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1.
Lab Chip ; 20(3): 548-557, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-31942592

RESUMO

Non-small cell lung cancer (NSCLC) is one of the leading causes of death from cancer worldwide. The delivery and controlled regulation of miRNAs via exosomes is known as a potential therapeutic approach in the treatment of cancer. In this study, human cell-derived exosomes were used as delivery vehicles for miRNAs, and we investigated their anti-tumor and anti-angiogenic effects on NSCLCs that were cultured in 2D and 3D microfluidic devices. We demonstrated that exosomes that contained miRNA-497 (miR-497) effectively suppressed tumor growth and the expression of their associated genes, i.e., yes-associated protein 1 (YAP1), hepatoma-derived growth factor (HDGF), cyclin E1 (CCNE1), and vascular endothelial growth factor-A (VEGF-A), in A549 cells. Also, the level of VEGF-A-mediated angiogenic sprouting was decreased drastically in human umbilical vein endothelial cells (HUVECs) cultured in a microfluidic device. To mimic the in vivo-like tumor microenvironment of NSCLC, A549 cells were co-cultured with HUVECs in a single device, and miR-497-loaded exosomes were delivered to both types of cells. As a result, both the tube formation of endothelial cells and the migration of tumor decreased dramatically compared to the control. This indicated that miR-497 has synergistic inhibitory effects that target tumor growth and angiogenesis, so exosome-mediated miRNA therapeutics combined with the microfluidic technology could be a predictive, cost-efficient translational tool for the development of targeted cancer therapy.

2.
J Nat Prod ; 2020 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-31944695

RESUMO

Seven new stilbene glycosides including three dimers (1-3) and four monomers (4-7) were isolated from the roots of Polygonum multiflorum along with nine previously identified stilbenes (8-16). In addition, two deglucosylated stilbenes, 2a and 3a, were also obtained as new dimeric stilbenes. The structures of the purified phytochemicals were elucidated by interpreting their spectroscopic data (NMR, HRMS, and ECD). To the best of our knowledge, this represents the first isolation of a phenylpropanoid (C6-C3) substituted with a stilbene unit (7) from the Polygonaceae family. In an in vitro enzyme assay with human recombinant protein tyrosine phosphatase-1B (PTP1B), compounds 2-5 showed weak PTP1B inhibition with an IC50 value range of 27.4-37.6 µM, while three deglucosylated stilbenes 2a, 3a, and 8a exhibited IC50 values of 2.1, 1.9, and 12.1 µM, respectively. The inhibition modes and binding mechanism of selected inhibitors (2a and 3a) were investigated using kinetic methods and molecular docking simulations.

3.
Clin Infect Dis ; 70(3): 464-473, 2020 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-30891596

RESUMO

BACKGROUND: Endemic outbreaks of hantaviruses pose a critical public health threat worldwide. Hantaan orthohantavirus (HTNV) causes hemorrhagic fever with renal syndrome (HFRS) in humans. Using comparative genomic analyses of partial and nearly complete sequences of HTNV from humans and rodents, we were able to localize, with limitations, the putative infection locations for HFRS patients. Partial sequences might not reflect precise phylogenetic positions over the whole-genome sequences; finer granularity of rodent sampling reflects more precisely the circulation of strains. METHODS: Five HFRS specimens were collected. Epidemiological surveys were conducted with the patients during hospitalization. We conducted active surveillance at suspected HFRS outbreak areas. We performed multiplex polymerase chain reaction-based next-generation sequencing to obtain the genomic sequence of HTNV from patients and rodents. The phylogeny of human- and rodent-derived HTNV was generated using the maximum likelihood method. For phylogeographic analyses, the tracing of HTNV genomes from HFRS patients was defined on the bases of epidemiological interviews, phylogenetic patterns of the viruses, and geographic locations of HTNV-positive rodents. RESULTS: The phylogeographic analyses demonstrated genetic clusters of HTNV strains from clinical specimens, with HTNV circulating in rodents at suspected sites of patient infections. CONCLUSIONS: This study demonstrates a major shift in molecular epidemiological surveillance of HTNV. Active targeted surveillance was performed at sites of suspected infections, allowing the high-resolution phylogeographic analysis to reveal the site of emergence of HTNV. We posit that this novel approach will make it possible to identify infectious sources, perform disease risk assessment, and implement preparedness against vector-borne viruses.

4.
Radiology ; 294(1): 31-41, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31769740

RESUMO

Background Previous studies have suggested that texture analysis is a promising tool in the diagnosis, characterization, and assessment of treatment response in various cancer types. Therefore, application of texture analysis may be helpful for early prediction of pathologic response in breast cancer. Purpose To investigate whether texture analysis of features from MRI is associated with pathologic complete response (pCR) to neoadjuvant chemotherapy (NAC) in breast cancer. Materials and Methods This retrospective study included 136 women (mean age, 47.9 years; range, 31-70 years) who underwent NAC and subsequent surgery for breast cancer between January 2012 and August 2017. Patients were monitored with 3.0-T MRI before (pretreatment) and after (midtreatment) three or four cycles of NAC. Texture analysis was performed at pre- and midtreatment T2-weighted MRI, contrast material-enhanced T1-weighted MRI, diffusion-weighted MRI, and apparent diffusion coefficient (ADC) mapping by using commercial software. A random forest method was applied to build a predictive model for classifying those with pCR with use of texture parameters. Diagnostic performance for predicting pCR was assessed and compared with that of six other machine learning classifiers (adaptive boosting, decision tree, k-nearest neighbor, linear support vector machine, naive Bayes, and linear discriminant analysis) by using the Wald test and DeLong method. Results Forty of the 136 patients (29%) achieved pCR after NAC. In the prediction of pCR, the random forest classifier showed the lowest diagnostic performance with pretreatment ADC (area under the receiver operating characteristic curve [AUC], 0.53; 95% confidence interval: 0.44, 0.61) and the highest diagnostic performance with midtreatment contrast-enhanced T1-weighted MRI (AUC, 0.82; 95% confidence interval: 0.74, 0.88) among pre- and midtreatment T2-weighted MRI, contrast-enhanced T1-weighted MRI, diffusion-weighted MRI, and ADC mapping. Conclusion Texture parameters using a random forest method of contrast-enhanced T1-weighted MRI at midtreatment of neoadjuvant chemotherapy were valuable and associated with pathologic complete response in breast cancer. © RSNA, 2019 Online supplemental material is available for this article.

5.
Cancer Immunol Res ; 8(1): 46-56, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31554638

RESUMO

Regulatory T cells (Treg) are targeted for cancer immunotherapy because they suppress antitumor immunity. Although the importance of neuropilin-1 (NRP1) in the stability and function of intratumoral Tregs is well-documented, targeting of NRP1+ Tregs for anticancer immunotherapy has not been well explored. Here, we found that an NRP1 antagonist [Fc(AAG)-TPP11], generated by fusion of the NRP1-specific binding peptide TPP11 with the C-terminus of an effector function-deficient immunoglobulin Fc(AAG) variant, inhibits intratumoral NRP1+ Treg function and stability. Fc(AAG)-TPP11 triggered the internalization of NRP1, reducing its surface expression on Tregs and thereby inhibiting the suppressive function of Tregs. In two murine syngeneic tumor models, Fc(AAG)-TPP11 retarded tumor growth, comparable with a Treg-depleting anti-CTLA-4 antibody, without noticeable toxicity. Fc(AAG)-TPP11 inhibited NRP1-dependent Treg function, inducing unstable intratumoral Tregs, with reduced expression of Foxp3 and enhanced production of IFNγ, which subsequently increased the functionality and frequency of intratumoral CD8+ T cells. We also observed selective expression of NRP1 on Tregs isolated from human tumors, but not from the blood of healthy donors and patients with cancer, as well as ex vivo inhibition of intratumoral NRP1+ Treg function by Fc(AAG)-TPP11. Our results suggest that the NRP1 antagonist Fc(AAG)-TPP11 has therapeutic potential for the inhibition of intratumoral NRP1+ Tregs with limited unfavorable effects on peripheral Tregs.

6.
Eur Radiol ; 2019 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-31802216

RESUMO

PURPOSE: To investigate whether monitoring with ultrasound and MR imaging before, during and after neoadjuvant chemotherapy (NAC) can predict axillary response in breast cancer patients. MATERIALS AND METHODS: A total of 131 breast cancer patients with clinically positive axillary lymph node (LN) who underwent NAC and subsequent surgery were enrolled. They had ultrasound and 3.0 T-MR examinations before, during and after NAC. After reviewing ultrasound and MR images, axillary LN features and tumour size (T size) were noted. According to LN status after surgery, imaging features and their diagnostic performances were analysed. RESULTS: Of the 131 patients, 60 (45.8%) had positive LNs after surgery. Pre-NAC T size at ultrasound and MR was different in positive LN status after surgery (p < 0.01). There were significant differences in mid- and post-NAC number, cortical thickness (CxT), T size and T size reduction at ultrasound and mid- and post-NAC CxT, hilum, T size and T size reduction, and post-NAC ratio of diameter at MR (p < 0.03). On multivariate analysis, pre-NAC MR T size (OR, 1.03), mid-NAC ultrasound T size (OR, 1.05) and CxT (OR, 1.53), and post-NAC MR T size (OR, 1.06) and CxT (OR, 1.64) were independently associated with positive LN (p < 0.004). Combined mid-NAC ultrasound T size and CxT showed the best diagnostic performance with AUC of 0.760. CONCLUSION: Monitoring ultrasound and MR axillary LNs and T size can be useful to predict axillary response to NAC in breast cancer patients. KEY POINTS: • Monitoring morphologic features of LNs is useful to predict axillary response. • Monitoring tumour size by imaging is useful to predict axillary response. • The axillary ultrasound during NAC showed the highest diagnostic performance.

8.
Korean J Radiol ; 20(12): 1646-1652, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31854152

RESUMO

OBJECTIVE: To develop a scoring system stratifying the malignancy risk of mammographic microcalcifications using the 5th edition of the Breast Imaging Reporting and Data System (BI-RADS). MATERIALS AND METHODS: One hundred ninety-four lesions with microcalcifications for which surgical excision was performed were independently reviewed by two radiologists according to the 5th edition of BI-RADS. Each category's positive predictive value (PPV) was calculated and a scoring system was developed using multivariate logistic regression. The scores for benign and malignant lesions or BI-RADS categories were compared using an independent t test or by ANOVA. The area under the receiver operating characteristic curve (AUROC) was assessed to determine the discriminatory ability of the scoring system. Our scoring system was validated using an external dataset. RESULTS: After excision, 69 lesions were malignant (36%). The PPV of BI-RADS descriptors and categories for calcification showed significant differences. Using the developed scoring system, mean scores for benign and malignant lesions or BI-RADS categories were significantly different (p < 0.001). The AUROC of our scoring system was 0.874 (95% confidence interval, 0.840-0.909) and the PPV of each BI-RADS category determined by the scoring system was as follows: category 3 (0%), 4A (6.8%), 4B (19.0%), 4C (68.2%), and 5 (100%). The validation set showed an AUROC of 0.905 and PPVs of 0%, 8.3%, 11.9%, 68.3%, and 94.7% for categories 3, 4A, 4B, 4C, and 5, respectively. CONCLUSION: A scoring system based on BI-RADS morphology and distribution descriptors could be used to stratify the malignancy risk of mammographic microcalcifications.

9.
Ultraschall Med ; 2019 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-31703239

RESUMO

PURPOSE: To identify and compare diagnostic performance of radiomic features between grayscale ultrasound (US) and shear-wave elastography (SWE) in breast masses. MATERIALS AND METHODS: We retrospectively collected 328 pathologically confirmed breast masses in 296 women who underwent grayscale US and SWE before biopsy or surgery. A representative SWE image of the mass displayed with a grayscale image in split-screen mode was selected. An ROI was delineated around the mass boundary on the grayscale image and copied and pasted to the SWE image by a dedicated breast radiologist for lesion segmentation. A total of 730 candidate radiomic features including first-order statistics and textural and wavelet features were extracted from each image. LASSO regression was used for data dimension reduction and feature selection. Univariate and multivariate logistic regression was performed to identify independent radiomic features, differentiating between benign and malignant masses with calculation of the AUC. RESULTS: Of 328 breast masses, 205 (62.5 %) were benign and 123 (37.5 %) were malignant. Following radiomic feature selection, 22 features from grayscale and 6 features from SWE remained. On univariate analysis, all 6 SWE radiomic features (P < 0.0001) and 21 of 22 grayscale radiomic features (P < 0.03) were significantly different between benign and malignant masses. After multivariate analysis, three grayscale radiomic features and two SWE radiomic features were independently associated with malignant breast masses. The AUC was 0.929 for grayscale US and 0.992 for SWE (P < 0.001). CONCLUSION: US radiomic features may have the potential to improve diagnostic performance for breast masses, but further investigation of independent and larger datasets is needed.

10.
Sci Rep ; 9(1): 16631, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31719616

RESUMO

Orthohantaviruses, negative-sense single-strand tripartite RNA viruses, are a global public health threat. In humans, orthohantavirus infection causes hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome. Whole-genome sequencing of the virus helps in identification and characterization of emerging or re-emerging viruses. Next-generation sequencing (NGS) is a potent method to sequence the viral genome, using molecular enrichment methods, from clinical specimens containing low virus titers. Hence, a comparative study on the target enrichment NGS methods is required for whole-genome sequencing of orthohantavirus in clinical samples. In this study, we used the sequence-independent, single-primer amplification, target capture, and amplicon NGS for whole-genome sequencing of Hantaan orthohantavirus (HTNV) from rodent specimens. We analyzed the coverage of the HTNV genome based on the viral RNA copy number, which is quantified by real-time quantitative PCR. Target capture and amplicon NGS demonstrated a high coverage rate of HTNV in Apodemus agrarius lung tissues containing up to 103-104 copies/µL of HTNV RNA. Furthermore, the amplicon NGS showed a 10-fold (102 copies/µL) higher sensitivity than the target capture NGS. This report provides useful insights into target enrichment NGS for whole-genome sequencing of orthohantaviruses without cultivating the viruses.

11.
World J Stem Cells ; 11(10): 803-816, 2019 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-31693013

RESUMO

Although the recent advances in stem cell engineering have gained a great deal of attention due to their high potential in clinical research, the applicability of stem cells for preclinical screening in the drug discovery process is still challenging due to difficulties in controlling the stem cell microenvironment and the limited availability of high-throughput systems. Recently, researchers have been actively developing and evaluating three-dimensional (3D) cell culture-based platforms using microfluidic technologies, such as organ-on-a-chip and organoid-on-a-chip platforms, and they have achieved promising breakthroughs in stem cell engineering. In this review, we start with a comprehensive discussion on the importance of microfluidic 3D cell culture techniques in stem cell research and their technical strategies in the field of drug discovery. In a subsequent section, we discuss microfluidic 3D cell culture techniques for high-throughput analysis for use in stem cell research. In addition, some potential and practical applications of organ-on-a-chip or organoid-on-a-chip platforms using stem cells as drug screening and disease models are highlighted.

12.
Adv Sci (Weinh) ; 6(20): 1900962, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31637161

RESUMO

Harmful materials in the blood are prevented from entering the healthy brain by a highly selective blood-brain barrier (BBB), and impairment of barrier function has been associated with a variety of neurological diseases. In Alzheimer's disease (AD), BBB breakdown has been shown to occur even before cognitive decline and brain pathology. To investigate the role of the cerebral vasculature in AD, a physiologically relevant 3D human neural cell culture microfluidic model is developed having a brain endothelial cell monolayer with a BBB-like phenotype. This model is shown to recapitulate several key aspects of BBB dysfunction observed in AD patients: increased BBB permeability, decreased expression of claudin-1, claudin-5, and VE-cadherin, increased expression of matrix-metalloproteinase-2 and reactive oxygen species, and deposition of ß-amyloid (Aß) peptides at the vascular endothelium. Thus, it provides a well-controlled platform for investigating BBB function as well as for screening of new drugs that need to pass the BBB to gain access to neural tissues.

13.
Am J Ophthalmol ; 208: 265-272, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31449792

RESUMO

PURPOSE: To evaluate the long-term incidence and risk of glaucoma after bilateral congenital cataract surgery in microphthalmic eyes. DESIGN: Retrospective, observational case series. METHODS: Subjects: Children with microphthalmic eyes who had undergone surgery for bilateral congenital cataract within 6 months of birth and been followed up for at least 5 years. PROCEDURES: Review of medical records at our institution. MAIN OUTCOME MEASURES: Probability of an eye's developing glaucoma after bilateral congenital cataract surgery and associated risk factors. RESULTS: Thirty-eight eyes of 19 children with bilateral congenital cataract were included. The mean age at surgery was 3.2 ± 1.7 months, and the mean follow-up duration was 7.79 ± 2.61 years. After cataract surgery, 11 eyes (29.0%) developed glaucoma at the age of 4.0 ± 1.4 years. Three of these eyes underwent Ahmed glaucoma valve implantation surgery. The probability of an eye's developing glaucoma was estimated to be 32.0% by 10 years after surgery. In a multivariate analysis, axial length was significantly associated with glaucoma development (odds ratio = 0.364, P = .025). Age at the time of cataract surgery, corneal diameter, and aphakia did not affect the risk of glaucoma (P > .10). Eyes without glaucoma had a better final visual outcome than those with glaucoma (0.75 ± 0.60 and 1.47 ± 1.10 logMAR, respectively, P = .049). CONCLUSIONS: The long-term cumulative risk of postoperative glaucoma development was 32.0% by 10 years after bilateral congenital cataract surgery. Because the risk of developing glaucoma persists for several years after surgery, careful monitoring and control of intraocular pressure is needed to preserve vision in such patients.

14.
Fitoterapia ; 137: 104261, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31284019

RESUMO

Three new sesquilignans, zijusesquilignans A-C (1-3), together with fifteen known compounds (4-18), were isolated from fruits of Ziziphus jujuba var. inermis Rehder (Rhamnaceae). Their chemical structures were established using spectroscopic analyses including 1D- and 2D-NMR, HR-EIMS, and ECD spectra. These compounds were assessed for their inhibitory effects on nitric oxide (NO) production. Of these compounds, 1-3 and 17 displayed inhibitory effects on NO production, with IC50 values ranging from 18.1 to 66.4 µM. Pretreatment with 1 and 17 significantly suppressed LPS-induced expression of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein in cells. Moreover, compounds 1-3, 7, 9, and 17 exhibited cytotoxic activities against three human tumor cell lines, with IC50 values ranging from 8.4 to 44.9 µM.


Assuntos
Anti-Inflamatórios/farmacologia , Frutas/química , Lignanas/farmacologia , Ziziphus/química , Animais , Anti-Inflamatórios/isolamento & purificação , Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/metabolismo , Humanos , Lignanas/isolamento & purificação , Camundongos , Estrutura Molecular , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Células RAW 264.7 , República da Coreia
15.
Invest Ophthalmol Vis Sci ; 60(7): 2423-2430, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31158274

RESUMO

Purpose: To investigate intereye differences in lamina cribrosa (LC) morphology in normal tension glaucoma (NTG) patients with unilateral damage. Methods: A total of 152 eyes of 76 treatment-naive NTG patients with unilateral damage from the ongoing Investigating Glaucoma Progression Study were included. Optic nerve heads were scanned using enhanced-depth spectral-domain optical coherence tomography. The magnitude of the LC curve and LC position were assessed by measuring the LC curve index (LCCI) and LC depth (LCD), respectively, at seven locations spaced equidistantly across the vertical optic disc diameter. LCCI and LCD were compared between glaucomatous and fellow healthy eyes. Results: Eyes with NTG had larger average LCCI and LCD than contralateral healthy eyes (for both P < 0.002). The LCCI was greater in the glaucomatous eyes at all seven locations (P < 0.001). Univariate conditional logistic regression analysis showed that higher baseline intraocular pressure (P = 0.010), deeper LCD (P = 0.007), and larger LCCI (P < 0.001) were significantly associated with the presence of glaucoma. In multivariate analysis, only larger LCCI was significantly associated with the presence of glaucoma (P < 0.001). Conclusions: Glaucomatous eyes have more steeply curved LC than fellow healthy eyes. This finding suggests that LC undergoes significant remodeling in NTG eyes.


Assuntos
Glaucoma de Baixa Tensão/diagnóstico , Disco Óptico/patologia , Doenças do Nervo Óptico/diagnóstico , Idoso , Estudos Transversais , Feminino , Gonioscopia , Humanos , Pressão Intraocular/fisiologia , Glaucoma de Baixa Tensão/fisiopatologia , Masculino , Pessoa de Meia-Idade , Disco Óptico/diagnóstico por imagem , Doenças do Nervo Óptico/fisiopatologia , Estudos Prospectivos , Tomografia de Coerência Óptica/métodos , Tonometria Ocular , Acuidade Visual/fisiologia , Testes de Campo Visual , Campos Visuais/fisiologia
16.
Xenobiotica ; : 1-9, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31233374

RESUMO

1. Glycyrol is a coumestan derivative that is isolated from roots of Glycyrrhiza uralensis. Glycyrol exhibits several biological effects, including anti-oxidative and anti-inflammatory effects. 2. Herein, we characterized glycyrol metabolism by cytochrome P450 enzymes (CYPs) and UDP-glucuronosyltransferases (UGTs) using human liver microsomes (HLM), human liver cytosol, human intestinal microsomes, or human recombinant cDNA-expressed CYPs and UGTs. The analysis was conducted using high resolution mass spectroscopy (HR-MS) on a Q ExactiveTM HF Hybride Quadrupole-Orbitrap mass spectrometer. 3. NADPH-supplemented HLM generated six glycyrol metabolites (M1-M6) via hydroxylation, oxidation, and hydration; both NADPH- and UDPGA-supplemented liver microsomes generated three glucuronides (M7-M9). Reaction phenotyping revealed that CYP1A2 is the primary enzyme responsible for phase I metabolism, with minor involvement of the CYP3A4/5, CYP2D6, and CYP2E1 enzymes. Glucuronidation of glycyrol was primarily mediated by UGT1A1, UGT1A3, UGT1A9, and UGT2B7. 4. In conclusion, glycyrol undergoes the efficient metabolic hydroxylation and glucuronidation reactions in human liver microsomes, which are predominantly catalyzed by CYP1A2, UGT1A1/3/9, and UGT2B7.

17.
Sci Rep ; 9(1): 7772, 2019 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-31123339

RESUMO

Development of antagonistic antibody (Ab) against interleukin-4 receptor alpha (IL-4Rα) subunit of IL-4/IL-13 receptors is a promising therapeutic strategy for T helper 2 (TH2)-mediated allergic diseases such as asthma and atopic dermatitis. Here we isolated anti-human IL-4Rα antagonistic Abs from a large yeast surface-displayed human Ab library and further engineered their complementarity-determining regions to improve the affinity using yeast display technology, finally generating a candidate Ab, 4R34.1.19. When reformatted as human IgG1 form, 4R34.1.19 specifically bound to IL-4Rα with a high affinity (KD ≈ 178 pM) and effectively blocked IL-4- and IL-13-dependent signaling in a reporter cell system at a comparable level to that of the clinically approved anti-IL-4Rα dupilumab Ab analogue. Epitope mapping by alanine scanning mutagenesis revealed that 4R34.1.19 mainly bound to IL-4 binding sites on IL-4Rα with different epitopes from those of dupilumab analogue. Further, 4R34.1.19 efficiently inhibited IL-4-dependent proliferation of T cells among human peripheral blood mononuclear cells and suppressed the differentiation of naïve CD4+ T cells from healthy donors and asthmatic patients into TH2 cells, the activities of which were comparable to those of dupilumab analogue. Our work demonstrates that both affinity and epitope are critical factors for the efficacy of anti-IL-4Rα antagonistic Abs.

18.
Mol Cells ; 42(5): 418-425, 2019 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-31085809

RESUMO

Multicistronic elements, such as the internal ribosome entry site (IRES) and 2A-like cleavage sequence, serve crucial roles in the eukaryotic ectopic expression of exogenous genes. For utilization of multicistronic elements, the cleavage efficiency and order of elements in multicistronic vectors have been investigated; however, the dynamics of multicistronic element-mediated expression remains unclear. Here, we investigated the dynamics of encephalomyocarditis virus (EMCV) IRES- and porcine teschovirus-1 2A (p2A)-mediated expression. By utilizing real-time fluorescent imaging at a minute-level resolution, we monitored the expression of fluorescent reporters bridged by either EMCV IRES or p2A in two independent cultured cell lines, HEK293 and Neuro2a. We observed significant correlations for the two fluorescent reporters in both multicistronic elements, with a higher correlation coefficient for p2A in HEK293 but similar coefficients for IRES-mediated expression and p2A-mediated expression in Neuro2a. We further analyzed the causal relationship of multicistronic elements by convergent cross mapping (CCM). CCM revealed that in all four conditions examined, the expression of the preceding gene causally affected the dynamics of the subsequent gene. As with the cross correlation, the predictive skill of p2A was higher than that of IRES in HEK293, while the predictive skills of the two multicistronic elements were indistinguishable in Neuro2a. To summarize, we report a significant temporal correlation in both EMCV IRES- and p2A-mediated expression based on the simple bicistronic vector and real-time fluorescent monitoring. The current system also provides a valuable platform to examine the dynamic aspects of expression mediated by diverse multicistronic elements under various physiological conditions.


Assuntos
Vírus da Encefalomiocardite/genética , Sítios Internos de Entrada Ribossomal/genética , Teschovirus/genética , Animais , Vírus da Encefalomiocardite/metabolismo , Regulação Viral da Expressão Gênica , Vetores Genéticos , Proteínas de Fluorescência Verde , Células HEK293 , Humanos , Proteínas Luminescentes , Camundongos , Modelos Moleculares , Teschovirus/metabolismo
19.
Toxicol In Vitro ; 59: 115-125, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30980863

RESUMO

Bisphenol A [BPA, 2,2-bis-(4-hydroxyphenyl)propane] is one of the most prevalent synthetic environmental estrogens; as an endocrine disruptor, it is associated with endocrine-related cancers including breast, ovarian, and prostate. However, the mechanisms by which BPA contributes to carcinogenesis are unclear. This study aims to clarify its toxic effects on mitotic cells and investigate the molecular mechanism. In vitro effects of BPA on mitotic progression were examined by performing experiments on HeLa cells. Proteins involved in mitotic processes were detected by Western blot, live cell imaging, and immunofluorescence staining. The results showed that BPA increased chromosomal instability by perturbing mitotic processes such as bipolar spindle formation and spindle microtubule attachment to the kinetochore. BPA prolonged mitotic progression by disturbing spindle attachment and concomitant activating spindle assembly checkpoint (SAC). Mechanistically, BPA interfered proper localization of HURP to the proximal ends of spindle microtubules, Kif2a to the minus ends of spindle microtubules, and TPX2 on the mitotic spindle. This mislocalization of microtubule associated proteins (MAPs) is postulated to lead to spindle attachment failure. Furthermore, BPA caused multipolar spindle by inducing centriole overduplication and premature disengagement. Although BPA acts as an estrogen receptor (ER) agonist, mitotic defects caused by BPA occurred in an ER-independent manner. Our findings indicate that BPA may stimulate carcinogenesis not only by acting as an endocrine disruptor but also by increasing chromosomal instability during mitosis.


Assuntos
Compostos Benzidrílicos/toxicidade , Disruptores Endócrinos/toxicidade , Mitose/efeitos dos fármacos , Fenóis/toxicidade , Carcinogênese/induzido quimicamente , Centríolos/efeitos dos fármacos , Instabilidade Cromossômica/efeitos dos fármacos , Células HeLa , Humanos , Cinetocoros/efeitos dos fármacos , Células MCF-7 , Proteínas de Neoplasias/metabolismo
20.
Nat Prod Res ; : 1-7, 2019 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-30961363

RESUMO

Four new diterpene-type compounds normiltirone (3) and isosalviamides F-H (14-16) together with twelve known compounds (1, 2, 4-13) were isolated from the roots of Salvia miltiorrhiza. Their structures were mainly elucidated from detailed spectroscopic data. All isolated compounds were evaluated for their ability to inhibit lipopolysaccharide-induced nitric oxide production in RAW264.7 macrophages. Compound 11 showed a strong inhibitory effect, with an IC50 value of 3.4 ± 1.2 µM.

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