in-vitro-biochemical-assays-using-biotin-labels-to-study-protein

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...

Nanjing Medical University

MOV10L1 and its paralog MOV10 are evolutionally conserved RNA helicases involved in distinct RNA regulatory pathways. The testis-specific MOV10L1 is essential for spermatogenesis and PIWI-interacting RNAs biogenesis, whereas MOV10 is ubiquitous and multifunctional. Although both proteins have been implied to correlate with RNA G-quadruplex (RG4) in vivo, their capabilities in binding and resolving RG4 and their respective biological significance remain unclear. Herein, we comprehensively characterize and compare the activities of these two helicases on various nucleic acid substrates in vitro, with a focus on RG4 structure. We find that both MOV10L1 and MOV10 are able to resolve RG4, with MOV10L1 being more efficient in that. In contrast to MOV10, MOV10L1 prefers to bind to a junction between single-stranded RNA and RG4, which is mediated by both its N and C termini. Furthermore, we show that RG4 unwinding by MOV10L1 facilitates the cleavage of this specific RNA structure by an endonuclease.

MOV10L1 Binds RNA G-Quadruplex in a Structure-Specific Manner and Resolves It More Efficiently Than MOV10.

Itaconate plays a prominent role in anti-inflammatory effects and has gradually been ushered as a promising drug candidate for treating inflammatory diseases. However, its significance and underlying mechanism for inflammatory pain remain unexplored. In the current study, we investigated the effects and mechanisms of Dimethyl Itaconate (DI, a derivative of itaconate) on Complete Freund's adjuvant (CFA)-induced inflammatory pain in a rodent model. Here, we demonstrated that DI significantly reduced mechanical allodynia and thermal hyperalgesia. The DI-attenuated neuroinflammation was evident with the amelioration of infiltrative macrophages in peripheral sites of the hind paw and the dorsal root ganglion. Concurrently, DI hindered the central microglia activation in the spinal cord. Mechanistically, DI inhibited the expression of pro-inflammatory factors interleukin (IL)-1β and tumor necrosis factor alpha (TNF-α) and upregulated anti-inflammatory factor IL-10. The analgesic mechanism of DI was related to the downregulation of the nod-like receptor protein 3 (NLRP3) inflammasome complex and IL-1β secretion. This study suggested possible novel evidence for prospective itaconate utilization in the management of inflammatory pain.

Dimethyl Itaconate Attenuates CFA-Induced Inflammatory Pain  the NLRP3/ IL-1β Signaling Pathway.

Neuropathic pain (NP) is a type of chronic pain that is different from the common type of pain. The mechanisms of NP are still poorly understood. Exploring the key genes and neurobiological changes in NP could provide important diagnostic and treatment tools for clinicians. GSE24982 is an mRNA-seq dataset that we downloaded from the Gene Expression Omnibus database to identify key genes in NP. Differentially expressed genes (DEGs) were identified using the BRB-ArrayTools software and R. Functional and pathway enrichment analyses of the DEGs were performed using Metascape. A protein-protein interaction network was created and visualized using Cytoscape. A total of 123 upregulated DEGs were obtained. Among these genes, p53 was the node with the highest degree; hence, we validated it experimentally using a chronic constriction injury mouse model. Our results showed that overexpression of the p53 gene, and the subsequent increase in caspase-3 expression, in dorsal root ganglion neurons led to increased apoptotic changes in these neurons. p53 may therefore be partly responsible for the development of chronic constriction injury-induced NP.

Bioinformatics Analysis Identifies p53 as a Candidate Prognostic Biomarker for Neuropathic Pain.

The aim of this study was to determine the role of spinal adenosine A1 receptors (A1Rs) in the analgesic effects of electroacupuncture (EA) for neuropathic pain.

Role of spinal adenosine A1 receptors in the analgesic effect of electroacupuncture in a rat model of neuropathic pain.

The influence of surgical delay on mortality and morbidity has been studied extensively among elderly hip fracture patients. However, most studies only focus on the timing of surgery when patients have already been hospitalized, without considering pre-admission waiting time. Therefore, the present study aims to explore the influence of admission delay on surgical outcomes.

Admission delay is associated with worse surgical outcomes for elderly hip fracture patients: A retrospective observational study.

Nerve injury-induced gene expression change in the spinal cord is critical for neuropathic pain genesis. RNA N-methyladenosine (mA) modification represents an additional layer of gene regulation. We showed that spinal nerve ligation (SNL) upregulated the expression of matrix metallopeptidase 24 (MMP24) protein, but not  mRNA, in the spinal cord neurons. Blocking the SNL-induced upregulation of spinal MMP24 attenuated local neuron sensitization, neuropathic pain development and maintenance. Conversely, mimicking MMP24 increase promoted the spinal ERK activation and produced evoked nociceptive hypersensitivity. Methylated RNA Immunoprecipitation Sequencing (MeRIP-seq) and RNA Immunoprecipitation (RIP) assay indicated the decreased mA enrichment in the  mRNA under neuropathic pain condition. Moreover, fat-mass and obesity-associated protein (FTO) was colocalized with MMP24 in spinal neurons and shown increased binding to the  mRNA in the spinal cord after SNL. Overexpression or suppression of FTO correlates with promotion or inhibition of MMP24 expression in cultured spinal cord neurons. In conclusion, SNL promoted the mA eraser FTO binding to the  mRNA, which subsequently facilitated the translation of MMP24 in the spinal cord, and ultimately contributed to neuropathic pain genesis.

MMP24 Contributes to Neuropathic Pain in an FTO-Dependent Manner in the Spinal Cord Neurons.

Nerve injury-induced changes of gene expression in dorsal root ganglion (DRG) are critical for neuropathic pain genesis. However, how these changes occur remains elusive. Here we report the down-regulation of zinc finger protein 382 (ZNF382) in injured DRG neurons after nerve injury. Rescuing this down-regulation attenuates nociceptive hypersensitivity. Conversely, mimicking this down-regulation produces neuropathic pain symptoms, which are alleviated by C-X-C motif chemokine 13 (CXCL13) knockdown or its receptor CXCR5 knockout. Mechanistically, an identified cis-acting silencer at distal upstream of the Cxcl13 promoter suppresses Cxcl13 transcription via binding to ZNF382. Blocking this binding or genetically deleting this silencer abolishes the ZNF382 suppression on Cxcl13 transcription and impairs ZNF382-induced antinociception. Moreover, ZNF382 down-regulation disrupts the repressive epigenetic complex containing histone deacetylase 1 and SET domain bifurcated 1 at the silencer-promoter loop, resulting in Cxcl13 transcriptional activation. Thus, ZNF382 down-regulation is required for neuropathic pain likely through silencer-based epigenetic disinhibition of CXCL13, a key neuropathic pain player, in DRG neurons.

ZNF382 controls mouse neuropathic pain via silencer-based epigenetic inhibition of Cxcl13 in DRG neurons.

Opioids are the last option for the pharmacological treatment of neuropathic pain, but their antinociceptive effects are limited. Decreased mu opioid receptor (MOR) expression in the peripheral nervous system may contribute to this. Here, we showed that nerve injury induced hypermethylation of the  gene promoter and an increased expression of methyl-CpG binding protein 2 (MeCP2) in injured dorsal root ganglion (DRG). The downregulation of MOR in the DRG is closely related to the augmentation of MeCP2, an epigenetic repressor, which could recruit HDAC1 and bind to the methylated regions of the  gene promoter. MeCP2 knockdown restored the expression of MOR in injured DRG and enhanced the analgesic effect of morphine, while the mimicking of this increase  the intrathecal infusion of viral vector-mediated MeCP2 was sufficient to reduce MOR in the DRG. Moreover, HDAC1 inhibition with suberoylanilide hydroxamic acid, an HDAC inhibitor, also prevented MOR reduction in the DRG of neuropathic pain mice, contributing to the augmentation of morphine analgesia effects. Mechanistically, upregulated MeCP2 promotes the binding of a high level of HDCA1 to hypermethylated regions of the  gene promoter, reduces the acetylation of histone H3 (acH3) levels of the  gene promoter, and attenuates  transcription in injured DRG. Thus, upregulated MeCP2 and HDAC1 in  gene promoter sites, negatively regulates MOR expression in injured DRG, mitigating the analgesic effect of the opioids. Targeting MeCP2/HDAC1 may thus provide a new solution for improving the therapeutic effect of opioids in a clinical setting.

MeCP2 Epigenetic Silencing of  Gene in Primary Sensory Neurons Under Neuropathic Pain Conditions.

A series of neuropathic pain conditions have a prevalence in older adults potentially associated with declined functioning of the peripheral and/or central nervous system. Neuropathic pain conditions demonstrate defective cortical excitability and intermissions, which raises questions of the impact of pain on cortical excitability changes and when to deliver repetitive transcranial magnetic stimulation (rTMS) to maximize the analgesic effects. Using prolonged continuous theta-burst stimulation (pcTBS), a relatively new rTMS protocol to increase excitability, this study was designed to investigate pcTBS analgesia and cortical excitability in the context of pain. With capsaicin application, twenty-nine healthy participants received pcTBS or Sham stimulation either in the phase of pain initialization (capsaicin applied) or pain ascending (20 min after capsaicin application). Pain intensity was measured with a visual-analogic scale (VAS). Cortical excitability was assessed by motor-evoked potential (MEP) and cortical silent period (CSP) which evaluates corticospinal excitability and GABAergic intracortical inhibition, respectively. Our data on pain dynamics demonstrated that pcTBS produced a consistent analgesic effect regardless of the time frame of pcTBS. More importantly, pcTBS delivered at pain initialization induced a larger pain reduction and a higher response rate compared to the stimulation during pain ascending. We further provide novel findings indicating distinct mechanisms of pcTBS analgesia dependent on the context of pain, in which pcTBS delivered at pain initialization was able to reverse depressed MEP, whereby pcTBS during pain ascending was associated with increased CSP. Overall, our data indicate pcTBS to be a potential protocol in pain management that could be delivered before the initialization of a pain episode to improve rTMS analgesia, potentially through inducing early corticospinal excitability changes that would be suppressed by nociceptive transmission.

Prolonged Continuous Theta Burst Stimulation to Demonstrate a Larger Analgesia as Well as Cortical Excitability Changes Dependent on the Context of a Pain Episode.

Nicotinamide adenine dinucleotide (NAD) is an omnipresent metabolite that participates in redox reactions. Multiple NAD-consuming enzymes are implicated in numerous biological processes, including transcription, signaling, and cell survival. Multiple pieces of evidence have demonstrated that NAD-consuming enzymes, including poly(ADP-ribose) polymerases (PARPs), sirtuins (SIRTs), and sterile alpha and TIR motif-containing 1 (SARM1), play major roles in peripheral neuropathic pain of various etiologies. These NAD consumers primarily participate in peripheral neuropathic pain via mechanisms such as mitochondrial dysfunction, oxidative stress, and inflammation. Furthermore, NAD synthase and nicotinamide phosphoribosyltransferase (NAMPT) have recently been found to contribute to the regulation of pain. Here, we review the evidence indicating the involvement of NAD metabolism in the pathological mechanisms of peripheral neuropathic pain. Advanced understanding of the molecular and cellular mechanisms associated with NAD in peripheral neuropathic pain will facilitate the development of novel treatment options for diverse types of peripheral neuropathic pain.

NAD metabolism in peripheral neuropathic pain.

Preoperative conditions in pediatric liver transplant recipients are understandably complex. Compared with adults, children have lesser compensatory abilities and demand greater precision during procedural executions. In the setting of end-stage liver disease, the heightened perioperative risk of coexistent cardiovascular pathology may impact graft survival as well. Requirements for anesthesia and perioperative management are thus more rigorous, calling for individualized treatments that reflect specific cardiovascular constraints and proposed surgical plans.

Perioperative anesthesia management in pediatric liver transplant recipient with atrial septal defect: A case report.

This study aimed to use machine learning algorithms to build an efficient forecasting model of atrial fibrillation after cardiac surgery, and to compare the predictive performance of machine learning to traditional logistic regression.

Article	Year
MOV10L1 Binds RNA G-Quadruplex in a Structure-Specific Manner and Resolves It More Efficiently Than MOV10. iScience\| PubMed ID: 31252377	2019
Dimethyl Itaconate Attenuates CFA-Induced Inflammatory Pain the NLRP3/ IL-1β Signaling Pathway. Frontiers in pharmacology\| PubMed ID: 35935847	2022
Bioinformatics Analysis Identifies p53 as a Candidate Prognostic Biomarker for Neuropathic Pain. Frontiers in genetics\| PubMed ID: 30233637	2018
Role of spinal adenosine A1 receptors in the analgesic effect of electroacupuncture in a rat model of neuropathic pain. The Journal of international medical research\| PubMed ID: 31868057	2020
Admission delay is associated with worse surgical outcomes for elderly hip fracture patients: A retrospective observational study. World journal of emergency medicine\| PubMed ID: 31893000	2020
MMP24 Contributes to Neuropathic Pain in an FTO-Dependent Manner in the Spinal Cord Neurons. Frontiers in pharmacology\| PubMed ID: 33995105	2021
ZNF382 controls mouse neuropathic pain via silencer-based epigenetic inhibition of Cxcl13 in DRG neurons. The Journal of experimental medicine\| PubMed ID: 34762123	2021
MeCP2 Epigenetic Silencing of Gene in Primary Sensory Neurons Under Neuropathic Pain Conditions. Frontiers in neuroscience\| PubMed ID: 34803588	2021
Prolonged Continuous Theta Burst Stimulation to Demonstrate a Larger Analgesia as Well as Cortical Excitability Changes Dependent on the Context of a Pain Episode. Frontiers in aging neuroscience\| PubMed ID: 35153723	2021
NAD metabolism in peripheral neuropathic pain. Neurochemistry international\| PubMed ID: 36273706	2022
Perioperative anesthesia management in pediatric liver transplant recipient with atrial septal defect: A case report. World journal of clinical cases\| PubMed ID: 36312503	2022
Machine Learning Models of Postoperative Atrial Fibrillation Prediction After Cardiac Surgery. Journal of cardiothoracic and vascular anesthesia\| PubMed ID: 36535840	2023

Lina Yu

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