Alfalfa MsbHLH115 confers tolerance to cadmium stress through activating the iron deficiency response in Arabidopsis thaliana.

Cadmium (Cd) pollution severely affects plant growth and development, posing risks to human health throughout the food chain. Improved iron (Fe) nutrients could mitigate Cd toxicity in plants, but the regulatory network involving Cd and Fe interplay remains unresolved. Here, a transcription factor gene of alfalfa, MsbHLH115 was verified to respond to iron deficiency and Cd stress. Overexpression of MsbHLH115 enhanced tolerance to Cd stress, showing better growth and less ROS accumulation in Arabidopsis thaliana. Overexpression of MsbHLH115 significantly enhanced Fe and Zn accumulation and did not affect Cd, Mn, and Cu concentration in Arabidopsis. Further investigations revealed that MsbHLH115 up-regulated iron homeostasis regulation genes, ROS-related genes, and metal chelation and detoxification genes, contributing to attenuating Cd toxicity. Y1H, EMSA, and LUC assays confirmed the physical interaction between MsbHLH115 and E-box, which is present in the promoter regions of most of the above-mentioned iron homeostasis regulatory genes. The transient expression experiment showed that MsbHLH115 interacted with MsbHLH121pro. The results suggest that MsbHLH115 may directly regulate the iron-deficiency response system and indirectly regulate the metal detoxification response mechanism, thereby enhancing plant Cd tolerance. In summary, enhancing iron accumulation through transcription factor regulation holds promise for improving plant tolerance to Cd toxicity, and MsbHLH115 is a potential candidate for addressing Cd toxicity issues.

MsYSL6, A Metal Transporter Gene of Alfalfa, Increases Iron Accumulation and Benefits Cadmium Resistance.

Iron (Fe) is necessary for plant growth and development. The mechanism of uptake and translocation in Cadmium (Cd) is similar to iron, which shares iron transporters. Yellow stripe-like transporter (YSL) plays a pivotal role in transporting iron and other metal ions in plants. In this study, MsYSL6 and its promoter were cloned from leguminous forage alfalfa. The transient expression of MsYSL6-GFP indicated that MsYSL6 was localized to the plasma membrane and cytoplasm. The expression of MsYSL6 was induced in alfalfa by iron deficiency and Cd stress, which was further proved by GUS activity driven by the MsYSL6 promoter. To further identify the function of MsYSL6, it was heterologously overexpressed in tobacco. MsYSL6-overexpressed tobacco showed better growth and less oxidative damage than WT under Cd stress. MsYSL6 overexpression elevated Fe and Cd contents and induced a relatively high Fe translocation rate in tobacco under Cd stress. The results suggest that MsYSL6 might have a dual function in the absorption of Fe and Cd, playing a role in the competitive absorption between Fe and Cd. MsYSL6 might be a regulatory factor in plants to counter Cd stress. This study provides a novel gene for application in heavy metal enrichment or phytoremediation and new insights into plant tolerance to toxic metals.

MsNRAMP2 Enhances Tolerance to Iron Excess Stress in Nicotiana tabacum and MsMYB Binds to Its Promoter.

Iron(Fe) is a trace metal element necessary for plant growth, but excess iron is harmful to plants. Natural resistance-associated macrophage proteins (NRAMPs) are important for divalent metal transport in plants. In this study, we isolated the MsNRAMP2 (MN_547960) gene from alfalfa, the perennial legume forage. The expression of MsNRAMP2 is specifically induced by iron excess. Overexpression of MsNRAMP2 conferred transgenic tobacco tolerance to iron excess, while it conferred yeast sensitivity to excess iron. Together with the MsNRAMP2 gene, MsMYB (MN_547959) expression is induced by excess iron. Y1H indicated that the MsMYB protein could bind to the "CTGTTG" cis element of the MsNRAMP2 promoter. The results indicated that MsNRAMP2 has a function in iron transport and its expression might be regulated by MsMYB. The excess iron tolerance ability enhancement of MsNRAMP2 may be involved in iron transport, sequestration, or redistribution.

The accuracy and influencing factors of sleep staging based on single-channel EEG via a deep neural network / 中华耳鼻咽喉头颈外科杂志

Objective: To investigate theaccuracy of artificial intelligence sleep staging model in patients with habitual snoring and obstructive sleep apnea hypopnea syndrome (OSAHS) based on single-channel EEG collected from different locations of the head. Methods: The clinical data of 114 adults with habitual snoring and OSAHS who visited to the Sleep Medicine Center of Beijing Tongren Hospital from September 2020 to March of 2021 were analyzed retrospectively, including 93 males and 21 females, aging from 20 to 64 years old. Eighty-five adults with OSAHS and 29 subjects with habitual snoring were included. Sleep staging analysis was performed on the single lead EEG signals of different locations (FP2-M1, C4-M1, F3-M2, ROG-M1, O1-M2) using the deep learning segmentation model trained by previous data. Manual scoring results were used as the gold standard to analyze the consistency rate of results and the influence of different categories of disease. Results: EEG data in 124 747 30-second epochs were taken as the testing dataset. The model accuracy of distinguishing wake/sleep was 92.3%,92.6%,93.5%,89.2% and 83.0% respectively,based on EEG channel Fp2-M1, C4-M1, F3-M2, REOG-M1 or O1-M2. The mode accuracy of distinguishing wake/REM/NREM and wake/REM/N1-2/SWS , was 84.7% and 80.1% respectively based on channel Fp2-M1, which located in forehead skin. The AHI calculated based on total sleep time derived from the model and gold standard were 13.6[4.30,42.5] and 14.2[4.8,42.7], respectively (Z=-2.477, P=0.013), and the kappa coefficient was 0.977. Conclusions: The autonomic sleep staging via a deep neural network model based on forehead single-channel EEG (Fp2-M1) has a good consistency in the identification sleep stage in a population with habitual snoring and OSAHS with different categories. The AHI calculated based on this model has high consistency with manual scoring.

Construction of Tumor Tissue Array on An Open-Access Microfluidic Chip / 分析化学

An open-access microfluidic chip which enabled automatic cell distribution and complex multi-step operations was developed.The microfluidic chip featured a key structure in which a nanoporous membrane was sandwiched by a cell culture chamber array layer and a corresponding media reservoir array layer.The microfluidic approach took advantage of the characteristics of the nanoporous membrane.On one side, this membrane permitted the flow of air but not liquid, thus acting as a flow-stop valve to enable automatic cell distribution.On the other side, it allowed diffusion-based media exchange and thus, mimicked the endothelial layer.In synergy with a liquid transferring platform, the open-access microfluidic system enabled complex multi-step operations involving medium exchange, drug treatment, and cell viability testing.By using this microfluidic protocol, a 10 × 10 tissue arrays was constructed in 90 s, followed by schedule-dependent drug testing.Morphological and immunohistochemical assays results indicated that the resultant tumor tissue was faithful to that in vivo.Drug testing assays showed that the microfluidic tissue array promised multi-step cell assays under biomimetic microenvironment, thus providing an advantageous tool for cell research.

Epigallocatechin-3-gallate inhibits proliferation and migration of human colon cancer SW620 cells in vitro.

AIM: Epigallocatechin-3-gallate (EGCG) is the major polyphenolic constituent in green tea. The aim of this study is to investigate the effects of EGCG on proliferation and migration of the human colon cancer SW620 cells. METHODS: Proliferation and migration of SW620 cells were induced by the protease-activated receptor 2-agonist peptide (PAR2-AP, 100 µmol/L) or factor VIIa (10 nmol/L), and analyzed using MTT and Transwell assays, respectively. The cellular cytoskeleton was stained with rhodamine-conjugated phalloidin and examined with a laser scanning confocal fluorescence microscope. The expression of caspase-7, tissue factor (TF) and matrix metalloproteinase (MMP)-9 in the cells was examined using QT-PCR, ELISA and Western blot assays. The activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and nuclear factor-kappa B (NF-κB) signaling pathways was analyzed with Western blot. RESULTS: Both PAR2-AP and factor VIIa promoted SW620 cell proliferation and migration, and caused cytoskeleton reorganization (increased filopodia and pseudopodia). Pretreatment with EGCG (25, 50, 75, and 100 µg/mL) dose-dependently blocked the cell proliferation and migration induced by PAR2-AP or factor VIIa. EGCG (100 µg/mL) prevented the cytoskeleton changes induced by PAR2-AP or factor VIIa. EGCG (100 µg/mL) counteracted the down-regulation of caspase-7 expression and up-regulation of TF and MMP-9 expression in the cells treated with PAR2-AP or factor VIIa. Furthermore, it blocked the activation of ERK1/2 and NF-κB (p65/RelA) induced by PAR2-AP or factor VIIa. CONCLUSION: EGCG blocks the proliferation and migration of SW620 cells induced by PAR2-AP and factor VIIa via inhibition of the ERK1/2 and NF-κB pathways. The compound may serve as a preventive and therapeutic agent for colon cancers.

[Activation of tumor necrosis factor receptor-associated factor 6 in anti-ß2GPI/ß2GPI-induced tissue factor expression on THP-1 cells].

AIM: To investigate whether tumor necrosis factor receptor-associated factor 6 (TRAF6) is involved in anti-ß2GPI/ß2GPI-induced tissue factor (TF) expression on THP-1 cells. METHODS: The total RNA was extracted and the protein lysates were collected from THP-1 cells stimulated with anti-ß2GPI/ß2GPI complex. And then the TF expression on THP-1 cells was detected by real-time quatitative PCR (RT-qPCR) and TF activity kit. TRAF6 mRNA and its protein expression were investigated by RT-qPCR and Western blotting, respectively. The proteasome inhibitor, MG-132, was used for inhibitory assays, in order to demonstrate the effect of anti-ß2GPI/ß2GPI complex on THP-1 cells. RESULTS: The TF expression (both mRNA and activity) on THP-1 cells was significantly up-regulated with the treatment of anti-ß2GPI/ß2GPI complex (100 mg/L), compared with untreated cells(P<0.05). The TRAF6 mRNA and protein levels in THP-1 cells were also significantly increased with the treatment of anti-ß2GPI/ß2GPI complex. The expression of TRAF6 was shown in a time-dependent manner, with the maximal level at 15 minutes (mRNA) and 30 minutes (protein) respectively. All the stimulating effects of anti-ß2GPI/ß2GPI complex (100 mg/L) on THP-1 cells were inhibited by MG-132 (5 µmol/L). CONCLUSION: TRAF6 is up-regulated and contributes to TF expression on THP-1 cells induced with anti-ß2GPI/ß2GPI complex.

[Role of NF-κB in factor VIIa-induced proliferation and migration of colon cancer cell line SW620 cells].

OBJECTIVE: To explore the roles of NF-κB in factor VIIa-induced proliferation and migration of a colon cancer cell line (SW620) in vitro and its possible mechanism. METHODS: The expression levels of NF-κB (p65), inhibitory protein of NF-κB (IκB-α), caspase-7, interleukin 8 (IL-8) and tissue factor (TF) in SW620 cells treated with factor VIIa, PDTC (an inhibitor of NF-κB) and other factors were measured by Western-blotting and real-time PCR. Proliferation and migration of the cells were analyzed by flow cytometry and Transwell assay, respectively. RESULTS: Factor VIIa down-regulated the IκB-α level in SW620 cells and increased the intranuclear level of NF-κB. Those effects of factor VIIa were blocked by anti-TF or anti-PAR2 antibodies. The effects of factor VIIa on proliferation and migration of SW620 cells, expression of IL-8, TF as well as caspase-7, were interfered by PDTC (the inhibitor of NF-κB). CONCLUSIONS: TF/VIIa complex activates NF-κB pathway via PAR2, thereby up-regulates IL-8 and down-regulates caspase-7 expression in SW620 cells, finally promotes proliferation and migration of colon cancer cells. In addition, TF/VIIa/PAR2/NF-κB pathway also upregulates TF expression, thus to create a positive feedback loop of TF/VIIa/PAR2/NF-κB/TF.

[Effects of chronic amiodarone therapy on L-type calcium current recovery and action potential duration of rabbit ventricular myocytes].

OBJECTIVE: To investigate the effects of chronic amiodarone therapy on L-type calcium current recovery and action potential duration of rabbit ventricular myocytes. METHODS: Healthy rabbits (1.6-1.8 kg) were treated with amiodarone (80 mg x kg(-1) x d(-1)) for four weeks. Action potential duration (APD) was recorded under isolated arterially perfused left ventricular wedge preparation, then single myocytes were isolated using enzyme digestion. L-type calcium current recovery (time constant, tau) were determined by fitting data with monoexponential. Tau/APD90 were compared in cells treated with saline, amiodarone and sotalol (3 x 10(-5) mmol/L). RESULTS: In chronic amiodarone treated myocytes, tau [(164 +/- 8) ms vs. (98 +/- 8) ms, P<0.05], APD90 [(321 +/- 12) ms vs. (220 +/- 10) ms, P<0.05] and tau/APD90 (0.51 +/- 0.03 vs. 0.44 +/- 0.03, P<0.05) were significantly increased than those in control myocytes. Sotalol significantly increased tau [(128 +/- 7) ms vs. (98 +/- 8) ms, P<0.05] and ADP90 [(405 +/- 13) ms vs. (220 +/- 10) ms, P<0.05] while reduced the tau/APD90 (0.32 +/- 0.05 vs. 0.44 +/- 0.03, P<0.05) compared to control myocytes. CONCLUSION: The differential effect of amiodarone and sotalol on ventricular myocytes tau/APD90 ratio might be responsible for the safety profile of these two drugs.

Comparative study on hepatoma cell line 7721 and human fetal hepatocytes for in vitro culture of hepatitis C virus / 第三军医大学学报

Objective　To study the similarities and differences on in vitro replication and expression of hepatitis C virus (HCV) between human fetal hepatocytes (HFH) and 7721 cell line. Methods　Human fetal hepatocytes and a hepatoma cell line 7721 were incubated with a serum from hepatitis C patient. After incubation, the presence of HCV RNA, the expression of HCV NS3 antigens in cells and/or supernatant were examined by RT-PCR, in situ hybridization and immunohistochemistry, respectively. Results　It was found that: ①The intracellular HCV RNA was first detected on d 2～3 post-incubation and then could be intermittently detected in cells and/or supernatant subsequently (HCV RNA could be detected in 7721 cells during a period of at least 66 days. In HFH, HCV RNA could be detected up to 25 days after incubation); ②HCV-NS3 antigen could be expressed in infected cells; ③Minus-strand RNA of HCV was mainly located within cytoplasm by in situ hybridization. Conclusion　The results suggest that both the fetal hpatocytes and the hepatoma cell line 7721 are susceptible to HCV, and especially 7721 cell line can stably support HCV replication in vitro and may be used as the target cell for long-term cultures of HCV.

Comparative study on hepatoma cell line 7721 and human fetal hepatocytes for in vitro culture of hepatitis C virus / 第三军医大学学报

Objective　To study the similarities and differences on in vitro replication and expression of hepatitis C virus (HCV) between human fetal hepatocytes (HFH) and 7721 cell line. Methods　Human fetal hepatocytes and a hepatoma cell line 7721 were incubated with a serum from hepatitis C patient. After incubation, the presence of HCV RNA, the expression of HCV NS3 antigens in cells and/or supernatant were examined by RT-PCR, in situ hybridization and immunohistochemistry, respectively. Results　It was found that: ①The intracellular HCV RNA was first detected on d 2～3 post-incubation and then could be intermittently detected in cells and/or supernatant subsequently (HCV RNA could be detected in 7721 cells during a period of at least 66 days. In HFH, HCV RNA could be detected up to 25 days after incubation); ②HCV-NS3 antigen could be expressed in infected cells; ③Minus-strand RNA of HCV was mainly located within cytoplasm by in situ hybridization. Conclusion　The results suggest that both the fetal hpatocytes and the hepatoma cell line 7721 are susceptible to HCV, and especially 7721 cell line can stably support HCV replication in vitro and may be used as the target cell for long-term cultures of HCV.