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2022 ◽  
Vol 2022 ◽  
pp. 1-6
Author(s):  
Wulin Wen ◽  
Fengxia Yang ◽  
Xueliang Shen ◽  
Ningyu Feng ◽  
Huiyu Ha ◽  
...  

Objective. We aimed to investigate the expression of serum zinc and cytokines interleukin- (IL-) 13 and IL-33 in patients with allergic rhinitis (AR) and observe the effects of zinc on cytokines and pathway proteins in P815 mast cells stimulated by Artemisia annua allergen (Art.) in the IL-33/suppression of the tumorigenicity 2 (ST2) pathway. We also aimed to explore the possible regulatory role of zinc in AR and provide new ideas to determine the etiology and treatment of AR. Methods. AR patients treated from March to September in 2018 were selected as the research participants, and 50 healthy people in the same period were selected as the control group. Serum samples of all patients were collected, and those of AR patients were tested for the presence of allergens. The expression of IL-13 and IL-33 was detected by performing an enzyme-linked immunosorbent assay, while the serum zinc level was detected by conducting an inductively coupled plasma mass spectrometry. The cell counting kit (CCK-8) was used to detect the proliferation of P815 mast cells, and western blot was used to detect the expression of ST2, p38, and p65 proteins. Results. A total of 92 AR patients were included in the study; of them, 52 had mild AR, while 40 had moderate AR. The primary allergen found in AR patients was Artemisia, and the positivity rate was 53.26%. The serum zinc ion level of AR patients decreased, and the expression of IL-13 and IL-33 increased. After Art. was used to treat P815 mast cells, the expression of IL-33 in the cell supernatant increased in a concentration-dependent manner, the expression of receptor ST2 increased, and the expression of downstream p38 and p65 proteins increased. However, after treatment with ZnSO4, the expression of IL-33 in the cell supernatant decreased, and the expression of ST2, p38, and p65 protein decreased. Conclusion. The serum zinc level of AR patients decreased. In the IL-33/ST2 pathway, ZnSO4 can reduce the hypersensitivity of mast cells induced by Art.


2021 ◽  
Author(s):  
Nicky Craig ◽  
Sarah Louise Fletcher ◽  
Alison Daniels ◽  
Caitlin Newman ◽  
Marie O'Shea ◽  
...  

An enormous global effort is being made to study SARS-CoV-2 and develop safe and effective treatments. Studying the entire virus replication cycle of SARS-CoV-2 is essential to identify host factors and treatments to combat the infection. However, quantification of released virus often requires lengthy procedures, such as endpoint dilution assays or reinfection with engineered reporter viruses. Quantification of viral RNA in cell supernatant is faster and can be performed on clinical isolates. However, viral RNA purification is expensive in time and resources and often unsuitable for high-throughput screening. Here, we show a direct lysis RT-qPCR method allowing sensitive, accurate, fast, and cheap quantification of SARS-CoV-2 in culture supernatant. During lysis, the virus is completely inactivated, allowing further processing in low containment areas. This protocol facilitates a wide array of high- and low-throughput applications from basic quantification to studying the biology of SARS-CoV-2 and to identify novel antiviral treatments in vitro.


2021 ◽  
Vol 11 (8) ◽  
pp. 1624-1629
Author(s):  
Nali Liu ◽  
Beijing Zhu ◽  
Xin Wei

Islet β-cell regeneration is beneficial for treating diabetic patients. Silencing information regulator (SIRT1) has a regulatory role in endocrine diseases. However, SIRT1’s role in islet β cells remains unclear. MIN6 cells were cultured and assigned into control group, high glucose group, and SIRT1 group (treated with SIRT1 agonist, Resveratrol) followed by analysis of SIRT1 expression by Real time PCR and ELISA, cell proliferation by MTT assay, apoptosis activity by Caspase3 activity kit, secretion of TNF-α and IL-2 by ELISA, insulin secretion, ROS and SOD generation and expression of PI3K/Akt/NF-κB signaling by Western blot. SIRT1 mRNA was decreased in high glucose environment and its secretion in cell supernatant was reduced, with inhibited cell proliferation, increased Caspase3 activity and secretion of TNF-α and IL-2, decreased insulin secretion and SOD activity, increased ROS content, pAKT phosphorylation and NF-κB expression. Resveratrol significantly promoted SIRT1 expression and cell proliferation, decreased Caspase3 activity and secretion of TNF-α and IL-2, increased insulin secretion and SOD activity, as well as decreased ROS content, pAKT phosphorylation and NF-κB expression (P <0.05). SIRT1 is decreased in high glucose environment, and SIRT1 expression can inhibit islet cell apoptosis, inhibit oxidative stress and inflammation, and promote islet cell proliferation and insulin secretion by regulating PI3K/Akt/NF-κB signaling.


2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Rui Hou ◽  
Luo Jiang

Abstract Objective Long non-coding RNAs (lncRNAs) and microRNAs (miRs) are differentially expressed in ovarian cancer (OC) cells and influence OC progression. This study intended to explore the underlying roles of LINC00115 and miR-30a in OC. Methods Gene Expression Omnibus database was used to find OC microarray datasets and bioinformatics analysis predicted the potential molecular mechanism of OC. OC stem cells (OCSCs) surface marker was isolated from human OC cell line and identified. CD133+ OCSCs were transfected with LINC00115, miR-30a and SOX9 alone or together to detect sphere-forming ability and apoptosis of OCSCs. Caspase-3 activity and DNA damage in cell supernatant were detected. The levels of CD44, NANOG, POU5F1, LINC00115, CD133, miR-30a and SOX9 were measured. Then sh-LNC00115-treated OCSCs were added with Wnt/β-catenin activator SKL2001 to observe the changes of cell stemness and activity. Finally, animal models were established to evaluate the effect of LINC00115 on OCSC in vivo. Results LINC00115 and SOX9 were highly expressed in OC, while miR-30a was lowly expressed. After silencing LINC00115 or overexpressing miR-30a, the sphere-forming rate of CD133+ OCSC and levels of CD133, CD44, NANOG and POU5F1 decreased, while apoptotic rate, Caspase-3 activity and histone-related DNA damage increased. SOX9 reversed these trends. Additionally, LINC00115 could bind to miR-30a and miR-30a could target SOX9. SKL2001 partially reversed cell stemness and activity in sh-LNC00115-treated OCSCs. Finally, silencing LINC00115 could inhibit OCSCs growth in vivo. Conclusion LINC00115 promoted stemness and inhibited apoptosis of OCSCs by upregulating SOX9 and in activating the Wnt/β-catenin pathway through competitively binding to miR-30a.


2021 ◽  
Vol 4 (6) ◽  
pp. e202000957
Author(s):  
Rachel E Heap ◽  
José Luis Marín-Rubio ◽  
Julien Peltier ◽  
Tiaan Heunis ◽  
Abeer Dannoura ◽  
...  

BMDMs are a key model system to study macrophage biology in vitro. Commonly used methods to differentiate macrophages from BM are treatment with either recombinant M-CSF or the supernatant of L929 cells, which secrete M-CSF. However, little is known about the composition of L929 cell-conditioned media (LCCM) and how it affects the BMDM phenotype. Here, we used quantitative mass spectrometry to characterise the kinetics of protein secretion from L929 cells over a 2-wk period, identifying 2,193 proteins. Whereas M-CSF is very abundant in LCCM, we identified several other immune-regulatory proteins such as macrophage migration inhibitory factor (MIF), osteopontin, and chemokines such as Ccl2 and Ccl7 at surprisingly high abundance levels. We therefore further characterised the proteomes of BMDMs after differentiation with M-CSF, M-CSF + MIF, or LCCM, respectively. Interestingly, macrophages differentiated with LCCM induced a stronger anti-inflammatory M1 phenotype that those differentiated with M-CSF. This resource will be valuable to all researchers using LCCM for the differentiation of BMDMs.


2021 ◽  
Author(s):  
Zhiying Chen ◽  
Huiyan Zhang ◽  
Jun Zhou ◽  
Xiaoqin Wu ◽  
Moxin Wu ◽  
...  

Abstract Objective: This study aimed to explore the mechanism of CORM-2 on attenuating bleeding-related inflammation. Methods: Microglia were isolated from the neonatal rats (1-2days old) and identified by the CD11b/c anti-body, and some microglia were co-cultured with RBCs marked with PKH26 fluorescent dye, and then treated with CORM-2. That is, the microglia cells were divided into the microglia, microglia+ PKH26+RBCs and microglia + PKH26+ RBCs+CORM-2 cell-groups. Microglial phagocytosis to RBCs PKH26+ was observed under an inverted fluorescence microscope; moreover, the fluorescence intensity of microglia that phagocytized PKH26+RBCs was detected through immunofluorescence. HO-1, NF-κB p65, and IL-1β expressions were detected using RT-qPCR, western blotting, and immunofluorescence, respectively. The levels of carbon monoxide hemoglobin (HbCO) in the cell supernatant in each group were detected with ELISA.Results. After 1- day of co-culturing, the number of residual PKH26+RBCs in the Microglia+ PKH26+RBCs+CORM-2 group decreased remarkably than that in the Microglia+ PKH26+RBCs groups (18 × 106 vs. 14 × 106, p=0.02), which revealed that microglia phagocytosis was stronger in CORM-2 treated group. More over, compared with microglia + PKH26+RBCs group, the microglia+ PKH26+RBCs +CORM-2 group showed higher levels of HO-1 mRNA and protein expressions at the 3rd day and the 5th day after co-culturing. Further more, CORM-2 significantly inhibited the expressions of mRNA and proteins of NF‐κB p65 and IL-1 after 3 days of co-culturing, meanwhile, CORM-2 did not increase the level of HbCO in the cell supernatant.Conclusions CORM-2 can inhibit inflammatory reactions in bleeding setting in vitro by promoting microglial phagocytosis to RBCs and decrease IL-1β and NF-κB; the mechanism may involve HO-1/CO system.


2021 ◽  
Vol 11 (01) ◽  
pp. 58-70
Author(s):  
Ryusuke Suzuki ◽  
Harue Suzuki ◽  
Maki Yokogawa ◽  
Junichi Motohashi

2020 ◽  
Vol 196 ◽  
pp. 543-549
Author(s):  
Kamila Czubak-Prowizor ◽  
Joanna Rywaniak ◽  
Halina Malgorzata Zbikowska

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