scholarly journals Long-lasting Analgesia via Targetedin vivoEpigenetic Repression of Nav1.7

2019 ◽  
Author(s):  
Ana M. Moreno ◽  
Glaucilene F. Catroli ◽  
Fernando Alemán ◽  
Andrew Pla ◽  
Sarah A. Woller ◽  
...  
Keyword(s):  
Chronic Pain ◽  
Genome Engineering ◽  
Therapeutic Potential ◽  
Sequence Similarity ◽  
Thermal Hyperalgesia ◽  
Loss Of Function ◽  
High Sequence Similarity ◽  
Inflammatory State

ABSTRACTCurrent treatments for chronic pain rely largely on opioids despite their unwanted side effects and risk of addiction. Genetic studies have identified in humans key targets pivotal to nociceptive processing, with the voltage-gated sodium channel, NaV1.7 (SCN9A), being perhaps the most promising candidate for analgesic drug development. Specifically, a hereditary loss-of-function mutation in NaV1.7 leads to insensitivity to pain without other neurodevelopmental alterations. However, the high sequence similarity between NaVsubtypes has frustrated efforts to develop selective inhibitors. Here, we investigated targeted epigenetic repression of NaV1.7 via genome engineering approaches based on clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9 and zinc finger proteins as a potential treatment for chronic pain. Towards this end, we first optimized the efficiency of NaV1.7 repressionin vitroin Neuro2A cells, and then by the lumbar intrathecal route delivered both genome-engineering platforms via adeno-associated viruses (AAVs) to assess their effects in three mouse models of pain: carrageenan-induced inflammatory pain, paclitaxel-induced neuropathic pain and BzATP-induced pain. Our results demonstrate: one, effective repression of NaV1.7 in lumbar dorsal root ganglia; two, reduced thermal hyperalgesia in the inflammatory state; three, decreased tactile allodynia in the neuropathic state; and four, no changes in normal motor function. We anticipate this genomically scarless and non-addictivepainamelioration approach enablingLong-lastingAnalgesia viaTargetedin vivoEpigeneticRepression of Nav1.7, a methodology we dubpain LATER, will have significant therapeutic potential, such as for preemptive administration in anticipation of a pain stimulus (pre-operatively), or during an established chronic pain state.One sentence summaryIn situepigenome engineering approach for genomically scarless, durable, and non-addictive management of pain.

scholarly journals Long-lasting analgesia via targeted in situ repression of NaV1.7 in mice

2021 ◽  
Vol 13 (584) ◽  
pp. eaay9056 ◽  
Author(s):  
Ana M. Moreno ◽  
Fernando Alemán ◽  
Glaucilene F. Catroli ◽  
Matthew Hunt ◽  
Michael Hu ◽  
...  
Keyword(s):  
Chronic Pain ◽  
Therapeutic Potential ◽  
Thermal Hyperalgesia ◽  
Structural Similarity ◽  
Loss Of Function ◽  
Sensory Afferents ◽  
Inflammatory State ◽  
Epigenetic Repression

Current treatments for chronic pain rely largely on opioids despite their substantial side effects and risk of addiction. Genetic studies have identified in humans key targets pivotal to nociceptive processing. In particular, a hereditary loss-of-function mutation in NaV1.7, a sodium channel protein associated with signaling in nociceptive sensory afferents, leads to insensitivity to pain without other neurodevelopmental alterations. However, the high sequence and structural similarity between NaV subtypes has frustrated efforts to develop selective inhibitors. Here, we investigated targeted epigenetic repression of NaV1.7 in primary afferents via epigenome engineering approaches based on clustered regularly interspaced short palindromic repeats (CRISPR)–dCas9 and zinc finger proteins at the spinal level as a potential treatment for chronic pain. Toward this end, we first optimized the efficiency of NaV1.7 repression in vitro in Neuro2A cells and then, by the lumbar intrathecal route, delivered both epigenome engineering platforms via adeno-associated viruses (AAVs) to assess their effects in three mouse models of pain: carrageenan-induced inflammatory pain, paclitaxel-induced neuropathic pain, and BzATP-induced pain. Our results show effective repression of NaV1.7 in lumbar dorsal root ganglia, reduced thermal hyperalgesia in the inflammatory state, decreased tactile allodynia in the neuropathic state, and no changes in normal motor function in mice. We anticipate that this long-lasting analgesia via targeted in vivo epigenetic repression of NaV1.7 methodology we dub pain LATER, might have therapeutic potential in management of persistent pain states.

scholarly journals Insights into Transcriptional Repression of the Homologous Toxin-Antitoxin Cassettes yefM-yoeB and axe-txe

2020 ◽  
Vol 21 (23) ◽  
pp. 9062
Author(s):  
Barbara Kędzierska ◽  
Katarzyna Potrykus ◽  
Agnieszka Szalewska-Pałasz ◽  
Beata Wodzikowska
Keyword(s):  
Transcriptional Repression ◽  
Transcription Initiation ◽  
Sequence Similarity ◽  
In Vitro Transcription ◽  
High Sequence Similarity ◽  
Sequence Composition ◽  
Repressor Complex ◽  
Transcriptional Fusions

Transcriptional repression is a mechanism which enables effective gene expression switch off. The activity of most of type II toxin-antitoxin (TA) cassettes is controlled in this way. These cassettes undergo negative autoregulation by the TA protein complex which binds to the promoter/operator sequence and blocks transcription initiation of the TA operon. Precise and tight control of this process is vital to avoid uncontrolled expression of the toxin component. Here, we employed a series of in vivo and in vitro experiments to establish the molecular basis for previously observed differences in transcriptional activity and repression levels of the pyy and pat promoters which control expression of two homologous TA systems, YefM-YoeB and Axe-Txe, respectively. Transcriptional fusions of promoters with a lux reporter, together with in vitro transcription, EMSA and footprinting assays revealed that: (1) the different sequence composition of the −35 promoter element is responsible for substantial divergence in strengths of the promoters; (2) variations in repression result from the TA repressor complex acting at different steps in the transcription initiation process; (3) transcription from an additional promoter upstream of pat also contributes to the observed inefficient repression of axe-txe module. This study provides evidence that even closely related TA cassettes with high sequence similarity in the promoter/operator region may employ diverse mechanisms for transcriptional regulation of their genes.

Biological and Therapeutic Potential of Mir-155, 585 and Let-7f in Myeloma in Vitro and In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 833-833
Author(s):  
Sophia Adamia ◽  
Mariateresa Fulciniti ◽  
Herve Avet-Loiseau ◽  
Samir B Amin ◽  
Parantu Shah ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Tumor Size ◽  
Therapeutic Potential ◽  
Mrna Translation ◽  
Biological Role ◽  
Loss Of Function

Abstract Abstract 833 A growing body of evidence suggests that the genome of a many organisms, particularly mammals is controlled not only by transcription factors but also by post-transcriptional programs that are modulated by the family of small RNA molecules including microRNAs (miRs). miRs can block mRNA translation and affect mRNA stability. We have evaluated profiles of 384 human miRs in CD138+ cells from 79 patients with multiple myeloma (MM), 11 MM cell lines and 9 healthy donors (HD) using qRT-PCR based microRNA array. This analysis has identified a MM specific miRNA signature that significantly correlates with OS (p=0.05) and EFS (p=0.017) of patients. Based on this signature one group of patients clustered with HD suggesting indolent disease while other with cell lines indicating aggressive disease. We identified significant modulation of expression of 61 microRNAs in MM cells compared to normal plasma cells. Specific miRs with established oncogenic and tumor suppressor functions such as miR-155, miR-585 and Let7-f were significantly dysregulated in MM (p<0.001). Modulation of miRs-155, -585 and Let7 were observed most frequently in the group of patients with poor OS and EFS suggesting their crucial role in MM. However biological role of these miRs have not yet been defined. To further evaluate biological function of these most recurrent miRs in MM, we evaluated role of miR-155, let-7f and mir-585 in MM cell lines by gain- and loss- of function experiments. We used locked nucleic acid (LNA) anti-miR probes for loss of function and pre-miR-155 for gain of function studies using them alone or in combination. Although manipulation of all 3 miRs induced 20-25% change in MM cell proliferation and/or induction of apoptosis, combination of anti-miR-let7f with pre-miR-155, and anti-miR-585 in combination with miR-155 had dramatic effects on MM cell proliferation and over 60% cells undergoing apoptosis. To evaluate the targets of these miRs, we have determined effects of these anti-miRs and pre-miR on global gene and miR expression profile in MM alone and in combinations. This analysis identified modulation of cluster of miRs as well as genes critical for cell growth and survival. Next, we have tested efficacy of these miRs in vivo in murine Xenograft model to evaluate their therapeutic potential. Tumor-bearing mice were treated intraperitoneal for four consecutively days with the LNA anti-miR-585 and Let-7 and pre-miR-155 probes and respective controls alone and in combination. We observed that the single LNA anti-miR-585 and let 7 and pre miR-155 treatment reduced tumor size by 36%, 31% and 155% in animal 7 days after treatment. However, significant tumor size reductions were achieved when animals were treated with combinations; anti-miR-Let 7f plus pre-miR-155 (58 %); LNA anti-miR-Let 7f plus LNA anti-miR-585 (56 %); LNA-anti-miR-585 plus pre-miR-155 (74 %).We did not observe any significant systemic toxicity in the animals. In conclusion our results suggest significant biological role for miR-585, let 7f and miR-155 in myeloma, both in vitro and in vivo; it highlights for the first time a concerted activity of combination of miRs and holds a great promise for developing novel therapeutic approach for myeloma. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Rapid Generation of Long Noncoding RNA Knockout Mice Using CRISPR/Cas9 Technology

2019 ◽  
Vol 5 (1) ◽  
pp. 12 ◽  
Author(s):  
Nils R. Hansmeier ◽  
Pia J. M. Widdershooven ◽  
Sajjad Khani ◽  
Jan-Wilhelm Kornfeld
Keyword(s):  
Gene Targeting ◽  
Mouse Models ◽  
Noncoding Rna ◽  
Genome Engineering ◽  
Model Organisms ◽  
Specific Gene ◽  
Loss Of Function ◽  
Mouse Lines

In recent years, long noncoding RNAs (lncRNAs) have emerged as multifaceted regulators of gene expression, controlling key developmental and disease pathogenesis processes. However, due to the paucity of lncRNA loss-of-function mouse models, key questions regarding the involvement of lncRNAs in organism homeostasis and (patho)-physiology remain difficult to address experimentally in vivo. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 platform provides a powerful genome-editing tool and has been successfully applied across model organisms to facilitate targeted genetic mutations, including Caenorhabditis elegans, Drosophila melanogaster, Danio rerio and Mus musculus. However, just a few lncRNA-deficient mouse lines have been created using CRISPR/Cas9-mediated genome engineering, presumably due to the need for lncRNA-specific gene targeting strategies considering the absence of open-reading frames in these loci. Here, we describe a step-wise procedure for the generation and validation of lncRNA loss-of-function mouse models using CRISPR/Cas9-mediated genome engineering. In a proof-of-principle approach, we generated mice deficient for the liver-enriched lncRNA Gm15441, which we found downregulated during development of metabolic disease and induced during the feeding/fasting transition. Further, we discuss guidelines for the selection of lncRNA targets and provide protocols for in vitro single guide RNA (sgRNA) validation, assessment of in vivo gene-targeting efficiency and knockout confirmation. The procedure from target selection to validation of lncRNA knockout mouse lines can be completed in 18–20 weeks, of which <10 days hands-on working time is required.

scholarly journals Viral Hormones: Expanding Dimensions in Endocrinology

Endocrinology ◽  
2019 ◽  
Vol 160 (9) ◽  
pp. 2165-2179 ◽  
Author(s):  
Qian Huang ◽  
C Ronald Kahn ◽  
Emrah Altindis
Keyword(s):  
Dna Sequences ◽  
Sequence Similarity ◽  
Endocrine System ◽  
Peptide Hormones ◽  
Rna Sequences ◽  
High Sequence Similarity ◽  
In Vivo Models ◽  
Viral Mimicry

AbstractViruses have developed different mechanisms to manipulate their hosts, including the process of viral mimicry in which viruses express important host proteins. Until recently, examples of viral mimicry were limited to mimics of growth factors and immunomodulatory proteins. Using a comprehensive bioinformatics approach, we have shown that viruses possess the DNA/RNA with potential to encode 16 different peptides with high sequence similarity to human peptide hormones and metabolically important regulatory proteins. We have characterized one of these families, the viral insulin/IGF-1–like peptides (VILPs), which we identified in four members of the Iridoviridae family. VILPs can bind to human insulin and IGF-1 receptors and stimulate classic postreceptor signaling pathways. Moreover, VILPs can stimulate glucose uptake in vitro and in vivo and stimulate DNA synthesis. DNA sequences of some VILP-carrying viruses have been identified in the human enteric virome. In addition to VILPs, sequences with homology to 15 other peptide hormones or cytokines can be identified in viral DNA/RNA sequences, some with a very high identity to hormones. Recent data by others has identified a peptide that resembles and mimics α-melanocyte-stimulating hormone’s anti-inflammatory effects in in vitro and in vivo models. Taken together, these studies reveal novel mechanisms of viral and bacterial pathogenesis in which the microbe can directly target or mimic the host endocrine system. These findings also introduce the concept of a system of microbial hormones that provides new insights into the evolution of peptide hormones, as well as potential new roles of microbial hormones in health and disease.

scholarly journals NPR3 inhibits EMT and Tumour Metastasis of Human Osteosarcoma by Activating AKT/mTOR-mediated Autophagy

2021 ◽  
Author(s):  
Zhihong Yao ◽  
Yihao Yang ◽  
Jiaxiang Chen ◽  
Ting Chen ◽  
Lei Han ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Therapeutic Potential ◽  
Loss Of Function ◽  
Over Expression ◽  
Functional Studies ◽  
Qrt Pcr ◽  
Tumour Metastasis ◽  
Lower Expression

Abstract BackgroundOsteosarcoma is an aggressive primary malignant cancer of bone mainly occurring in adolescence with a characteristic of high metastasis and relapse rate. In our previous study, we first identified that NPR3 was significantly decreased in OS samples. Here, we purposed to investigate the effect and the possible mechanisms of NPR3 on the progression of human OS. MethodsThe expression of NPR3 in OS patients and cells was detected by qRT-PCR, and IHC analysis. The effect of the expression of NPR3 on tumour metastasis was examined in vitro and in vivo. The molecular mechanisms of the regulation of NPR3 were evaluated in vitro and in vivo. The clinical relevance of 5-year overall survival with the expression of NPR3 was evaluated in 294 patients with OS. ResultsFirstly, we indicated that NPR3 was substantially downregulated expression in OS tissues and cells by qRT-PCR and IHC assay. And the patients with lower expression of NPR3 have a poor prognosis. Functional studies revealed that over-expression of NPR3 inhibited the proliferation and invasion of cells. Meanwhile, over-expression of NPR3 markedly inhibited tumorigenesis and weakened tumour metastasis in vivo. Interestingly, we found that over-expression of NPR3 could induce autophagy, promote apoptosis and inhibit EMT. Additionally, overexpression of NPR3 decreased the phosphorylation levels of AKT and mTOR. Loss-of-function experiments displayed that effects of NPR3 were weakened by treatment with the specific autophagy inhibitor Baf-A1 and CQ. ConclusionsTaken together, these results demonstrated that down-regulation of NPR3 promote lung metastasis of human OS by promoting EMT in part through the AKT/mTOR mediated autophagy, suggesting that NPR3 has therapeutic potential for OS patients with metastasis.

scholarly journals Long noncoding RNA FAM225B facilitates proliferation and metastasis of nasopharyngeal carcinoma cells by regulating miR-613/CCND2 axis

2021 ◽  
Author(s):  
Weijun Dai ◽  
Yi Shi ◽  
Weiqi Hu ◽  
Chenjie Xu
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Noncoding Rna ◽  
Function Analysis ◽  
Sequence Similarity ◽  
Migration And Invasion ◽  
Loss Of Function ◽  
Poor Overall Survival ◽  
Proliferation And Metastasis

Growing evidence has suggested that abnormally expressed long non-coding RNAs (lncRNAs) play critical regulatory roles in nasopharyngeal carcinoma (NPC)  pathogenesis. Family with sequence similarity 225 member B (FAM225B) is a novel lncRNA that has been implicated in several human cancers, yet its role in the context of NPC remains largely unclear. The aim of this study was to determine the expression level of FAM225B and its clinical significance in NPC patients. We observed a remarkable increase of FAM225B in NPC tissues and cell lines compared with controls. Also, highly expressed FAM225B was closely correlated with advanced TNM stage, distant metastasis, and poor overall survival. Interestingly, loss-of-function analysis revealed that FAM225B knockdown significantly inhibited tumor growth in vitro and in vivo, and decreased the migratory and invasive capacity of NPC cells. Mechanically, FAM225B functioned as an endogenous sponge by competing for miR-613 binding to up-regulate CCND2 expression. More importantly, rescue experiments further demonstrated that the suppressive impacts of FAM225B knockdown on cell proliferation, migration and invasion were significantly reversed after CCND2 overexpression. Taken all together, these findings highlight FAM225B as an oncogene that promotes NPC proliferation and metastasis through miR-613/CCND2 axis.

scholarly journals Resistance to UV Irradiation Caused by Inactivation ofnurAandherAGenes inThermus thermophilus

2018 ◽  
Vol 200 (16) ◽  
Author(s):  
Yuki Fujii ◽  
Masao Inoue ◽  
Kenji Fukui ◽  
Seiki Kuramitsu ◽  
Ryoji Masui
Keyword(s):  
Homologous Recombination ◽  
Uv Irradiation ◽  
Sequence Similarity ◽  
Thermus Thermophilus ◽  
Cellular Function ◽  
Loss Of Function ◽  
Phenotypic Analysis ◽  
Content Type

ABSTRACTNurA and HerA are thought to be essential proteins for DNA end resection in archaeal homologous recombination systems.Thermus thermophilus, an extremely thermophilic eubacterium, has proteins that exhibit significant sequence similarity to archaeal NurA and HerA. To unveil the cellular function of NurA and HerA inT. thermophilus, we performed phenotypic analysis of disruptant mutants ofnurAandherAwith or without DNA-damaging agents. ThenurAandherAgenes were not essential for survival, and their deletion had no effect on cell growth and genome integrity. Unexpectedly, these disruptants ofT. thermophilusshowed increased resistance to UV irradiation and mitomycin C treatment. Further, these disruptants and the wild type displayed no difference in sensitivity to oxidative stress and a DNA replication inhibitor.T. thermophilusNurA had nuclease activity, and HerA had ATPase. The overexpression of loss-of-function mutants ofnurAandherAin the respective disruptants showed no complementation, suggesting their enzymatic activities were involved in the UV sensitivity. In addition,T. thermophilusNurA and HerA interacted with each otherin vitroandin vivo, forming a complex with 2:6 stoichiometry. These results suggest that the NurA-HerA complex has an architecture similar to that of archaeal counterparts but that it impairs, rather than promotes, the repair of photoproducts and DNA cross-links inT. thermophiluscells. This cellular function is distinctly different from that of archaeal NurA and HerA.IMPORTANCEMany nucleases and helicases are engaged in homologous recombination-mediated DNA repair. Previousin vitroanalyses in archaea indicated that NurA and HerA are the recombination-related nuclease and helicase. However, their cellular function had not been fully understood, especially in bacterial cells. In this study, we performedin vivoanalyses to address the cellular function ofnurAandherAin an extremely thermophilic bacterium,Thermus thermophilus. As a result,T. thermophilusNurA and HerA exhibited an interfering effect on the repair of several instances of DNA damage in the cell, which is in contrast to the results in archaea. This finding will facilitate our understanding of the diverse cellular functions of the recombination-related nucleases and helicases.

A Comparison of the Effect of Decorsin and Two Disintegrins, Albolabrin and Eristostatin, on Platelet Function

1995 ◽  
Vol 74 (05) ◽  
pp. 1316-1322 ◽  
Author(s):  
Mary Ann McLane ◽  
Jagadeesh Gabbeta ◽  
A Koneti Rao ◽  
Lucia Beviglia ◽  
Robert A Lazarus ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Sequence Similarity ◽  
Platelet Aggregation Inhibitors ◽  
Antiplatelet Drug ◽  
Rgd Sequence ◽  
Fibrinogen Receptor ◽  
Activated Platelets

SummaryNaturally-occurring fibrinogen receptor antagonists and platelet aggregation inhibitors that are found in snake venom (disintegrins) and leeches share many common features, including an RGD sequence, high cysteine content, and low molecular weight. There are, however, significant selectivity and potency differences. We compared the effect of three proteins on platelet function: albolabrin, a 7.5 kDa disintegrin, eristostatin, a 5.4 kDa disintegrin in which part of the disintegrin domain is deleted, and decorsin, a 4.5 kDa non-disintegrin derived from the leech Macrobdella decora, which has very little sequence similarity with either disintegrin. Decorsin was about two times less potent than albolabrin and six times less potent than eristostatin in inhibiting ADP- induced human platelet aggregation. It had a different pattern of interaction with glycoprotein IIb/IIIa as compared to the two disintegrins. Decorsin bound with a low affinity to resting platelets (409 nM) and to ADP-activated platelets (270 nM), and with high affinity to thrombin- activated platelets (74 nM). At concentrations up to 685 nM, it did not cause expression of a ligand-induced binding site epitope on the (β3 subunit of the GPIIb/IIIa complex. It did not significantly inhibit isolated GPIIb/IIIa binding to immobilized von Willebrand Factor. At low doses (1.5-3.0 μg/mouse), decorsin protected mice against death from pulmonary thromboembolism, showing an effect similar to eristostatin. This suggested that decorsin is a much more potent inhibitor of platelet aggregation in vivo than in vitro, and it may have potential as an antiplatelet drug.

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