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1.
Protein Expr Purif ; 178: 105783, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33122138

RESUMO

Natural killer (NK) cells are potent cytotoxic effector cells of the innate immune system and play an important role in tumor immunosurveillance and control. NKG2D is an activating receptor of NK cells. The NKG2D receptor-ligand system has contributed to immune cells recognizing tumor cells and the tumor microenvironment. In order to stretch the application of NK cells on adoptive immunotherapy for B-cell malignancies, we designed and produced a novel bispecific ULBP1×CD19-scFv fusion protein, in which the extracellular domain of NKG2D ligand ULBP1 was fused to a single chain variable fragment (scFv) of anti-CD19. The vector expressing ULBP1×CD19-scFv protein was constructed and expressed in Pichia pastoris. Effects of medium composition, concentration of methanol as the inducer, induction time and broth content in shake flask on the expression of the recombinant protein were investigated. The results showed that the optimized conditions for ULBP1×CD19-scFv expression were 1% methanol induction for 96 h with 15% broth content. The secreted recombinant protein was purified using ammonium sulfate fractionation and Ni-NTA affinity chromatography and the purity is about 93%. The cytotoxicity of NK92-MI cells against CD19+ Raji cells was enhanced in the presence of purified ULBP1×CD19-scFv protein. These results indicated that ULBP1 could be used as an activating element of bispecific killer engagers (BiKEs) and Pichia pastoris yeast might be an alternative expression host for BiKEs production.

2.
Int J Biol Macromol ; 2020 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-33157139

RESUMO

Alpha-fetoprotein (AFP) is one of the most important biomarkers associated with primary liver cancer, and the main approaches for diagnosis are based on immunoassay. Affibody is a 58 amino acids peptide derived from the Z domain of staphylococcal protein A and generally applied in imaging diagnosis, clinical therapeutics and biotechnology research. The aim of this study was therefore to develop a novel affibody-based ELISA for detection of AFP. After three rounds of biopanning, six AFP-binding affibody peptides were selected using phage display technology, among them affibody ZAFPD2 showed high and specific binding affinity to AFP. An affibody dimer of ZAFPD2 was created, named (ZAFP D2)2, expressed in E.coli and the purified (ZAFP D2)2 recombinant protein showed higher binding affinity to AFP, as well as high thermal stability. A novel affibody-based two-site ELISA method using ZAFPD2 or (ZAFP D2)2 and polyclonal antibody to detect AFP was developed, the detection limit of the immunoassay using (ZAFP D2)2 was 2 ng mL-1 that was 4 times lower than ZAFPD2, which meets the requirements for practical application. Therefore, this concept of affibody-based ELISA may provide a new method for the detection of various cancer biomarkers.

3.
Int J Mol Med ; 46(6): 2102-2114, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33125094

RESUMO

Annexin IV (ANXA4) is highly expressed in ovarian clear cell carcinoma (OCCC); however, its underlying molecular mechanism in OCCC remains unknown. The present study aimed to identify the molecule that ANXA4 may act on and to determine its underlying molecular mechanism. Immunohistochemistry, co­immunoprecipitation and western blotting were performed to detect the expression and interaction of ANXA4, and its associated proteins. Furthermore, MTT assay, flow cytometry, western blotting and gene expression profile enrichment analysis were performed to identify the potential role and molecular mechanism of ANXA4 in OCCC. The results demonstrated that ANXA4 and nuclear factor­κ­light­chain­enhancer of activated B cells (NF­κB) p50 nuclear expression levels were significantly higher in OCCC tissues compared with other subtypes of ovarian cancer, such as serous and mucinous. In addition, a significantly positive correlation was observed between ANXA4 and NF­κB p50 expression in OCCC; however, the expression levels of mutant p53 and ANXA4 were negatively correlated in a linear manner. These results suggest that ANXA4 and NF­κB p50 may be potential independent risk factors for poor prognosis. ANXA4 and NF­κB p50 were demonstrated to interact and their expression was co­localized. The cBioPortal database was used to construct a protein­protein interaction network between ANXA4, NF­κB p50 and p53, and functional pathway analysis indicated that the genes were predominantly enriched in the cell cycle and during apoptosis. Transfection of the ANXA4 gene increased the expression of NF­κB p50, as well as its downstream targets, Cyclin D1 and B­cell lymphoma­2 (Bcl­2). Furthermore, transfection of the ANXA4 gene increased proliferation and decreased apoptosis of OCCC cells. Treatment with the NF­κB inhibitor, BAY 11­7082, decreased Cyclin D1 and Bcl­2 expression levels. Collectively, the results of the present study suggest that wild p53 activates ANXA4 transcription, promotes its expression and enhances NF­κB p50 and ANXA4 interaction. This in turn activates the NF­κB signaling pathway, promotes cell cycle progression and inhibits apoptosis, thus contributing to the malignant progression of OCCC. Thus, ANXA4 and NF­κB p50 may be used as prognostic biomarkers, and may be molecular therapeutic targets in OCCC.

4.
J Mater Chem B ; 8(38): 8845-8852, 2020 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-33026405

RESUMO

Using a regular CMOS sensor as a template, we are able to fabricate a simple but highly effective superhydrophobic SERS substrate. Specifically, we decorated the microlens layer of the sensor with 7 µm polystyrene beads to obtain a PDMS patterned replica. The process resulted in a uniform pattern of voids in the PDMS (denoted nanobowls) that are intercalated with a few larger voids (denoted here microbowls). The voids act as superhydrophobic substrates with analyte concentration capabilities in bigger bowl-like structures. Silver nanoparticles were directly grown on the patterned PDMS substrate inside both the nano- and microbowls, and serve as strong electromagnetic field enhancers for the SERS substrate. After systematic characterization of the fabricated SERS substrate by atomic force microscopy and scanning electron microscopy, we demonstrated its SERS performance using 4-aminothiophenol as a reporter molecule. Finally, we employed this innovative substrate to concentrate and analyze extracellular vesicles (EVs) isolated from an MC65 neural cell line in an ultralow sample volume. This substrate can be further exploited for the investigation of various EV biomarkers for early diagnosis of different diseases using liquid biopsy.

5.
Pediatr Infect Dis J ; 2020 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-33021592

RESUMO

OBJECTIVE: The purpose of the current study is to investigate the bactericidal effect of macrolides and ß-lactams on Bordetella pertussis (B. pertussis) in the nasopharynx and provide guidance for treating macrolides-resistant B. pertussis infections. METHODS: Patients with whooping cough was diagnosed by culture of nasopharynx swabs between January 2016 to December 2018. B. pertussis was identified using specific antisera against pertussis and parapertussis. Drug susceptibility test was carried out using the E-test method. The clearance of B. pertussis in nasopharynx at 7 and 14 days into and posttreatment with macrolides, and ß-lactams was compared. RESULTS: A total of 125 B. pertussis samples were collected from patients who received single antibiotic treatment. Among those isolates, 62.4% (78/125) had high resistance with minimum inhibitory concentrations greater than 256 mg/L for erythromycin and azithromycin. The MIC90 of piperacillin, cefoperazone-sulbactam, meropenem, ampicillin, ceftriaxone, ceftazidime and trimethoprim-sulfamethoxazole for these isolates was <0.016, 0.094, 0.094, 0.19, 0.19, 0.25 and 0.75 mg/L, respectively. The clearance rate with ß-lactams treatment (68.8%, 44/64) was significantly higher than that with macrolides treatment at 14 days posttreatment (50.8%, 31/61) (χ = 4.18, P = 0.04). Macrolides had a better clearance rate at 7 days posttreatment than ß-lactams (χ = 4.49, P = 0.03) for macrolides-sensitive isolates and a worse clearance rate for macrolides-resistant isolates. CONCLUSION: B. pertussis isolates had a high-resistant rate for macrolides in our study. Macrolides are the first choice for treating pertussis caused by macrolides-sensitive strains, and some ß-lactams such as piperacillin should be considered as alternative antibiotics for treatment of macrolides-resistant B. pertussis infection.

6.
BMC Plant Biol ; 20(1): 416, 2020 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-32894064

RESUMO

BACKGROUND: Cotton (Gossypium spp.) fiber yield is one of the key target traits, and improved fiber yield has always been thought of as an important objective in the breeding programs and production. Although some studies had been reported for the understanding of genetic bases for cotton yield-related traits, the detected quantitative trait loci (QTL) for the traits is still very limited. To uncover the whole-genome QTL controlling three yield-related traits in upland cotton (Gossypium hirsutum L.), phenotypic traits were investigated under four planting environments and 9244 single-nucleotide polymorphism linkage disequilibrium block (SNPLDB) markers were developed in an association panel consisting of 315 accessions. RESULTS: A total of 53, 70 and 68 significant SNPLDB loci associated with boll number (BN), boll weight (BW) and lint percentage (LP), were respectively detected through a restricted two-stage multi-locus multi-allele genome-wide association study (RTM-GWAS) procedure in multiple environments. The haplotype/allele effects of the significant SNPLDB loci were estimated and the QTL-allele matrices were organized for offering the abbreviated genetic composition of the population. Among the significant SNPLDB loci, six of them were simultaneously identified in two or more single planting environments and were thought of as the stable SNPLDB loci. Additionally, a total of 115 genes were annotated in the nearby regions of the six stable SNPLDB loci, and 16 common potential candidate genes controlling target traits of them were predicted by two RNA-seq data. One of 16 genes (GH_D06G2161) was mainly expressed in the early ovule-development stages, and the stable SNPLDB locus (LDB_19_62926589) was mapped in its promoter region. CONCLUSION: This study identified the QTL alleles and candidate genes that could provide important insights into the genetic basis of yield-related traits in upland cotton and might facilitate breeding cotton varieties with high yield.

7.
ACS Sens ; 5(9): 2820-2833, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32935542

RESUMO

For more effective early-stage cancer diagnostics, there is a need to develop sensitive and specific, non- or minimally invasive, and cost-effective methods for identifying circulating nanoscale extracellular vesicles (EVs). Here, we report the utilization of a simple plasmonic scaffold composed of a microscale biosilicate substrate embedded with silver nanoparticles for surface-enhanced Raman scattering (SERS) analysis of ovarian and endometrial cancer EVs. These substrates are rapidly and inexpensively produced without any complex equipment or lithography. We extensively characterize the substrates with electron microscopy and outline a reproducible methodology for their use in analyzing EVs from in vitro and in vivo biofluids. We report effective chemical treatments for (i) decoration of metal surfaces with cysteamine to nonspecifically pull down EVs to SERS hotspots and (ii) enzymatic cleavage of extraluminal moieties at the surface of EVs that prevent localization of complementary chemical features (lipids/proteins) to the vicinity of the metal-enhanced fields. We observe a major loss of sensitivity for ovarian and endometrial cancer following enzymatic cleavage of EVs' extraluminal domain, suggesting its critical significance for diagnostic platforms. We demonstrate that the SERS technique represents an ideal tool to assess and measure the high heterogeneity of EVs isolated from clinical samples in an inexpensive, rapid, and label-free assay.

8.
In Vitro Cell Dev Biol Anim ; 56(8): 650-658, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32888116

RESUMO

Yangtze sturgeon (Acipenser dabryanus) is an endangered endemic freshwater fish of China. Cell-line is a potential means used for long-term preservation of germplasm resources and an ideal in vitro model in place of living organisms for biological studies. Here, culture condition and characterization of fin-derived cell in Yangtze sturgeon were carried out. Tissue explant techniques have been efficiently used in the Yangtze sturgeon caudal fin (YSCF) culture. The YSCF cell line showed a fibroblast-like morphology and stable growth in minimum essential medium eagle's (MEME) supplemented with 10-20% fetal bovine serum at 25°C. Cells were cryopreserved with preservative DMSO in liquid nitrogen and grew normally after recovery. No bacterial, fungal, or mycoplasma contamination was detected in the YSCF cells. Karyotype analysis of the YSCF cells showed that the chromosome numbers of the YSCF ranged from 242 to 273, and the modal chromosome number was identified as 264 at passage 9. The YSCF cells were confirmed from A. dabryanus by assay of 16S rRNA and COI. Furthermore, GFP reporter gene was successfully transferred into YSCF cells and expressed. The established YSCF cell lines will contribute to the preservation of germplasm resources and provide a useful vitro tool for further biological studies in sturgeon species.

9.
Pathol Res Pract ; 216(11): 153148, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32823233

RESUMO

Ovarian cancer is the most lethal gynecological malignancy worldwide. A better understanding of the pathogenesis of ovarian cancer may help to improve the overall survival. Our previous studies have demonstrated that alpha-(1,2)-fucosyltransferase 1 (FUT1) is an oncogenic glycogene in ovarian cancer. However, the underlying mechanism is not fully clarified. In this study, we identified a microRNA as an important downstream regulator for the carcinogenic effect of FUT1 in ovarian cancer. miR-5193 was found down-regulated in ovarian cancer cells, FUT1-overexpression ovarian cancer cells and ovarian tumor samples. MTT, flow cytometry and Transwell assays demonstrated that miR-5193 inhibited the proliferation and migration, and induced the cell cycle arrest and apoptosis of ovarian cancer cells. Real-time PCR and western blot assays showed that miR-5193 downregulated the expression of TRIM11 and upregulated the expression of p53 and p21. Dual luciferase reporter assay indicated that TRIM11 was a direct target of miR­5193. Rescue experiments confirmed that miR-5193 functioned in ovarian cancer cells by directly targeting TRIM11. Moreover, transfection with miR-5193 mimic in FUT1-overexpression ovarian cancer cells reversed the carcinogenic effect of FUT1. Taken together, our results suggest that miR-5193 is an essential suppressor of human ovarian cancer development, and is an important downstream regulator regarding the carcinogenesis of FUT1 in ovarian cancer.

10.
Sci Rep ; 10(1): 11626, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669571

RESUMO

In migratory insects, increasing evidence has demonstrated juvenile hormone (JH) is involved in regulating adult reproduction and flight. Our previous study demonstrated that the switch from migrants to residents in Mythimna separata could be induced by adverse environmental conditions during a sensitive period in adulthood (the first day post-emergence), but the role of JH in this switch is not clear. Here, we found a significantly different pattern of JH titers between migrants and residents, with migrants showing a slower release of JH during adulthood than residents. Application of JH analogue (JHA) in the 1-day-old adults, significantly accelerated adult reproduction and suppressed flight capacity. The pre-oviposition period and period of first oviposition of migrants treated with JHA were significantly shorter, while the total lifetime fecundity and mating percentage increased. The flight capacity and dorso-longitudinal muscle size of the migrants were decreased significantly when treated with JHA. The effect of JHA on reproduction and flight capacity indicate that JH titers during the sensitive period (first day post-emergence) regulates the shift from migrants to residents in M. separata.

11.
Microbiol Res ; 240: 126532, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32622100

RESUMO

Bitespiramycin (biotechnological spiramycin, Bsm) is a new 16-membered macrolide antibiotic produced by Streptomyces spiramyceticus WSJ-1 integrated exogenous genes. The gene cluster for Bsm biosynthesis consists of two parts: spiramycin biosynthetic gene cluster (92 kb) and two exogenous genes including 4"-O-isovaleryltransferase gene (ist) and a positive regulatory gene (acyB2) from S. thermotolerans. Four putative regulatory genes, bsm2, bsm23, bsm27 and bsm42, were identified by sequence analysis in the spiramycin gene cluster. The inactivation of bsm23 or bsm42 in S. spiramyceticus eliminated spiramycin production, while the deletion of bsm2 and bsm27 did not abolish spiramycin biosynthesis. The acyB2 gene, homologous with bsm42 gene, cannot recover the spiramycin production in Δbsm42 mutant. The high expression of bsm42 significantly increased the spiramycin production, but overexpression of bsm23 inhibited its production in Δbsm23 and wild-type strain. Bsm23 was shown to be involved in the regulation of the expression of bsm42 and acyB2 by electrophoretic mobility shift assays. The bsm42 gene was also positive regulator for ist expression inferred from the improved yield of 4"-isovalerylspiramycins in the S. lividans TK24 biotransformation test, but adding bsm23 decreased the production of 4''-isovalerylspiramycins. These results demonstrated Bsm42 was a pathway-specific activator for spiramycin or Bsm biosynthesis, but overexpression of Bsm23 alone was adverse to produce these antibiotics although Bsm23 was essential for positive regulation of spiramycin production.

12.
Sci Rep ; 10(1): 9527, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32533102

RESUMO

We developed an inexpensive, portable platform for urea detection via electrochemistry by depositing silver nanoparticles (AgNPs) on a commercial glucose test strip. We modified this strip by first removing the enzymes from the surface, followed by electrodeposition of AgNPs on one channel (working electrode). The morphology of the modified test strip was characterized by Scanning Electron Microscopy (SEM), and its electrochemical performance was evaluated via Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS). We evaluated the performance of the device for urea detection via measurements of the dependency of peak currents vs the analyte concentration and from the relationship between the peak current and the square root of the scan rates. The observed linear range is 1-8 mM (corresponding to the physiological range of urea concentration in human blood), and the limit of detection (LOD) is 0.14 mM. The selectivity, reproducibility, reusability, and storage stability of the modified test strips are also reported. Additional tests were performed to validate the ability to measure urea in the presence of confounding factors such as spiked plasma and milk. The results demonstrate the potential of this simple and portable EC platform to be used in applications such as medical diagnosis and food safety.

13.
Med Mycol ; 2020 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-32453815

RESUMO

Candida albicans and Candida glabrata are frequently coisolated from the oral cavity in immunosuppressive or immunocompromised individuals. Their relationship is usually defined as competition as C. glabrata can inhibit growth of C. albicans in cohabitation. In this study, eight C. albicans isolates as well as two C. glabrata strains were used to investigate the effects of culture medium (Roswell Park Memorial Institute [RPMI]-1640, YPD, YND), incubation time (24 h, 48 h, 72 h, 96 h), initial inoculum (C. glabrata: C. albicans = 2:1, 1:1, 1:2), and medium state (static and dynamic states) on viable cell enumeration and relative abundance in both Candida SB and MB. The results showed that in most cases, C. glabrata and C. albicans SB and MB flourished in RPMI-1640 at 24 h under dynamic state compared with other conditions. Except YPD medium, there were high proportions of preponderance of C. albicans over C. glabrata in MB compared with SB. High initial inoculum promoted corresponding Candida number in both SB and MB and its abundance in MB relative to SB. This study revealed an impact of several environmental conditions on the formation of C. albicans and C. glabrata SB and MB and their abundance in MB in comparison with SB, deepening our understanding of both Candida interaction and their resistance mechanism in MB. LAY SUMMARY: This study described the effects of diverse experimental conditions on the numbers of Candida albicans and Candida glabrata single biofilms and mixed biofilms and their abundance.

14.
Nurs Health Sci ; 2020 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-32449839

RESUMO

Family caregivers play an important role in the rehabilitation of stroke survivors. The aims of this study were to describe preparedness, uncertainty, and knowledge regarding stroke in family caregivers of people who have undergone strokes and to investigate factors influencing preparedness. A total of 306 caregivers completed the questionnaires, including the Caregiver Preparedness Scale, the Mishel Uncertainty in Illness Scale, and Knowledge of Stroke. The result showed that the mean score of the family caregivers' preparedness was 14.42, the mean score of disease uncertainty was 75.62, and the mean score of stroke knowledge was 10.41. Caregiver preparedness was negatively correlated with disease uncertainty and positively correlated with knowledge. Multivariate stepwise regression analysis showed that educational background, profession, caregiving experience and uncertainty degree of the family caregivers, gender, and age of stroke survivor could predict 45.90% of the total variation in preparedness. The findings suggest that demographic characteristics of stroke survivor and family caregiver, as well as caregivers' disease uncertainty, enable predict the level of caregiver preparedness. Medical professionals should pay attention to providing personalized and targeted approaches to maximize caregivers' preparedness.

15.
Biofouling ; 36(3): 319-331, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32410461

RESUMO

Traditional herbal monomers (THMs) are widely distributed in many traditional Chinese formulas (TCFs) and decoctions (TCDs) and are frequently used for the prevention and treatment of fungal infections. The antifungal activities of five common THMs, including sodium houttuyfonate (SH), berberine (BER), palmatine (PAL), jatrorrhizine (JAT) and cinnamaldehyde (CIN), and their potential for inducing cell wall remodeling (CWR), were evaluated against Candida albicans SC5314 and Candida auris 12372. SH/CIN plus BER/PAL/JAT showed synergistic antifungal activity against both Candida isolates. Furthermore, SH-associated combinations (SH plus BER/PAL/JAT) induced stronger exposure of ß-glucan and chitin than their counterparts, while CIN triggered more marked exposure compared with CIN-associated combinations (CIN plus BER/PAL/JAT). Collectively, this study demonstrated the anti-Candida effect and the CWR induction potential of the five THMs and their associated combinations, providing a possibility of their in vivo application against fungal-associated infections.


Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candida/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Acroleína/análogos & derivados , Acroleína/farmacologia , Alcanos/farmacologia , Berberina/análogos & derivados , Berberina/farmacologia , Alcaloides de Berberina/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Sulfitos/farmacologia
16.
Microb Pathog ; 145: 104213, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32333954

RESUMO

Current problem of antibiotic resistance and the high incidence of bacterial diseases has brought huge losses to the yak breeding industry in Tibet. Therefore, the purpose of this study was to isolate Lactobacillus with safety and beneficial probiotic potential for the prophylaxis of intestinal diseases in yaks. After 16S rDNA sequence, four strains i.e. Lactobacillus sakei (named L4), Enterococcus hirae (named E5), Pediococcus acidilactici (named P7), Weissella confusa (named W8) were isolated from feces of yaks. The results of tolerance to acid, bile salt, enzyme and temperature showed that P7 was highly tolerant to acid, bile salt and digestive enzyme, while E5 was more resistant to temperature. The antibacterial assay showed L4 had a strong inhibitory effect against Staphylococcus aureus (BNCC186335), and E5, P7, W8 had effective antibacterial ability against Escherichia coli (C83902). In addition, L4, E5, P7 and W8 mainly produced organic acids and bacteriocin production to inhibit common intestinal pathogens. The results of antibiotic susceptibility assay indicated that L4, E5, P7 and W8 were highly sensitive to most clinically used antibiotics and didn't contain the VanA and VanB genes on the basis of PCR amplification, and L4, E5, P7 and W8 didn't exhibit hemolytic activity. The animal toxicity experiment results showed that no obvious pathological change was found in intestinal tissue sections, and L4, E5 and W8 strains also promoted the growth performance of mice, consequently, the L4, E5, P7 and W8 had no toxic effect on mice. In conclusion, lactobacillus isolated from feces of yaks not only have potential probiotics and strong antibacterial ability in vitro, but also are safe. Therefore, they have the potential to reduce the occurrence of bacterial diseases as new feed additives.

17.
IEEE Trans Cybern ; 2020 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-32224472

RESUMO

In the recently published paper, a switching method has been proposed to deal with the time derivative of the membership functions and less conservative results can be obtained due to this method; however, this method is based on the assumption that the switching times are finite. In this article, this method is further studied and the average dwell-time (ADT) switching technique is applied to ensure the stability if there is no such assumption. In addition, an algorithm is proposed to find the switching controller gains. The final simulation demonstrates the effectiveness of the developed new results.

18.
Mol Cell Endocrinol ; 508: 110791, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32173349

RESUMO

Calcium/calmodulin-dependent protein kinases (CaMKs) are a group of important molecules mediating calcium signal transmission and have been proved to participate in osteoclastogenesis regulation. CaMKII, a subtype of CaMKs is expressed during osteoclast differentiation, but its role in osteoclastogenesis regulation remains controversial. In the present study, we identified that both mRNA and protein levels of CaMKII (δ) were upregulated in a time-dependent manner during osteoclast differentiation. CaMKII (δ) gene silencing significantly inhibited osteoclast formation, bone resorption, and expression of osteoclast-related genes, including nuclear factor of activated T cells c1 (NFATc1), tartrate-resistant acid phosphatase (TRAP), and c-Src. Furthermore, CaMKII (δ) gene silencing downregulated phosphorylation of mitogen-activated protein kinases (MAPKs), including JNK, ERK, and p38, which were transiently activated by RANKL. Specific inhibitors of ERK, JNK, and p38 also markedly inhibited expression of osteoclast-related genes, osteoclast formation, and bone resorption like CaMKII (δ) gene silencing. Additionally, CaMKII (δ) gene silencing also suppressed RANKL-triggered CREB phosphorylation. Collectively, these data demonstrate the important role of CaMKII (δ) in osteoclastogenesis regulation through JNK, ERK, and p38 MAPKs and CREB pathway.

19.
Biochem Biophys Res Commun ; 524(4): 970-976, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32059847

RESUMO

Transforming growth factor-ß, a cell secretion factor of the TGF-ß superfamily, is involved in the regulation of cell proliferation, differentiation, cytoskeleton formation, migration, invasion and other biological behaviors. Autophagy and mitophagy play an important role in tumor progression by regulating self-digestion, and degradation and reuse of cells and mitochondria. In this study, changes in autophagy and mitophagy processes in ovarian cancer cells under TGF-ß1 treatment were detected via Western blot and immunofluorescence, as well as the role of fucosylation modification. Changes in mitochondrial membrane potential in response to TGF-ß1 and fucosylation were detected via immunofluorescence. The effects of TGF-ß1 and its fucosylation on autophagic flux were further determined by transient transfection of cells with Ad-mRFP-GFP-LC3 adenovirus. TGF-ß1 clearly promoted autophagy and mitophagy in ovarian cancer cells. TGF-ß1 fucosylation stimulated these regulatory effects on ovarian cancer cells via modulation of PI3K/Akt and Ras-Raf-MEK-ERK pathways through TAK1. Our collective data support the physiological significance of TGF-ß1 and provide a novel direction for targeted therapy for ovarian cancer.


Assuntos
Autofagia , Fucose/metabolismo , Mitofagia , Neoplasias Ovarianas/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Linhagem Celular Tumoral , Feminino , Glicosilação , Humanos , Sistema de Sinalização das MAP Quinases , Neoplasias Ovarianas/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Quinases raf/metabolismo , Proteínas ras/metabolismo
20.
Virology ; 541: 63-74, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32056716

RESUMO

GCNT3 (core 2ß-1,6-acetylglucosaminyltransferase) is a novel core mucin synthase. It is known that abnormal expression of GCNT3 promotes the progression of several human cancers. However, its relationship with Epstein-Barr virus (EBV) has not been comprehensively studied. We found GCNT3 expression in EBV-associated gastric cancer cells and tissues to be lower than in EBV-negative gastric cancer cells and tissues, and high expression was significantly associated with advanced tumor-lymph node metastasis. Luciferase reporter assay revealed that miR-BART1-5p directly targeted GCNT3. In addition, miR-BART1-5p mimics transfection was observed to reduce cell proliferation and migration, while miR-BART1-5p inhibitor increased cell proliferation and migration following transfection. In conclusion, both miR-BART1-5p and knockdown of GCNT3 inhibited cell proliferation and migration. In addition, EBV may regulate GCNT3 by affecting the NF-kB signaling pathway. E-cadherin, N-cadherin, vimentin, and p-ERK were found to be downstream molecules of the miR-BART1-5p/GCNT3 pathway.


Assuntos
N-Acetilglucosaminiltransferases/antagonistas & inibidores , Neoplasias Gástricas/patologia , Adulto , Idoso , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Herpesvirus Humano 4 , Humanos , Metástase Linfática , Masculino , MicroRNAs/fisiologia , Pessoa de Meia-Idade , N-Acetilglucosaminiltransferases/fisiologia , NF-kappa B/fisiologia , Transdução de Sinais/fisiologia , Neoplasias Gástricas/virologia
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