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1.
Biomed Res Int ; 2020: 4351671, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32025520

RESUMO

Cervical cancer is one of the malignant tumors that seriously threaten women's health. The mechanism of development needs to be deeply studied. In recent years, lncRNA has been identified as one of the important factors affecting the malignant progression of tumors. In this study, we illustrated the important mechanism of lncRNA CAR10 in the development of cervical cancer. We found that CAR10 is significantly increased in4 cervical cancer tissues and cells, which can promote the proliferation of cervical cancer cells in vitro and in vivo, indicating that CAR10 is involved in the progression of cervical cancer as an oncogene. Further studies showed that CAR10 is a target gene of miR-125b-5p, and miR-125b-5p can inhibit the effect of CAR10 on the proliferation of cervical cancer cells. In addition, we also found that 3-phosphoinositide-dependent protein kinase 1 (PDPK1) is also a target gene of miR-125b-5p, and CAR10 can upregulate the expression level of PDPK1. The results showed that CAR10 acts as a ceRNA to upregulate the expression of PDPK1 by sponging miR-125b-5p. Knockdown of PDPK1 can inhibit the effect of CAR10 on cervical cancer cells. Our study demonstrates that, based on ceRNA mechanism, CAR10/miR-125b-5p/PDPK1 network can regulate the proliferation of cervical cancer cells and play an important role in the development of cervical cancer. In addition, our study also suggests that intervention of CAR10/miR-125b-5p/PDPK1 network may be a new strategy for targeted therapy of cervical cancer.

2.
Stem Cells ; 2020 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-32052915

RESUMO

The intricate balance of neural stem cell (NSC) amplification and neurogenesis is central to nervous system development. Dopamine D1 receptor (DRD1) is a typical G protein-coupled receptor (GPCR) mainly expressed in neurogenic area, with high constitutive activity. The receptor appears in the embryonic period before the formation of mature synaptic contacts, which indicates that dopamine receptor and its constitutive activity play crucial roles in the embryonic brain development. Here, we found that DRD1 was enriched in human NSCs. Inhibition of the receptor activity by its inverse agonists promoted human NSCs proliferation and impeded its differentiation. These results were also mimicked by genetic knockdown of DRD1, which also blocked the effects of inverse agonists, suggesting a receptor-dependent manner. More interestingly, knock-in A229T mutant with reduced DRD1 constitutive activity by CRISPR-Cas9 genome editing technology resulted into increased endogenous human NSCs proliferation. These results were well reproduced in human cerebral organoids, and inhibition of the DRD1 constitutive activity by its inverse agonists induced the expansion and folding of human cerebral organoids. The anatomic analysis uncovered that decreasing the constitutive activity of DRD1 by its inverse agonists promoted the NSCs proliferation and maintenance that led to hindered cortical neurogenesis. Further mechanistic studies revealed that the PKC-CBP pathway was involved in the regulation by DRD1. Thus, our findings indicate that the constitutive activity of DRD1 and possibly other GPCRs plays an important role in the development of human nervous system.

3.
Nat Commun ; 11(1): 923, 2020 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-32066723

RESUMO

The precise circuit of the substantia nigra pars reticulata (SNr) involved in temporal lobe epilepsy (TLE) is still unclear. Here we found that optogenetic or chemogenetic activation of SNr parvalbumin+ (PV) GABAergic neurons amplifies seizure activities in kindling- and kainic acid-induced TLE models, whereas selective inhibition of these neurons alleviates seizure activities. The severity of seizures is bidirectionally regulated by optogenetic manipulation of SNr PV fibers projecting to the parafascicular nucleus (PF). Electrophysiology combined with rabies virus-assisted circuit mapping shows that SNr PV neurons directly project to and functionally inhibit posterior PF GABAergic neurons. Activity of these neurons also regulates seizure activity. Collectively, our results reveal that a long-range SNr-PF disinhibitory circuit participates in regulating seizure in TLE and inactivation of this circuit can alleviate severity of epileptic seizures. These findings provide a better understanding of pathological changes from a circuit perspective and suggest a possibility to precisely control epilepsy.

4.
Anal Bioanal Chem ; 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-32076791

RESUMO

The activity of proteins rather than the concentration of proteins in biopharmaceutical and in vitro diagnostics are often the primary focus. Nonetheless, development of a calibration-free concentration analysis (CFCA) approach that accurately quantifies the concentration of proteins based on molecular interactions with specific monoclonal antibodies and without the requirement of external calibrators would be beneficial to diagnostics. Generally, only analytes that interact with the antibody (Ab) are quantified by CFCA. Moreover, protein concentrations measured by CFCA usually vary when different Abs are used, and are lower than those obtained by amino acid analysis because any non-native state population of the target protein is not captured by the Ab. To achieve comparable results between CFCA and traditional amino acid analysis (AAA), an Ab that recognizes the target protein irrespective of its conformation should be used. In this report, three different monoclonal antibodies were used to quantify purified human myoglobin in solution by CFCA. The concentrations obtain by the Abs (i.e., 2.985, 2.912, 3.032 mg mL-1) were comparable with that obtained by AAA. Moreover, isotope dilution mass spectrometry (IDMS) gave a human myoglobin concentration of 2.851 mg mL-1, which is also in agreement with the results from CFCA. The performance of CFCA was evaluated by measuring various parameters, including within-day and between-day precision. The results demonstrated that the active concentration measured by CFCA is comparable with that of IDMS when the appropriate Ab is used. Recommended procedures for performing the new CFCA approach are provided. This study shows that CFCA represents a primary method for accurate protein concentration determination, which should aid the development of certified reference materials. Graphical abstract.

5.
Toxicology ; 431: 152366, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31926187

RESUMO

Kidney injury is a major adverse effect of cisplatin use. Metabolomics has been used to characterize physiological or pathological conditions through identification of metabolites and characterization of the metabolic pathway. Metabolomics profiling could allow for identification of nephrotoxic mechanisms of cisplatin and identification of biomarkers of cisplatin-induced injury. In this study, we performed metabolomics analysis to characterize key changes in metabolite levels during cisplatin-induced acute kidney injury (AKI) in rats, and screened for sensitive biomarkers for early diagnosis using HPLC-TOF/MS. Rats were intraperitoneally injected with 7.5 mg/kg or 15 mg/kg of cisplatin, or normal saline, and 12 h urine and kidney samples were collected after 72 h. Serum biochemical parameters and kidney histological evaluations showed dose-dependent AKI in response to cisplatin. Metabolomics analysis showed that 37 and 35 endogenous metabolite levels changed in rat urine and kidneys, respectively. Seven key metabolic pathways were disrupted, including the tricarboxylic acid cycle (TCA cycle), phenylalanine, tyrosine, and tryptophan biosynthesis, phenylalanine metabolism, glycerophospholipid metabolism, taurine and hypotaurine metabolism, d-glutamine and d-glutamate metabolism, and nicotinate and nicotinamide metabolism. These pathways are involved in energy generation, and amino acid and lipid metabolism, and disruption of these pathways could contribute to oxidative stress injury, inflammation, and cell membrane damage. Furthermore, 11 sensitive metabolites in urine were screened as potential biomarkers of AKI. To validate these biomarkers, we quantified 4 off these biomarkers, and confirmed that levels of these metabolites were altered in urine of rats treated with CDDP.

6.
J Mater Chem B ; 8(7): 1445-1455, 2020 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-31993613

RESUMO

The tumor microenvironment (TME), which is characterised by high H2O2 and glutathione (GSH) levels, low pH value and hypoxia, imposes crucial influences on tumor therapeutic outcomes. Rational design and preparation of nanomaterial systems that are responsive to the intrinsic properties of the TME open a promising avenue towards tumor-specific treatment. Herein, CoMn-layered double hydroxide (CoMn-LDH) nanosheets were synthesized via a bottom-up method followed by surface modification with a photosensitizer, chlorin e6 (Ce6), which exhibited TME-responsive imaging as well as photodynamic and chemodynamic synergistic therapy (PDT/CDT). Due to their ultralow bond energy and large adsorption energy, CoMn-LDH nanosheets show fast self-degradability in a GSH (10 mM) microenvironment, giving an excellent CDT activity in mildly acidic conditions (pH = 6.5), superior GSH removal ability (99.82%) and O2 production (35.37 µg L-1 s-1). Moreover, Ce6/CoMn-LDH nanosheets display satisfactory photoacoustic (PA) imaging and GSH-enhanced magnetic resonance imaging (MRI) with a 45.1-fold T1-enhancement. In addition, both in vitro and in vivo therapeutic tests based on Ce6/CoMn-LDH demonstrate a satisfactory anticancer activity with complete cancer cell apoptosis and dramatic tumor elimination. This work provides a new perspective for the design of multifunctional 2D nanosheets towards a fully promoted TME-responsive synergistic therapy, which holds great promise for future clinical diagnosis and treatment.

7.
Artigo em Inglês | MEDLINE | ID: mdl-31934690

RESUMO

Diffusive gradients in thin films (DGT) have gained wide attention for in situ measurement of reactive phosphorus species (PO4) in natural water, sediments and potentially soils. In this study, a novel Mg(OH)2 binding gel was formed using magnesium hydroxide obtained by in situ hydration of calcined magnesium oxide. Laboratory scale experiments showed that the novel Mg(OH)2 gel had a homogeneous dispersion of fine particles of Mg(OH)2 with a particle size of 2-5 µm. With 10 mL of 2.0 mol L-1 NaOH as the eluting agent, the optimal elution efficiency of PO4 on the Mg(OH)2 gel was 72 ± 5%. There were linear relationships between the accumulated PO4 mass and the applied PO4 concentration (0.1 to 20 mg P per L), time (0 to 24 h) and temperature (22 to 40 °C). The capacity of the Mg(OH)2 binding layer was determined to be 99.5 µg P per disc. Tests in synthetic seawater, Chaohu Lake and Yihai Pond confirmed that Mg(OH)2-DGT was able to accurately measure phosphorus up to 10 days. This was indicated by the good agreements between the concentrations measured by DGT (CDGT) technology and by an ex situ chemical method in solution (Csoln), with a CDGT/Csoln ratio between 0.91 and 1.09.

8.
Aging (Albany NY) ; 12(1): 481-501, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31901901

RESUMO

The accumulation of amyloid-ß (Aß), considered as the major cause of Alzheimer's disease (AD) pathogenesis, relays on the rate of its biosynthesis and degradation. Aß degradation is a common overture to late-onset AD and targeting the impairment of Aß degradation has gained attention in the recent years. In this study, we demonstrated a rhamnoside derivative PL402 suppressed Aß level in cell models without changing the expression or activity of Aß generation-related secretases. However, the levels of matrix metalloproteinase (MMP) 3 and 9, belonging to amyloid-degrading enzymes (ADEs), were up-regulated by PL402. The inhibition or the knockdown of these two enzymes abolished the effect of PL402, indicating that PL402 may reduce Aß via MMP3/9-mediated Aß degradation. Notably, administration of PL402 significantly attenuated Aß pathology and cognitive defects in APP/PS1 transgenic mice with the consistent promotion of ADEs expression. Thus, our study suggests that targeting Aß degradation could be an effective strategy against AD and the rhamnoside derivatives may have therapeutic effects.

9.
Clin Biochem ; 76: 11-16, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31765637

RESUMO

OBJECTIVE: The clinical and hematologic features of thalassemia are due to different factors, and patients with identical genotypes may regularly exhibit variable severity. In the present work, one homozygous Chinese Gγ+(Aγδß)0-thalassemia case with an asymptomatic phenotype, which is contrary to traditional views, was identified. Analysis of the underlying causes of this rare clinical phenotype involved accurate genetic diagnosis and detection of several genetic modifications. METHODS: Six members of the proband's family were enrolled in the study. Hematological parameters and hemoglobin analysis results were recorded. A suspension-array system, multiplex gap-polymerase chain reaction (gap-PCR) and multiplex ligation-dependent probe amplification (MLPA) were used together to characterize genotypes. Sanger sequencing was utilized to examine the KLF1 gene and four primary fetal hemoglobin (Hb F)-associated single-nucleotide polymorphisms (SNPs). RESULTS: Four family members carried the Chinese Gγ+(Aγδß)0-thalassemia mutation, and a homozygous state was ultimately diagnosed for the proband. All of the Chinese Gγ+(Aγδß)0 mutation-positive cases were coinherited with the Southern Asian α-thalassemia deletion (- - SEA/αα). Two SNP variants, rs7776054 and rs9399137, in the HBS1L-MYB locus were detected in the proband. CONCLUSIONS: Thus far, this is the first study to describe the molecular characterization of a homozygous Chinese Gγ+(Aγδß)0-thalassemia patient who exhibits no clinical symptoms. Our findings suggest that coinheritance of α-thalassemia or HBS1L-MYB locus variants may affect the clinical severity of Chinese Gγ+(Aγδß)0-thalassemia. We conclude that the molecular examination of genetic determinants known to be associated with clinical outcomes in Chinese Gγ+(Aγδß)0-thalassemia should be emphasized.

10.
Biochem Biophys Res Commun ; 521(2): 420-426, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31672275

RESUMO

Previous studies showed that miR-124 had a protective role by reducing oxidant stress and preventing cell apoptosis and autophagy. However, its role in doxorubicin-induced cardiomyopathy was less known. In our study, we confirmed increased ROS and decreased expression of miR-124 in doxorubicin-treated heart tissues and primary cardiomyocytes. The oxidative stress and cell apoptosis were alleviated by overexpressing miR-124, characterized by decreased activity of MDA and increased activity of SOD. While inhibiting miR-124 generated opposed effects. Mechanistically, our bioinformatic prediction and luciferase assay confirmed that miR-124 inhibited the expression of p66Shc, a proapoptotic signaling pathway. Our results suggested that miR-124 was hopeful to become a therapeutic target in doxorubicin-related cardiomyopathy.

11.
Int J Biol Macromol ; 144: 1004-1012, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31715236

RESUMO

Alzheimer's disease (AD) is the most common degenerative disease of the central nervous system. It is associated with abnormal accumulation of amyloid-ß (Aß) plaques, impaired neurogenesis, and damaged cognitive functions. We have known for a long time that natural compounds and their derivatives have gained increasing attention in AD drug research due to their multiple effects and inherently enormous chemicals. In this study, we will demonstrate that polysaccharides from L. barbarum (LBP1), a traditional natural compound, can reduce Aß level and improve the cognitive functions in APP/PS1 transgenic mouse. LBP1 can enhance neurogenesis as indicated by BrdU/NeuN double labeling. Furthermore, it can restore synaptic dysfunction at hippocampus CA3-CA1 pathway. Additionally, in vitro cell assay indicates that LBP1 may affect Aß processing. In conclusion, our study indicates that LBP1 might be a potential therapeutic agent for the treatment of AD against multiple targets that include synaptic plasticity, Aß pathology and neuropathology.

12.
Cancer Lett ; 469: 481-489, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31738958

RESUMO

Docetaxel-mediated chemotherapy is the first line therapy for metastatic castration-resistant prostate cancer (CRPC) patients, but its therapeutic benefit is limited by the development of resistance. Although Forkhead box protein M1 (FOXM1) has been implicated in prostate tumorigenesis and metastasis, its role in docetaxel resistance has not been studied. Here, we showed that FOXM1 expression was upregulated in the docetaxel resistant CRPC cell lines (PC3-DR and VCaP-DR) and knockdown of FOXM1 sensitized the cells to docetaxel both in vitro and in vivo. In addition, autophagy was found to be significantly enhanced in resistant cells. Moreover, FOXM1 overexpression cells showed increased autophagic flux and higher numbers of autophagosomes. Knockdown of ATG7, beclin-1 or cotreatment with chloroquine, partly restored sensitivity to docetaxel in the FOXM1-overexpressing cells. Mechanistically, FOXM1 targeted AMPK/mTOR to activate the autophagy pathway and altered docetaxel response in CRPC. These findings identify the role of FOXM1 as well as the mechanism underlying FOXM1 action in docetaxel sensitivity and may, therefore, aid in design of CRPC therapies.

13.
Environ Pollut ; 258: 113758, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31881510

RESUMO

Natural halloysite nanotubes (HNTs) with a hollow lumen are already applied in numerous fields and enter the environment in increasing quantities, which may have effects on animal and human health. However their in vivo toxicity in mammals is still largely unclear. The aim of this study is to assess acute oral toxicity of HNTs in the stomach of mice and recovery. Oral HNTs at low dose (5 mg HNTs/kg BW) for 30 days increased in daily food and water intake and promoted mouse growth with no obvious adverse effect on the stomach. The promotive effect on mouse growth disappeared after cessation of oral administration of the nanotubes. Oral HNTs for 30 days at high dose (50 mg HNTs/kg BW) induced Si and Al accumulation in the stomach, which caused oxidative stress, inflammation and iNOS-mediated damage in the organ. The damage in the stomach led to slight atrophic gastritis and reduced mouse growth. Oral HNTs-induced changes at high dose were not observed after a 30-days recovery period. The findings provided the evidence that oral HNTs-induced acute toxicity in the stomach was reversible. More importantly, this research showed that Al and Si were cleared out of the mice by hepatic excretion and renal excretion, respectively, during the recovery period. The results suggest that HNTs at low concentration in environments have no adverse effect on mice, while there are health risks to mice under severe contamination by HNTs.

15.
Onco Targets Ther ; 12: 8301-8310, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31632079

RESUMO

Purpose: Restoring lost function to suppressor gene products has captured the interest of the research community in the field of gene therapy. AKAP12, also known as Gravin/AKAP250, is a tumor suppressor gene, and its deregulation may be responsible for cancer progression. The aim of this study was to investigate whether AKAP12 mRNA has an anti-cancer function by regulating onco-miRNA expression in colorectal cancer (CRC) cells. Methods: miRNAs targeting AKAP12 were predicted by bioinformatics analysis and further confirmed by dual-luciferase reporter assays and RT-qPCR. The altered expression of microRNA was validated in early-stage CRC tumor tissues by miRseq. Cell proliferation was measured by Cell Counting Kit-8 (CCK-8) assay. Cell invasion and migration were detected by transwell and wound healing assays, respectively. In vivo experiments were conducted to confirm the in vitro findings. Results: Among all miRNAs, reversed correlation between AKAP12 expression and miRNA-183-5p expression was most significant. Luciferase assays revealed that AKAP12 directly targeted miR-183-5p. The miRseq data showed that miR-183 was also dysregulated at the early stage of tumor development and upregulated in late sub-stage II CRC patients (P<0.01). Mechanistic analysis both in vitro and in vivo demonstrated that anti-miR-183-5p depressed cell proliferation, migration, and invasion in CRC cells while miR-183-5p overexpression resulted in opposite effects. Conclusion: Our findings suggested that oncomiR-183-5p promoted the proliferation, migration, and invasion of CRC cells. AKAP12 miRNA-binding elements (MREs) suppressed miRNA-183-5p activities. Any change in expression of AKAP12 thus affected miRNA-183-5p. This may be another anti-tumor mechanism in addition to protein-mediation that regulates tumor suppressor genes.

16.
Artigo em Inglês | MEDLINE | ID: mdl-31650709

RESUMO

Different from conventional zero-dimensional (0D) and one-dimensional (1D) counterparts, two-dimensional (2D) nanomaterials show unique properties resulting from their specific structure and morphology. In recent years, broad interest has been focused on the exploration of 2D nanomaterials for drug delivery, which benefits greatly to various disease treatments due to the superior properties of 2D nanomaterials. The fast development of 2D-based drug delivery systems provides great potential for biomedical studies. In this review, a case-by-case analysis was carried out on the state-of-the-art 2D nanomaterials-based drug delivery systems, which possesses great significance to the further biomedical development of 2D nanomaterials. For the purpose of discussing the special advantages of these novel drug delivery systems, this review is organized according to the different types of the latest 2D nanomaterials and their loading capacity towards various cargos. Special emphasis will be located on the application of these 2D nanomaterials-based drug delivery systems in chemotherapy, gene therapy, and immunotherapy. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies.

17.
mSphere ; 4(5)2019 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-31619503

RESUMO

Gellan gum is a microbial exopolysaccharide, produced after aerobic fermentation using the Gram-negative bacterium strain Sphingomonas elodea ATCC 31461. Due to its unique structure and excellent physical characteristics, gellan gum has a broad range of applications in food, pharmaceutical, and other industries where it is used for stabilizing, emulsifying, thickening, and suspending. During the fermentative production of gellan, strain ATCC 31461 also accumulates large amounts of the metabolic by-products yellow carotenoid pigments and poly-ß-hydroxybutyrate (PHB), which is decreasing the gellan production and increasing processing costs. A pigment PHB-free mutant was obtained by knocking out the phytoene desaturase gene (crtI) in the carotenoid biosynthetic pathway and the phaC gene, encoding a PHB synthase for the polymerization of PHB. Unfortunately, the double gene knockout mutant produced only 0.56 g liter-1 gellan. Furthermore, blocking PHB and carotenoid synthesis resulted in the accumulation of pyruvate, which reduced gellan production. To elevate gellan production, combined UV irradiation and ethyl methanesulfonate (EMS) mutagenesis treatment were used. A mutant strain with the same level of pyruvate as that of the wild-type strain and higher gellan production was isolated (1.35 g liter-1, 132.8% higher than the double gene knockout mutant and 14.4% higher than the wild-type strain ATCC 31461). In addition, a new gellan gum recovery method based on the new mutant strain was investigated, in which only 30% isopropanol was required, which is twice for the wild-type strains, and the performance of the final product was improved. Thus, the mutant strain could be an ideal strain for the commercial production of gellan.IMPORTANCE A carotenoid- and PHB-free double gene knockout strain mutant was constructed to simplify the purification steps normally involved in gellan production. However, the production of gellan gum was unexpectedly reduced. A mutant with 14.4% higher gellan production than that of the wild-type strain was obtained and isolated after employing UV and EMS combined mutagenesis. Based on this high-yield and low-impurity-producing mutant, a new recovery method requiring less organic solvent and fewer operating steps was developed. This method will effectively reduce the production costs and improve the economic benefits of large-scale gellan production.

18.
Analyst ; 144(22): 6689-6697, 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31598619

RESUMO

A sensitive and label-free fluorometric method has been developed for the determination of polynucleotide kinase (PNK) activity, by employing exonuclease III (Exo III)-assisted cyclic signal amplification and poly(thymine)-templated copper nanoparticles (polyT-CuNPs). In the presence of PNK, cDNA with 5'-hydroxyl termini was phosphorylated and then hybridized with tDNA to form the cDNA/tDNA duplex, which subsequently triggered the λ exonuclease cleavage reaction, eventually resulting in the release of tDNA. The released tDNA could unfold the hairpin structure of HP DNA to generate partially complementary duplex (tDNA/HP DNA), wherein the HP DNA possessed T-rich sequences (T30) and tDNA recognition sequence. With the help of Exo III digestion, the tDNA was able to initiate the cycle for the generation of T-rich sequences, the template for the formation of fluorescent CuNPs. Conversely, the cDNA could not be cleaved by λ exonuclease without PNK and individual HP DNA could not be hydrolyzed by Exo III. The T-rich sequence was caged in HP DNA, resulting in a weak fluorescence signal. Under optimized conditions, the fluorescence intensity was linearly correlated to a concentration range of 0.001 to 1 U mL-1 with a low detection limit of 2 × 10-4 U mL-1. Considering the intriguing analytical performance, this approach could be explored to screen T4 PNK inhibitors and hold promising applications in drug discovery and disease therapy.

19.
J Neurosci ; 39(46): 9130-9144, 2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31604834

RESUMO

Neuropathic pain is one of the most common and notorious neurological diseases. The changes in cerebral structures after nerve injury and the corresponding contributions to neuropathic pain are not well understood. Here we found that the majority of glutamatergic neurons in the area 2 of midcingulate cortex (MCC Cg2Glu) were inhibited by painful stimulation in male mice. Optogenetic manipulation revealed that these neurons were tonically involved in the inhibitory modulation of multimodal nociception. We further identified the projections to GABAergic neurons in the zona incerta (ZIGABA) mediated the pain inhibitory role. However, MCC Cg2Glu became hypoactive after nerve injury. Although a brief activation of the MCC Cg2Glu to ZIGABA circuit was able to relieve the aversiveness associated with spontaneous ongoing pain, consecutive activation of the circuit was required to alleviate neuropathic allodynia. In contrast, glutamatergic neurons in the area 1 of MCC played opposite roles in pain modulation. They became hyperactive after nerve injury and only consecutive inhibition of their activity relieved allodynia. These results demonstrate that MCC Cg2Glu constitute a component of intrinsic pain inhibitory circuitry and their hypoactivity underlies neuropathic pain. We propose that selective and persistent activation of the MCC Cg2Glu to ZIGABA circuit may serve as a potential therapeutic strategy for this disease.SIGNIFICANCE STATEMENT Glutamatergic neurons in the area 2 of midcingulate cortex (MCC Cg2Glu) are tonically involved in the intrinsic pain inhibition via projecting to GABAergic neurons in the zona incerta. They are hypoactive after nerve injury. Selective activation of the circuit compensates the reduction of its analgesic strength and relieves neuropathic pain. Therefore, MCC Cg2Glu and the related analgesic circuit may serve as therapeutic targets for neuropathic pain. In contrast, MCC Cg1Glu have an opposite role in pain modulation and become hyperactive after nerve injury. The present study provides novel evidence for the concept that neuropathic pain is associated with the dysfunction of endogenous pain modulatory system and new perspective on the treatment of neuropathic pain.

20.
Front Microbiol ; 10: 2079, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31555255

RESUMO

The bacterium Pseudomonas species sp. AP-3 is one of several microorganisms that are capable of using 2-aminophenol as its sole source of carbon, nitrogen and energy. Several 2-aminophenol-metabolizing enzymes have pivotal roles in the biodegradation of aniline and its derivatives as environmental pollutants in Pseudomonas. The bacterium Pseudomonas sp. AP-3 recruits a unique 2-aminomuconate deaminase (AmnE) to hydrolyze 2-aminomuconate to 4-oxalocrotonate, and releases ammonia in the modified meta-cleavage pathway by forming various compounds-including acetaldehyde, pyruvic acid, acetyl-CoA, and succinate-that may enter the Krebs cycle. AmnE also belongs to the YjgF/YER057c/UK114 family (also known as the Rid family), which is conserved in all domains of life and prefers structurally homotrimeric forms with diverse functional purposes. To study the mechanism of the modified meta-cleavage pathway in Pseudomonas sp. AP-3, we determined the first crystal structure of AmnE from Pseudomonas sp. AP-3 at 1.75 Å. AmnE forms a unique homohexamer instead of a trimer which is normally adopted by the members of YjgF/YER057c/UK114 family. Based on the structure of the AmnE hexamer, we observed a hydrophobic base composed of six Lp3 loops (residues 122-131) in each of the AmnE protomers that have pivotal roles in the assembly of the hexamer. Eighteen hydrogen bonds formed by the residues Met96, Pro126, and Arg56, which surround the hydrophobic base, allowed the combination of the two trimers into a stable hexamer. The single mutant of AmnE R56A lost the ability to maintain the hexameric conformation, and revealed that the hydrogen bonds between residues Arg56 and Met96 have pivotal roles in the AmnE hexameric assembly.

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