstruction of most of the human gene regulatory network. As an illustration, the predicted DNA binding site for the poorly characterized T-cell leukemia homeobox 3 (TLX3) TF was confirmed with gel shift assay experiments. TLX3 motif searches in human promoter regions identified a group of genes enriched in functions relating to hematopoiesis, tissue morphology, endocrine system and connective tissue development and function.