MOLECULES
·
2018
·Vol. 23
·期 7
·1594
·
IF 3.098
Jiaoyu Wang,Xiaoyu Guo,Ling Li,Haiping Qiu,Zhen Zhang,Yanli Wang,Guochang Sun
Rice blast is one of the most serious diseases affecting rice yield which is caused byMagnaporthe oryzae, a model organism for studies on plant pathogenic fungi. Lipids stored inM. oryzaecells have been shown to be crucial for the development of appressorium turgor and the ability of the pathogen to cause infection. Nile red staining is a common method to study lipid dynamics in phytopathogenic fungi. However, the disadvantages of this dye include its wide spectrum, poor water solubility, and susceptibility to quenching. Boron dipyrromethene (BODIPY) is a new type of fluorescent dye that has a different emission wavelength to that of Nile red as well as many desirable spectral and chemical properties. In this study, we used BODIPY to stain the lipids inM. oryzaecells to seek a possible substitute to Nile red in the study of lipid dynamics in plant pathogenic fungi. Our data showed that through simple and routine procedures, BODIPY was able to distinctly label lipids in the cells of mycelia and conidia. The positions of lipids labeled by BODIPY were essentially identical to those labeled by Nile red, but with more clear fluorescence labelling, lower background, and higher specificity. The use of BODIPY to stain germinatingM. oryzaeconidia allowed the lipid dynamics to be clearly tracked during this process. We also achieved double and multiple fluorescent staining conidia by combining BODIPY with the red fluorescent protein mCherry and other fluorescent dyes, such as Calcofluor white and DAPI, in conidia, mycelia, and sexual structures ofM. oryzae. These results indicate that BODIPY is an ideal fluorescent dye for staining fungal lipids and provide a method for the study of the lipid dynamics and lipid metabolism in plant pathogenic fungi.Keywords:Magnaporthe oryzae;lipid dynamics;BODIPY;fluorescent staining
研究领域:
植物病理学
·
真菌学
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DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF PHARMACEUTICS
·
2021
·
IF 5.875
Teng Guo,Jianying Lu,Yunlong Fan,Yongtai Zhang,Shuo Yin,Xianyi Sha,Nianping Feng
Combined therapy with anti-inflammatory drugs is preferred for the topical treatment of psoriasis , but the codelivery of drugs is restricted due to the lack of a suitable delivery system. Ethosomes with excellent transdermal properties are perfect as carriers for hyperplastic skin. Therefore, glycyrrhetinic acid-D-α-tocopherol acid polyethylene glycol succinate (GA-TPGS) was synthesized, which prevented the inflammation and lipid peroxidation damage, thus effectively stabilizing the psoriasis . Then GA-TPGS was surface-modified on the curcumin (Cur) loaded ethosomes to construct curcumin-loaded GA-TPGS-modified multifunctional ethosomes (Cur@GA-TPGS-ES), exerting synergistic treatment for psoriasis. Using an interleukin-6-induced cell model, we found that Cur@GA-TPGS-ES displayed desirable suppression of inflammation response and oxidative stress damage. Compared with the ethanol solution, the percutaneous penetration rates of Cur and GA in Cur@GA-TPGS-ES were superior. In vivo microdialysis revealed similar results, suggesting an increase of transcutaneous absorption in Cur@GA-TPGS-ES. Fluorescence staining revealed that the cellular uptake and skin distribution were distinctly enhanced with the delivery by Cur@GA-TPGS-ES. After topical administration to imiquimod-induced psoriatic mice, the Cur@GA-TPGS-ES group showed powerful treatment from inflammatory infiltration inhibition of Cur, glucocorticoid-like effects of GA and anti-lipid peroxidation of TPGS. Overall, GA-TPGS mediated ethosomes possess more advantageous transdermal properties and synergistic antipsoriatic efficacy.
研究领域:
药剂学
·
皮肤病学
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产品关联
DOI
PubMed
高影响因子
STAR Protocols
·
2021
·
IF 0
Yuqian Shen,Wenhua Liu,Jian Zuo,Junhai Han,Zi Chao Zhang
Summary Selective identification of newly synthesized proteins is challenging because all proteins, both existing and nascent, have the same amino acid pool and are therefore chemically indistinguishable. L-homopropargylglycine is an amino acid analog of methionine containing an alkyne moiety that can undergo a classic click chemical reaction with azide containing Alexa Fluor. Here, we present an integrated tool based on immunofluorescence staining to accurately trace and localize the newly synthesized protein in isolated primary mouse hepatocytes. For complete details on the use and execution of this protocol, please refer to Shen et al. (2021) .
研究领域:
生物化学
·
细胞生物学
✓ 已验证
DOI
PubMed
PLANT CELL REPORTS
·
2024
·Vol. 43
·期 7
·1-15
·
IF 5.3
Wang Lulu,Zeng Fang,Jiao Yongjuan,Zhou Qingqing,An Jinjie,Gao Hongbo
Key message Immunofluorescence staining with frozen sections of plant tissues and a nest tube is convenient and effective, and broadens the applicability of immunofluorescence staining. Abstract Immunofluorescence staining is an indispensable and extensively employed technique for determining the subcellular localization of chloroplast division proteins. At present, it is difficult to effectively observe the localization of target proteins in leaves that are hard, or very thin, or have epidermal hair or glands with the current immunofluorescence staining methods. Moreover, signals of target proteins were predominantly detected in mesophyll cells, not the cells of other types. Thus, the method of immunofluorescence staining was further explored for improvement in this study. The plant tissue was embedded with 50% PEG4000 at −60℃, which was then cut into sections by a cryomacrotome. The sections were immediately immersed in fixation solution. Then, the sample was transferred into a special nested plastic tube, which facilitated the fixation and immunofluorescence staining procedures. The use of frozen sections in this method enabled a short processing time and reduced material requirements. By optimizing the thickness of the sections, a large proportion of the cells could be well stained. With this method, we observed the localization of a chloroplast division protein FtsZ1 in the wild-type Arabidopsis and various chloroplast division mutants. Meanwhile, the localization of FtsZ1 was also observed not only in mesophyll cells, but also in guard cells and epidermal cells in a lot of other plant species, including many species with hard leaf tissues. This method is not only easy to use, but also expands the scope of applicability for immunofluorescence staining.
研究领域:
植物生物学
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细胞生物学
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分子生物学
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产品关联
DOI
PubMed
高影响因子
MICROVASCULAR RESEARCH
·
2021
·
IF 3.514
Keke Ge,Yatao Wang,Pan Li,Miao Li,Wenhua Zhang,Handong Dan,Xumeng Hu,Jiamu Zhou,Qixiang Yang,Jiaojiao Wang,Zongming Song
The investigation aimed to evaluate the effects of Mcc950, an inhibitor of the NLRP3 inflammasome , on diabetic retinopathy (DR) mice. The general physiological condition of each group of mice was recorded. Retinal blood vessels were stained for observation of the density of blood vessels, and retinas were used for further morphological examination and fluorescent staining after the intravitreal injection of Mcc950. Mcc950 partially reversed hyperglycemia-induced vascular damage and had reduced histological changes compared to DR mice. IL-1β production in mice retinas in the diabetic model (DM) group increased, but pretreatment with Mcc950 significantly reversed these changes. Additionally, Mcc950 engineered reduced FITC dextran extravasation and vascular leakage. Therefore, it played an apparent protective role in DR and could be a new treatment strategy for DR.
研究领域:
医学
·
眼科
·
免疫学
·
药理学
✓ 已验证
DOI
PubMed
高影响因子
Frontiers in Pharmacology
·
2023
·
IF 5.6
Li Jingyan,Wu Bingmin,Zeng Lishan,Lin Ying,Chen Qiuhe,Wang Haixia,An Lin,Zhang Jiajun,Chen Siyan,Huang Junying,Zhan Ruoting,Zhang Guifang
Background: The present study aimed to investigate the protective effect of the water extract of Amydrium sinense (Engl.) H. Li (ASWE) against hepatic fibrosis (HF) and clarify the underlying mechanism.Methods: The chemical components of ASWE were analysed by a Q-Orbitrap high-resolution mass spectrometer. In our study, an in vivo hepatic fibrosis mouse model was established via an intraperitoneal injection of olive oil containing 20% CCl4. In vitro experiments were conducted using a hepatic stellate cell line (HSC-T6) and RAW 264.7 cell line. A CCK-8 assay was performed to assess the cell viability of HSC-T6 and RAW264.7 cells treated with ASWE. Immunofluorescence staining was used to examine the intracellular localization of signal transducer and activator of transcription 3 (Stat3). Stat3 was overexpressed to analyse the role of Stat3 in the effect of ASWE on HF.Results: Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that candidate targets of ASWE, associated with protective effects against hepatic fibrosis, were related to inflammation response. ASWE ameliorated CCl4-induced liver pathological damage and reduced the liver index and alanine transaminase (ALT) and aspartate transaminase (AST) levels. ASWE also decreased the serum levels of collagen Ⅰ (Col Ⅰ) and hydroxyproline (Hyp) in CCl4-treated mice. In addition, the expression of fibrosis markers, including α-SMA protein and Acta2, Col1a1, and Col3a1 mRNA, was downregulated by ASWE treatment in vivo. The expression of these fibrosis markers was also decreased by treatment with ASWE in HSC-T6 cells. Moreover, ASWE decreased the expression of inflammatory markers, including the Tnf-α, Il6 and Il1β, in RAW264.7 cells. ASWE decreased the phosphorylation of Stat3 and total Stat3 expression and reduced the mRNA expression of the Stat3 gene in vivo and in vitro. ASWE also inhibited the nuclear shuttling of Stat3. Overexpression of Stat3 weakened the therapeutic effect of ASWE and accelerated the progression of HF.Conclusion: The results show that ASWE protects against CCl4-induced liver injury by suppressing fibrosis, inflammation, HSC activation and the Stat3 signaling pathway, which might lead to a new approach for preventing HF.
研究领域:
药理学
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肝病学
·
分子生物学
·
细胞生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-ANIMAL
·
2023
·
IF 2.1
Ke Shenghui,Feng Yiwei,Luo Liying,Qin Wanzhao,Liu Huayu,Nie Jingchong,Liang Beijiang,Ma Hongjie,Xie Mao,Li Jingyu,Niu Zhijie,Li Guojian,Tang Anzhou,Xia Wei,He Guangyao
A method for the in vitro isolation, purification, identification, and induced differentiation of satellite cells from adult tree shrew skeletal muscle was established. The mixed enzyme digestion method and differential adhesion method were used to obtain skeletal muscle satellite cells, which were identified and induced to differentiate to verify their pluripotency. The use of a mixture of collagenase II, hyaluronidase IV, and DNase I is an efficient method for isolating adult tree shrew skeletal muscle satellite cells. The P3 generation of cells had good morphology, rapid proliferation, high viability, and an “S”-shaped growth curve. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence staining indicated that marker genes or proteins were expressed in skeletal muscle satellite cells. After myogenic differentiation was induced, multiple-nucleated myotubes were observed, and the MyHC protein was expressed. The expression of myogenic marker genes changed with the differentiation process. After the induction of adipogenic differentiation, orange-red lipid droplets were observed, and the expression of adipogenic marker genes increased gradually with the differentiation process. In summary, satellite cells from adult tree shrew skeletal muscle were successfully isolated using a mixed enzyme digestion method, and their potential for differentiation into myogenic and adipogenic cells was confirmed, laying a foundation for further in vitro study of tree shrew muscle damage.
研究领域:
细胞生物学
·
肌肉生理学
·
干细胞研究
✓ 已验证
产品关联
DOI
PubMed
高影响因子
CHEMICAL ENGINEERING JOURNAL
·
2022
·
IF 16.744
Yun Zeng,Yonghua Zhan,Xinyue Liu,Jingwen Ma,Huifang Liu,Hanrui Li,Tong Yi,Qingxia Zhu,Getao Du,Linfei Zhao,Dan Chen,Xueli Chen
Tumor metastasis is a major cause of poor cancer prognosis. Photothermal therapy (PTT) is a non-invasive, non-oxygen-dependent, and highly spatiotemporally precise treatment that may aid chemotherapy in suppressing tumor growth and metastasis. This article aims to develop and modify a CuS-based nanoplatform linking RGD-Acrk to actively target and internalize doxorubicin (DOX) into tumor cells, resulting in an alternating strategy of synergistic chemo/PTT. The nanoparticle’s shape, spectra, drug encapsulation efficiency, loading content, and controlled release are characterized, and a comparison of 808/980 nm lasers is used to irradiate the nanoplatform. The cell survival rate is used to determine the cytotoxicity of DOX and PTT, and the chemo/PTT is used to treat 4T1-Luc tumor-bearing mice. Simultaneously, bioluminescence and photoacoustic imaging are employed to assess therapy efficacy, as well as tumor size, weight, density, the bodyweight of mice, and survival curves. H&E, immunohistochemistry for HIF-1α, and TUNEL fluorescence staining confirm these findings, suggesting that the nanoplatform has a good active-targeted capacity and perform high-efficiency PTT caused by 980 nm laser irradiation. The synergistic effect is at least 3.53 times higher than chemo/PTT without synergistic effect and tumor liver metastasis is successfully suppressed, showing that it has the potential to be used for cancer treatment and prognosis improvement.
研究领域:
肿瘤学
·
纳米技术
·
诊疗学
✓ 已验证
DOI
高影响因子
JOURNAL OF ETHNOPHARMACOLOGY
·
2023
·
IF 5.4
Pan Su,Mengdi Wu,Xiaoting Yin,Mengyuan Li,Yucheng Li,Ming Bai,Baoying Wang,Erping Xu
Ethnopharmacological relevance Modified Xiaoyao San (MXYS), a clinical empirical modified formula based on famous traditional Chinese herbal prescription Xiaoyao San, according to the “yu syndrome” theory of traditional Chinese medicine . MXYS has been shown to be an excellent effective therapy for depression patients in clinic, but the antidepressant mechanisms remain unclear. Aim of the study A growing body of evidence indicates the microglia autophagy and M1 polarized microglia (proinflammatory phenotype)-mediated neuroinflammation act critical roles in the pathogenesis of depression. This study aimed to investigate whether MXYS exerts antidepressant efficacy through inhibition of M1 polarized microglia-mediated neuroinflammation and modulation of autophagy involved in PI3K/Akt/mTOR pathway. Materials and methods In present research, the lipopolysaccharide (LPS)-induced depressive mice and LPS-stimulated N9 microglia cell line were utilized. Behavioral tests (sucrose preference, tail suspension and open field tests) were carried out to evaluate the antidepressant effect of MXYS. The neuronal damage was measured by Nissl's staining in LPS-treated mice. The proinflammatory cytokine levels, the autophagic markers, microglia M1 polarization as well as the PI3K/Akt/mTOR pathway related proteins of MXYS treatment were analyzed by ELISA kits, Western blot and immunofluorescence staining in vivo and vitro . Finally, the influence of autophagy antagonist (3-MA) on the protective effect of MXYS-containing serum in the LPS-stimulated N9 microglia was investigated. Results Treatment of LPS-induced depressive mice with MXYS significantly reversed depression-like behaviors, accompanied by reduction of proinflammatory cytokine levels (TNF-α, IL-1β) and amelioration of neuronal damage in prefrontal cortex. MXYS suppressed microglia M1 polarization and promoted autophagy in prefrontal cortex and LPS-stimulated N9 cells. Importantly, the remarkable inhibitory effect of the MXYS-medicated serum on microglia M1 polarization was blocked by autophagy antagonist 3-MA in LPS-stimulated N9 cells. Meanwhile, the MXYS treatment exhibited an excellent inhibition effect of PI3K/Akt/mTOR pathway in vivo and vitro . Conclusion Our research suggests that the antidepressant effect of MXYS in LPS-induced depressive mice may be related to alleviate neuroinflammation through suppression of microglia M1 polarization via enhancing autophagy involved in inactivation of the PI3K/Akt/mTOR pathway.
研究领域:
药理学
·
神经科学
·
中医
·
免疫学
·
细胞生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Nano Energy
·
2024
·
IF 16.8
Zuheng Jin,Xiao Zheng,Zhihong Zhu,Chuan Jiang,Sha Wu,Changzheng Hu,Laijun Liu,Liang Fang,Zhenxiang Cheng
Considering advanced oxidation techniques offer new opportunities for microbial degradation and inactivation, in this study, Z-type heterojunctions of Ba 1.4 Sr 3.6 NdNb 7 Ti 3 O 30 -N 2 /g-C 3 N 4 (BN 2 /CN) were successfully prepared using a solid-phase method and explored for their potential applications in tribocatalytic bactericidal inhibition. These results demonstrate that BN 2 /CN exhibits excellent inhibitory and inactivating effects on E. coli under tribocatalytic conditions. The UV–visible spectrum showed a gradual decrease in the survival rate of E. coli with the increase of tribocatalysis time, reaching an inactivation effect of 91% after 1 h, while fluorescence staining also confirmed that E. coli were essentially eliminated, with hardly any surviving bacteria observed under a laser confocal microscope . Hence, this study presents a novel catalytic approach for sterilization. Moreover, inhibition experiments demonstrated that the BN 2 /CN catalyst did not inhibit the growth of E. coli, tribocatalysis played a crucial role in the disinfection and sterilization processes . The inactivation of bacteria by BN 2 /CN catalyst was still above 90% after multiple cycles of sterilization experiments. Finally, we demonstrated that the production of •OH and •O 2 - during tribocatalysis is key to the inactivation of E. coli, thus achieving the desired effect of tribocatalytic sterilization.
研究领域:
催化
·
材料科学
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微生物学
·
环境科学
✓ 已验证
DOI
高影响因子
INFLAMMATION
·
2020
·Vol. 43
·期 5
·1751-1758
·
IF 3.212
Zhaohui Chen,Shuihua Wu
Accumulating evidence supports that Sirtuin 6 (SIRT6) may play a vital role in the pathogenesis of spinal cord injury. The current study was designed to investigate the specific effects of SIRT6 on spinal cord injury (SCI). HE and Nissl staining were performed for pathological analysis in SCI rats. SIRT6 expression was detected by RT-qPCR. CCK8 assay was applied for the detection of cell viability of LPS-injured PC12 cells. TNF-a, IL-1β, IL-6, MCP-1 levels and ROS, MPO, SOD levels were assessed to evaluate inflammation and oxidative stress in spinal cord injury. Cell apoptosis were evaluated by morphological examination using AO/EB fluorescent staining methods and key proteins related to apoptosis were explored via western blot. HE staining revealed increased cavity involving the dorsal white matter and central gray matter, and Nissl staining discovered the loss of motor neurons in the ventral horn in SCI rats. SIRT6 had lower expression in SCI rats. Lipopolysaccharide (LPS) exposure induced cell apoptosis and reduced the expression of SIRT6. Mechanistically, we revealed that up-regulation of SIRT6 alleviated inflammation and oxidative stress and inhibited cell apoptosis in spinal cord injury. Together, our findings indicated that SIRT6 attenuated spinal cord injury by suppressing inflammation, oxidative stress, and cell apoptosis. This study demonstrates that SIRT6 may represent a protective effect against spinal cord injury.
研究领域:
神经科学
·
分子生物学
·
病理学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Science of Advanced Materials
·
2024
·Vol. 16
·期 11
·1157-1166
·
IF 0.9
Li Yezhou,Tang Chengjian
This research investigated the application of alginate-collagen hydrogel (ACH) in rat cartilage injury (CI) repair and its related mechanisms through in vitro and in vivo experiments. Alginate solution and modified collagen solution were mixed to prepare the ACH. The obtained
samples were characterized and their sustained release properties were determined. L929 mouse fibroblasts were assigned into a blank control group (BC group, untreated), a positive control group (PC group, treated with 6.4% phenol), a negative control group (NC group, treated with tissue culture
polystyrene), and an ACH group (treated with ACH). Cell viability was assessed using the MTT assay. Meanwhile, the cytotoxicity of the ACH and its impact on cell proliferation and cell attachment capabilities were analyzed. Thirty 4-week-old specific pathogen-free (SPF) Sprague Dawley (SD)
rats were randomly grouped into a chondrocyte group (5 rats, chondrocytes collected and cultured), a cPAGE group (5 rats, chondrocytes treated with ACH), a model group (10 rats, CI model), and a mPAGE group (10 rats, CI model treated with CAH). The influence of CAH on chondrocyte proliferation
and the type II collagen (COLII) expression were assessed using the cell counting kit 8 (CCK-8) assay and immunofluorescence staining. Furthermore, the impacts of CAH on chondrocyte tissue and COL-II expression were analyzed using Safranin O-Fast Green (SOFG) staining, the International Cartilage
Repair Society (ICRS) score, and COL-II tissue staining. Immunoblotting was utilized to detect the TNF-α, NF-κB, matrix metalloproteinase 13 (MMP-13), and COL-II proteins in cartilage tissue. The results showed that release rate (RR) of the constructed ACH reached
over 85% within 72 hours and achieved degradation equilibrium within 5 days with a weight loss (WL) rate of over 60%. L929 fibroblasts culture results showed that the ACH posed no significant cytotoxicity, and cell attachment results were not obviously different from those on tissue culture
plates (P >0.05). MTT assay results demonstrated that the ACH exerted no adverse effects on L929 cell proliferation and growth, indicating a good biocompatibility. CCK-8 and COL-II results indicated that chondrocyte proliferation in cPAGE group was much higher in contrast to that in chondrocyte
group (P <0.05). Besides, the animal experiments unveiled that in comparison to the CI group, the mPAGE group had more chondrocytes, upregulated COL-II, and higher ICRS scores (P <0.05). MMP-13, NF-κB, and TNF-α were downregulated, and COL-II was
upshifted in the mPAGE group, with remarkable differences (P <0.05). In conclusion, ACH exhibited excellent biocompatibility and low cytotoxicity, promoted cell proliferation and growth, facilitated chondrocyte proliferation in rat CI repair, offering a valuable research foundation
for CI treatment.
研究领域:
生物医学工程
·
组织工程
·
再生医学
·
骨科
✓ 已验证
DOI
高影响因子
危险品化学品经营许可证（带存储）