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1.
Am J Hum Genet ; 108(2): 337-345, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33434492

RESUMO

Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) is associated with congenital absence of the uterus, cervix, and the upper part of the vagina; it is a sex-limited trait. Disrupted development of the Müllerian ducts (MD)/Wölffian ducts (WD) through multifactorial mechanisms has been proposed to underlie MRKHS. In this study, exome sequencing (ES) was performed on a Chinese discovery cohort (442 affected subjects and 941 female control subjects) and a replication MRKHS cohort (150 affected subjects of mixed ethnicity from North America, South America, and Europe). Phenotypic follow-up of the female reproductive system was performed on an additional cohort of PAX8-associated congenital hypothyroidism (CH) (n = 5, Chinese). By analyzing 19 candidate genes essential for MD/WD development, we identified 12 likely gene-disrupting (LGD) variants in 7 genes: PAX8 (n = 4), BMP4 (n = 2), BMP7 (n = 2), TBX6 (n = 1), HOXA10 (n = 1), EMX2 (n = 1), and WNT9B (n = 1), while LGD variants in these genes were not detected in control samples (p = 1.27E-06). Interestingly, a sex-limited penetrance with paternal inheritance was observed in multiple families. One additional PAX8 LGD variant from the replication cohort and two missense variants from both cohorts were revealed to cause loss-of-function of the protein. From the PAX8-associated CH cohort, we identified one individual presenting a syndromic condition characterized by CH and MRKHS (CH-MRKHS). Our study demonstrates the comprehensive utilization of knowledge from developmental biology toward elucidating genetic perturbations, i.e., rare pathogenic alleles involving the same loci, contributing to human birth defects.


Assuntos
Transtornos 46, XX do Desenvolvimento Sexual/genética , Anormalidades Congênitas/genética , Ductos Paramesonéfricos/anormalidades , Ductos Paramesonéfricos/crescimento & desenvolvimento , Mutação , Ductos Mesonéfricos/crescimento & desenvolvimento , Adulto , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 7/genética , Códon sem Sentido , Feminino , Estudos de Associação Genética , Pleiotropia Genética , Proteínas Homeobox A10/genética , Proteínas de Homeodomínio/genética , Humanos , Fator de Transcrição PAX8/genética , Herança Paterna , Penetrância , Proteínas com Domínio T/genética , Fatores de Transcrição/genética , Proteínas Wnt/genética , Ductos Mesonéfricos/anormalidades
2.
Stem Cell Reports ; 15(6): 1362-1376, 2020 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-33186539

RESUMO

Insulin is an essential growth factor for the survival and self-renewal of human embryonic stem cells (hESCs). Although it is best known as the principal hormone promoting glycolysis in somatic cells, insulin's roles in hESC energy metabolism remain unclear. In this report, we demonstrate that insulin is essential to sustain hESC mitochondrial respiration that is rapidly decreased upon insulin removal. Insulin-dependent mitochondrial respiration is stem cell specific, and mainly relies on pyruvate and glutamine, while glucose suppresses excessive oxidative phosphorylation. Pharmacologic and genetic manipulations reveal that continuous insulin signal sustains mitochondrial respiration through PI3K/AKT activation and downstream GSK3 inhibition. We further show that insulin acts through GSK3 inhibition to suppress caspase activation and rescue cell survival. This study uncovers a critical role of the AKT/GSK3 pathway in the regulation of mitochondrial respiration and cell survival, highlighting insulin as an essential factor for accurate assessment of mitochondrial respiration in hESCs.

3.
Artigo em Inglês | MEDLINE | ID: mdl-33128537

RESUMO

Objectives: To explore and establish the diagnosis, syndrome classification and syndrome differentiation criteria of palpitations below the heart in traditional Chinese medicine (TCM) in order to lay a foundation for the clinical diagnosis, treatment and research of palpitations below the heart. Methods: In the early stage of this study, we searched the literature related to palpitations below the heart in TCM in domestic and foreign databases, analyzed the results and developed an expert consultation questionnaire. Using the Delphi method, a survey was conducted by 19 expert TCM practitioners. The survey results were statistically analyzed, aggregated and categorized to create the next round of questionnaires, and the process was repeated was repeated for a total of 4 rounds of expert opinions and until a consensus was achieved. Finally, the questionnaire items were classified into the main diagnosis (primary disease) and secondary diagnosis (secondary disease) for each syndrome. Results: This study was completed ahead of schedule after 2 rounds of expert questionnaire surveys. A total of 19 exceptional TCM experts from all over China reached a consensus on 1 diagnostic standard and 4 syndrome types. The main diagnoses of palpitations below the heart included "conscious sub cardiac epigastric beating," "throbbing at the lower part of the heart" and "palpitation rhythm consistent with the pulse and obvious pulsation in the heart area," while the secondary diagnosis was "palpitation obvious after nervous tension, fatigue, drinking water or changing body position." Based on the balance of TCM Yin (negative, dark, feminine) and Yang (positive, bright, masculine) energy, TCM syndrome differentiation (Bian Zheng - the comprehensive analysis of clinical information obtained from the 4 main diagnostic TCM procedures: observation, listening, questioning, and pulse analysis, that is used to guide the choice of treatment by acupuncture and/or TCM) of palpitations below the heart are differentiated as heart yang deficiency syndrome, middle yang deficiency syndrome, kidney yang deficiency syndrome and phlegm drink syndrome, and the main and secondary syndromes of each are established. In the consultation process, the expert opinions were highly correlated, questionnaire reliability was strong and the results were credible. Conclusions: The criteria proposed in this study do not claim to be the best or the most accurate, but they do provide some guidelines for practitioners, a basis for clinical differentiation and treatment with TCM and a basis for further randomized controlled trials in the future. Further research is needed in order to reach a consensus regarding TCM treatment of palpitations below the heart.

4.
Neurosci Lett ; 739: 135429, 2020 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-33069813

RESUMO

BACKGROUND: Bone fracture may subsequently cause chronic postoperative pain after orthopedic surgery, but mechanisms remain elusive. The necessity of caspase-3 in neuroinflammation and synaptic plasticity has been summarized in pathological pain. Leucine-rich repeat transmembrane protein 1 (LRRTM1) mediates synaptic delivery of AMPA receptor and synaptogenesis. This study evaluated whether caspase-3 and LRRTM1 are required for fracture-associated postoperative allodynia. METHODS: A model of tibial fracture with intramedullary pinning in mice was established for the induction of postoperative pain, verified by measurement of mechanical paw withdrawal threshold and cold scores response to acetone. The caspase-3 specific inhibitor, recombinant caspase-3 and LRRTM1 knockdown by shRNA were utilized for the investigation of pathogenesis as well as the prevention of allodynia. Also, the activity of caspase-3 and the expression of LRRTM1 in the spinal dorsal horn were examined by Western blot and RT-qPCR. RESULTS: This study reported that tibial fracture and orthopedic surgery produced long-lasting mechanical allodynia and cold allodynia, along with the up-modulation of spinal caspase-3 activity (but not caspase-3 expression) and LRRTM1 expression. Spinal caspase-3 inhibition prevented fracture-associated behavioral allodynia in a dose-dependent manner. Caspase-3 inhibitor also reduced the spinal increased LRRTM1 level after tibial fracture with pinning. Spinal LRRTM1 deficiency impaired fracture-caused postoperative pain. Intrathecal recombinant caspase-3 facilitated acute pain hypersensitivity and spinal LRRTM1 expression in naïve mice, reversing by LRRTM1 knockdown. CONCLUSION: Our current results demonstrate the spinal up-regulation of LRRTM1 by caspase-3 activation in the development of tibial fracture-associated postoperative pain in mice.

5.
J Ginseng Res ; 44(5): 717-724, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32913401

RESUMO

Background: Malignant arrhythmias require drug therapy. However, most of the currently available antiarrhythmic drugs have significant side effects. Ginsenoside Rg2 exhibits excellent cardioprotective effects and appears to be a promising candidate for cardiovascular drug development. So far, the oral toxicity and antiarrhythmic effects of Rg2 have not been evaluated. Methods: Acute oral toxicity of Rg2 was assessed by the Limit Test method in mice. Subchronic oral toxicity was determined by repeated dose 28-day toxicity study in rats. Antiarrhythmic activities of Rg2 were evaluated in calcium chloride-induced arrhythmic rats. Antiarrhythmic mechanism of Rg2 was investigated in arrhythmic rats and H9c2 cardiomyocytes. Results: The results of toxicity studies indicated that Rg2 exhibited no single-dose (10 g/kg) acute oral toxicity. And 28-day repeated dose treatment with Rg2 (1.75, 3.5 and 5 g/kg/d) demonstrated minimal, if any, subchronic toxicity. Serum biochemical examination showed that total cholesterol in the high-dose cohort was dramatically decreased, whereas prothrombin time was increased at Day 28, suggesting that Rg2 might regulate lipid metabolism and have a potential anticoagulant effect. Moreover, pretreatment with Rg2 showed antiarrhythmic effects on the rat model of calcium chloride induced arrhythmia, in terms of the reduced duration time, mortality, and incidence of malignant arrhythmias. The antiarrhythmic mechanism of Rg2 might be the inhibition of calcium influx through L-type calcium channels by suppressing the phosphorylation of Ca2+/calmodulin-dependent protein kinase II. Conclusion: Our findings support the development of Rg2 as a promising antiarrhythmic drug with fewer side effects for clinical use.

6.
Pain ; 2020 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-32701657

RESUMO

Chronic postoperative pain hinders functional recovery after bone fracture and orthopedic surgery. Recently reported evidence indicates that caspase-6 is important in excitatory synaptic plasticity and pathological pain. Meanwhile, netrin-1 controls postsynaptic recruitment of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and synaptogenesis. The present work aimed to examine whether caspase-6 and netrin-1 contribute to fracture-induced postoperative allodynia. A mouse model of tibial fracture by intramedullary pinning was generated for inducing postoperative pain. Then, paw withdrawal threshold, spinal caspase-6 activity, netrin-1 secretion, AMPAR trafficking, and spine morphology were examined. Caspase-6 inhibition and netrin-1 knockdown by shRNA were performed to elucidate the pathogenetic mechanism of allodynia and its prevention. Whole-cell patch-clamp recording was performed to assess caspase-6's function in spinal AMPAR-induced current. Tibial fractures after orthopedic operation initiated persistent postsurgical mechanical and cold allodynia, accompanied by increased spinal active caspase-6, netrin-1 release, GluA1-containing AMPAR trafficking, spine density, and AMPAR-induced current in dorsal horn neurons. Caspase-6 inhibition reduced fracture-associated allodynia, netrin-1 secretion, and GluA1 trafficking. Netrin-1 deficiency impaired fracture-caused allodynia, postsynaptic GluA1 recruitment, and spine plasticity. The specific GluA2-lacking AMPAR antagonist NASPM also dose dependently prevented postoperative pain. The reduction of fracture-mediated postoperative excitatory synaptic AMPAR current in the dorsal horn by caspase-6 inhibition was compromised by recombinant netrin-1. Exogenous caspase-6 induced pain hypersensitivity, reversing by netrin-1 knockdown or coapplication of NASPM. Thus, spinal caspase-6 modulation of GluA1-containing AMPAR activation and spine morphology through netrin-1 secretion is important in the development of fracture-related postsurgical pain in the mouse.

7.
Kidney Int ; 98(4): 1020-1030, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32450157

RESUMO

Congenital anomalies of the kidney and urinary tract (CAKUTs) are the most common cause of chronic kidney disease in children. Human 16p11.2 deletions have been associated with CAKUT, but the responsible molecular mechanism remains to be illuminated. To explore this, we investigated 102 carriers of 16p11.2 deletion from multi-center cohorts, among which we retrospectively ascertained kidney morphologic and functional data from 37 individuals (12 Chinese and 25 Caucasian/Hispanic). Significantly higher CAKUT rates were observed in 16p11.2 deletion carriers (about 25% in Chinese and 16% in Caucasian/Hispanic) than those found in the non-clinically ascertained general populations (about 1/1000 found at autopsy). Furthermore, we identified seven additional individuals with heterozygous loss-of-function variants in TBX6, a gene that maps to the 16p11.2 region. Four of these seven cases showed obvious CAKUT. To further investigate the role of TBX6 in kidney development, we engineered mice with mutated Tbx6 alleles. The Tbx6 heterozygous null (i.e., loss-of-function) mutant (Tbx6+/‒) resulted in 13% solitary kidneys. Remarkably, this incidence increased to 29% in a compound heterozygous model (Tbx6mh/‒) that reduced Tbx6 gene dosage to below haploinsufficiency, by combining the null allele with a novel mild hypomorphic allele (mh). Renal hypoplasia was also frequently observed in these Tbx6-mutated mouse models. Thus, our findings in patients and mice establish TBX6 as a novel gene involved in CAKUT and its gene dosage insufficiency as a potential driver for kidney defects observed in the 16p11.2 microdeletion syndrome.

8.
Genomics ; 112(4): 2784-2793, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32209379

RESUMO

Acinetobacter haemolyticus (A. haemolyticus) is a significant Acinetobacter pathogen, and the resistance of A. haemolyticus continues to rise due to abuse of antibiotics and the frequent gene exchange between bacteria in hospital. In this study, we performed complete genome sequencing of two A. haemolyticus strains TJR01 and TJS01 to improve our understanding of pathogenic and resistance of A. haemolyticus. Both TJR01 and TJS01 contain one chromosome and two plasmids. Compared to TJS01, more virulence factors (VFs) associated pathogenicity and resistant genes were predicted in TJR01 due to T4SS and integron associated with combination and transport. Antimicrobial susceptibility results were consistent with sequencing. We suppose TJS01 was a susceptive strain and TJR01 was an acquired multidrug resistance strain due to plasmid-mediated horizontal gene transfer. We hope these findings may be helpful for clinical treatment of A. haemolyticus infection and reduce the risk of potential outbreak infection.

9.
Neurosci Lett ; 722: 134855, 2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-32088196

RESUMO

INTRODUCTION: Hevin, a matricellular protein involved in tissue repair and remodeling, is crucial for initiation and development of excitatory synapses. Besides, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA) is an ionotropic transmembrane receptor for glutamate that mediates fast synaptic transmission in the central nervous system (CNS). This study aimed to investigate the correlation between spinal Hevin and AMPA receptors in remifentanil-induced postoperative hyperalgesia in mice. METHODS: Remifentanil (1.33 µg/kg/min for 60 min) was subcutaneously injected into a mouse model of postoperative pain. The von Frey and hot plate tests were performed to assess mechanical and thermal hyperalgesia. The gene and protein expression of Hevin and the membrane trafficking of GluA1-containing AMPA receptors in the dorsal horn of spinal cord were detected by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot analysis. In addition, Hevin-shRNA, exogenous Hevin, and 1-naphtylacetyl-spermine (NASPM) were administrated intrathecally to assess the relationship between spinal Hevin and AMPA receptors. RESULTS: Perioperative administration of remifentanil can aggravate and prolong incision-induced mechanical and thermal hyperalgesia. Treatment with remifentanil increased the expression of spinal Hevin and the membrane trafficking of AMPA receptors. Additionally, knockdown of spinal Hevin attenuated pain hypersensitivity and downregulated membrane trafficking of AMPA receptors after treatment with remifentanil. Meanwhile, preadministration of NASPM reversed spontaneous pain and membrane trafficking of spinal GluA1-containing AMPA receptors induced by exogenous Hevin in naïve mice. CONCLUSIONS: Spinal Hevin was involved in the maintenance of remifentanil-induced postoperative hyperalgesia via modulating membrane trafficking of AMPA receptors.

11.
Neurosci Lett ; 715: 134616, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31705923

RESUMO

INTRODUCTION: Synaptosomal associated proteins of 25 kDa (SNAP-25), as a member of stable soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex, is critical for membrane fusion and required for the release of neurotransmitters. The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor is implicated in pathologic pain. This study aimed to investigate whether and how SNAP-25 regulated AMPA receptors in neuropathic pain. METHODS: Male Sprague-Dawley rats underwent L4 spinal nerve ligation (SNL) or the sham procedure. After assessing mechanical allodynia and thermal sensitivity, the ipsilateral portion of the L4-5 spinal cord was harvested. The expression level of SNAP-25 was analyzed by Western blot analysis and real-time quantitative polymerase chain reaction. SNAP-25 phosphorylation and AMPA receptor membrane trafficking levels were evaluated with Western blot analysis. An association between SNAP-25 and AMPA membrane trafficking was confirmed by SNAP-25 expression or phosphorylation inhibition. RESULTS: The SNL procedure induced and maintained mechanical allodynia and thermal hyperalgesia. SNL increased the expression and phosphorylation of SNAP-25 and the membrane trafficking of AMPA receptors in the spinal cord. SNAP-25 expression or phosphorylation inhibition alleviated neuropathic pain and downregulated membrane trafficking of AMPA receptors after SNL. GluA1-containing AMPA receptor inhibition relieved mechanical allodynia and thermal hyperalgesia after SNL. CONCLUSIONS: The upregulation of SNAP-25-dependent membrane trafficking of AMPA receptors via SNAP-25 phosphorylation at Ser187 contributed to SNL-induced neuropathic pain. Thus, the inhibition of SNAP-25 expression or phosphorylation might serve as a treatment for neuropathic pain. However, the mechanism of GluA1-containing AMPA receptor membrane trafficking mediated by SNAP-25 phosphorylation in neuropathic pain deserves further exploration.

12.
Reg Anesth Pain Med ; 2020 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-33443215

RESUMO

BACKGROUND: Prolonged postoperative pain is a major concern and occurs more frequently in women, but mechanisms remain elusive. NR2B-containging N-methyl-d-aspartate (NMDA) receptor is a key component of nociception transduction. Divalent metal transporter 1 (DMT1)-mediated iron overload involves NMDA-induced neurotoxicity in males. Kalirin-7 is vital in synaptic plasticity underlying pathological pain in males. Herein, the requirement for kalirin-7 in NR2B phosphorylation-dependent iron accumulation and spine plasticity in postoperative pain after tibial fracture in female mice has been examined. METHODS: Pain-related behavior, spinal NR2B phosphorylation at Tyr1472, kalirin-7 expression, DMT1 with/without iron-responsive element (IRE (+) DMT1 and IRE (-) DMT1) level, iron concentration and spine morphology were assessed in females. NR2B antagonist Ro25-6981, iron chelator deferoxamine and kalirin-7 knockdown by short hairpin RNA were employed to assess the potential cascade. RESULTS: Tibial fracture initiates long-term allodynia lasting at least 21 days postoperatively, and upregulates spinal NR2B phosphorylation, kalirin-7 and IRE (-) DMT1 expression, iron overload and spine density. Ro25-6981 reduces postoperative mechanical and cold allodynia, spinal NR2B phosphorylation, kalirin-7 level and IRE (-) DMT1-mediated iron overload. Kalirin-7 knockdown impairs fracture-associated allodynia, IRE (-) DMT1-mediated iron overload and spine plasticity. Deferoxamine also attenuates behavioral allodynia and spine plasticity. Spinal NMDA application elicits NR2B-dependent mechanical allodynia and iron overload, which is reversed by kalirin-7 knockdown or coadministration of deferoxamine. CONCLUSION: Spinal NR2B phosphorylation at Tyr1472 upregulates kalirin-7 expression to facilitate IRE (-) DMT1-mediated iron accumulation and spine morphogenesis in the development of fracture-associated postoperative pain in female mice.

13.
Front Chem ; 7: 746, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31781538

RESUMO

In this study, we investigated the structure-absorption relationship of common surface modifiers of chitosan (CTS), polyvinyl acetate (PVA), and titanium dioxide (TiO2) with α-quartz surface using molecular dynamics simulation. And the orientations and combinations derived from structures between modified α-quartz and ZSM-5 crystallites were also investigated. The results show that PVA is a non-linear organic macromolecule with a large amount of hydroxyl groups on its surface, which easily adhere to the surface of the substrate and agglomerate. CTS is a straight-chain structure containing a large number of hydroxyl and amino groups, which easily accumulate and spread on the surface of the substrate. TiO2 not only forms hydrogen bonds and complexes with the substrate but also interacts with each other to form a dense modifier layer. We observed that a large number of stable Ti-O-Si chemical bonds formed between the modified layer of inorganic small-molecule TiO2 and the surface of α-quartz, which compacted and stabilized the attached ZSM-5 film. Moreover, the orientation angle of the ZSM-5 nanocrystalline nucleus on the modified α-quartz was computed, which confirmed that the b-axis orientation of the ZSM-5 nanocrystalline nucleus was the highest on the surface of the substrate modified by TiO2. We discussed the influence of the modified temperature of modifiers in the constructed materials, and we have observed the adsorption state differences of TiO2 at different modified temperatures. We also discussed the catalytic properties of the materials prepared by the corresponding methods in conversion of methanol-to-aromatics (MTA) reaction. These results agree with our previous experimental data. By employing molecular dynamics simulation, we have obtained more precise conclusive information of the b-oriented growth of ZSM-5 crystallites, which highly depends on the surface modifiers.

14.
Stem Cell Reports ; 13(2): 338-351, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31353224

RESUMO

Pyruvate is a key metabolite in glycolysis and the tricarboxylic acid (TCA) cycle. Exogenous pyruvate modulates metabolism, provides cellular protection, and is essential for the maintenance of human preimplantation embryos and human embryonic stem cells (hESCs). However, little is known about how pyruvate contributes to cell-fate determination during epiblast stage. In this study, we used hESCs as a model to demonstrate that elevated exogenous pyruvate shifts metabolic balance toward oxidative phosphorylation in both maintenance and differentiation conditions. During differentiation, pyruvate potentiates mesoderm and endoderm lineage specification. Pyruvate production and its mitochondrial metabolism are required in BMP4-induced mesoderm differentiation. However, the TCA-cycle metabolites do not have the same effect as pyruvate on differentiation. Further study shows that pyruvate increases AMP/ATP ratio, activates AMPK, and modulates the mTOR pathway to enhance mesoderm differentiation. This study reveals that exogenous pyruvate not only controls metabolism but also modulates signaling pathways in hESC differentiation.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Mesoderma/citologia , Ácido Pirúvico/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Proteína Morfogenética Óssea 4/farmacologia , Linhagem da Célula , Ciclo do Ácido Cítrico/efeitos dos fármacos , Endoderma/citologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Mesoderma/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Regulação para Cima/efeitos dos fármacos
15.
Glycoconj J ; 36(5): 399-408, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31267246

RESUMO

Even though a vaccine that targets tumor-associated carbohydrate antigens on epithelial carcinoma cells presents an attractive therapeutic approach, relatively poor immunogenicity limits its development. In this study, we investigated the immunological activity of a fluoro-substituted Sialyl-Tn (F-STn) analogue coupled to the non-toxic cross-reactive material of diphtheria toxin197 (CRM197). Our results indicate that F-STn-CRM197 promotes a greater immunogenicity than non-fluorinated STn-CRM197. In the presence or absence of adjuvant, F-STn-CRM197 remarkably enhances both cellular and humoral immunity against STn by increasing antigen-specific lymphocyte proliferation and inducing a mixed Th1/Th2 response leading to production of IFN-γ and IL-4 cytokines, as well as STn-specific antibodies. Furthermore, antisera produced from F-STn-CRM197 immunization significantly recognizes STn-positive tumor cells and increases cancer cell lysis induced by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) pathways. Our data suggest that this F-STn vaccine may be useful for cancer immunotherapy and possibly for prophylactic prevention of cancer.


Assuntos
Anticorpos Antineoplásicos/farmacologia , Antígenos Glicosídicos Associados a Tumores/química , Proteínas de Bactérias/farmacologia , Vacinas Anticâncer/farmacologia , Neoplasias do Colo/terapia , Glicoconjugados/farmacologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antineoplásicos/isolamento & purificação , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Antígenos Glicosídicos Associados a Tumores/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Vacinas Anticâncer/síntese química , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Feminino , Expressão Gênica , Glicoconjugados/síntese química , Glicoconjugados/imunologia , Halogenação , Humanos , Soros Imunes/química , Soros Imunes/farmacologia , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Imunização , Imunogenicidade da Vacina , Interferon gama/genética , Interferon gama/imunologia , Interleucina-4/genética , Interleucina-4/imunologia , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Baço/efeitos dos fármacos , Baço/imunologia , Equilíbrio Th1-Th2
16.
Indian J Dermatol ; 64(3): 251, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31148876

RESUMO

Background: Herpes zoster (HZ) is identified to induce postherpetic neuralgia (PHN) which is difficult to cure. PHN-related pain brings patients not only physical discomfort but also mental depression and anxiety. Currently, the main purpose of PHN treatment is to reduce patients' pain. Now treatment combining some international pain management and drug therapy has come up. Aims and Objective: This study aims to evaluate the effect of interventional management through meta-analysis. Materials and Methods: Interventional pain management was defined as a direct strategy on nerve through physical or chemical method. Drug therapy was always regarded as control. Potentially relevant articles were searched in PubMed, EMBASE, and the Cochrane Library through key words by consensus. Pain severity was evaluated by a validated visual analog scale (VAS). Moreover, the weighted mean difference was used to calculate pain intensity. Some trails recorded the efficiency rate and odds ratio was used to calculate the effectiveness. Statistical heterogeneity was measured by the value of I 2, and when statistical I 2 > 50%, subgroup analysis was used to seek for the source of heterogeneity. Results: Pulsed radiofrequency (PRF) combined with medication reduced the VAS scores at 1, 2, 4, and 8 weeks after treatment. The nerve block combined with medication reduced VAS scores at 8 weeks after treatment, but there is no difference between the results of medication alone at 1, 2, and 4 weeks after treatment. Conclusion: The interventional mean of PRF combined with medication has a good effect on PHN. The effect of nerve block combined with medication on PHN seems to be the same as that of medication alone. Besides, a long period with high-quality randomized controlled trial should be done to verify the results.

17.
Carbohydr Polym ; 219: 121-129, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31151509

RESUMO

Galectin-3 (Gal-3) can induce T-cell activation and apoptosis and plays a role in tumor immune tolerance. Here, we demonstrate that ginseng pectins selectively inhibit Gal-3-induced T-cell apoptosis, while not affecting T-cell activation. This finding stands in contrast to that from the use of modified citrus pectin (MCP) and potato galactan (P-galactan) that inhibit both. Whereas PKC/ERK and ROS/ERK pathways are involved in both T-cell activation and apoptosis, the Ras/PI3K/Akt pathway is unique to T-cell activation. Ginseng pectins selectively inhibit the ROS/ERK pathway. Using the Sarcomar-180 mouse model in which Gal-3 expression is increased, we found that ginseng pectins (but not MCP or P-galactan) significantly promote T-cell proliferation and IL-2 expression, and inhibit tumor growth by 45%. These in vivo data correlate well with selective effects of pectins on Gal-3-mediated T-cell apoptosis and activation. Our study suggests a novel approach for the development of polysaccharide-based agents that target Gal-3 function.


Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Galactanos/farmacologia , Galectina 3/metabolismo , Panax/metabolismo , Pectinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Linfócitos T , Animais , Linhagem Celular Tumoral , Humanos , Interleucina-2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo
18.
Glycobiology ; 29(8): 608-618, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31094416

RESUMO

Galectin-3 (Gal-3) binds to cell adhesion glycoprotein CD146 to promote cytokine secretion and mediate endothelial cell migration. Here, we used Nuclear Magnetic Resonance (NMR) 15N-Heteronuclear Single Quantum Coherence (HSQC) spectroscopy to investigate binding between 15N-labeled Gal-3 and the extracellular domain (eFL) of purified CD146 (five Ig-like ectodomains D1-D5) and a shorter, D5-deleted version of CD146 (D1-D4). Binding of Gal-3 and its carbohydrate recognition domain (CRD) to CD146 D1-D4 is greatly reduced vis-à-vis CD146 eFL, supporting the proposal of a larger number of glycosylation sites on D5. Even though the canonical sugar-binding ß-sheet S-face (ß-strands 1, 10, 3, 4, 5, 6) of the Gal-3 ß-sandwich is involved in interactions with CD146 (e.g. N-linked glycosylation sites), equivalent HSQC spectral perturbations at residues on the opposing Gal-3 F-face ß-sheet (ß-strands 11, 2, 7, 8, 9) indicate involvement of the Gal-3 F-face in binding CD146. This is supported by the observation that addition of lactose, while significantly attenuating Gal-3 binding (primarily with the S-face) to CD146 eFL, does not abolish it. Bio-Layer Interferometry studies with Gal-3 F-face mutants yield KD values to demonstrate a significant decrease (L203A) or increase (V204A, L218A, T243A) in net binding to CD146 eFL compared to wild type Gal-3. However, HSQC lactose titrations show no highly significant effects on sugar binding to the Gal-3 CRD S-face. Overall, our findings indicate that Gal-3 binding to CD146 is more involved than simple interactions with ß-galactoside epitopes on the cell receptor, and that there is a direct role for the lectin's CRD F-face in the CD146 binding process.


Assuntos
Antígeno CD146/metabolismo , Galectina 3/química , Sítios de Ligação , Galectina 3/genética , Galectina 3/metabolismo , Células HEK293 , Humanos , Lactose/análogos & derivados , Mutação , Ligação Proteica
19.
Molecules ; 24(7)2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30974879

RESUMO

Isomaltose-oligosaccharides (IMOs), as food ingredients with prebiotic functionality, can be prepared via enzymatic synthesis using α-glucosidase. In the present study, the α-glucosidase (GSJ) from Geobacillus sp. strain HTA-462 was cloned and expressed in Escherichia coli BL21 (DE3). Recombinant GSJ was purified and biochemically characterized. The optimum temperature condition of the recombinant enzyme was 65 °C, and the half-life was 84 h at 60 °C, whereas the enzyme was active over the range of pH 6.0-10.0 with maximal activity at pH 7.0. The α-glucosidase activity in shake flasks reached 107.9 U/mL and using 4-Nitrophenyl ß-D-glucopyranoside (pNPG) as substrate, the Km and Vmax values were 2.321 mM and 306.3 U/mg, respectively. The divalent ions Mn2+ and Ca2+ could improve GSJ activity by 32.1% and 13.8%. Moreover, the hydrolysis ability of recombinant α-glucosidase was almost the same as that of the commercial α-glucosidase (Bacillus stearothermophilus). In terms of the transglycosylation reaction, with 30% maltose syrup under the condition of 60 °C and pH 7.0, IMOs were synthesized with a conversion rate of 37%. These studies lay the basis for the industrial application of recombinant α-glucosidase.


Assuntos
Proteínas de Bactérias/química , Escherichia coli/metabolismo , Geobacillus/genética , Isomaltose/química , alfa-Glucosidases/química , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Geobacillus/enzimologia , Oligossacarídeos/síntese química , Oligossacarídeos/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade por Substrato , alfa-Glucosidases/biossíntese , alfa-Glucosidases/genética
20.
Carbohydr Polym ; 214: 34-43, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30926005

RESUMO

A novel polysaccharide (WCCP-N-b) with a molecular weight of 18 kDa was isolated and purified from the fruiting bodies of Cantharellus cibarius. Monosaccharide composition, methylation analysis and NMR spectra indicated that WCCP-N-b was a linear α-1,6-galactan, partially methylated at O-3 of galactose. The molar ratio of Gal, 3-methylated-Gal, Glc and Man was 14.4:4.6:1.0:1.2. WCCP-N-b could significantly increase macrophage phagocytosis, release of NO and secretion of TNF-α, IL-6 and IL-1ß. On a cellular mechanistic level, WCCP-N-b activated MAPKs and NF-κB signaling pathway via Toll-like receptor 2 (TLR2). To further elucidate the structure-function relationship, WCCP-N-b was hydrolyzed by acid. Four degraded fragments were obtained, with molecular weights of 16.1 kDa, 11.2 kDa, 5 kDa and 3.5 kDa, respectively. Their macrophage activation effects were significantly decreased along with the molecular weight decrease. Collectively, WCCP-N-b could activate RAW264.7 cells, and the activation effect was related to its molecular weight.


Assuntos
Galactanos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Agaricales/química , Animais , Carpóforos/química , Galactanos/química , Galactanos/isolamento & purificação , Técnicas de Silenciamento de Genes , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Estrutura Molecular , Peso Molecular , Óxido Nítrico/metabolismo , Fagocitose/efeitos dos fármacos , Células RAW 264.7 , Relação Estrutura-Atividade , Receptor 2 Toll-Like/genética , Fator de Necrose Tumoral alfa/metabolismo
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