LncRNA MEG3 suppresses PI3K/AKT/mTOR signalling pathway to enhance autophagy and inhibit inflammation in TNF-α-treated keratinocytes and psoriatic mice.

BACKGROUND: Psoriasis is a common chronic inflammatory skin disorder that causes patches of thick red skin and silvery scales and affects 1-3% of the population, which reduces patient's quality of life. Understanding the pathogenesis of psoriasis is crucial for developing novel therapeutic strategies. METHODS: HaCaT and NHEK cells were treated with TNF-α in vitro. A mouse model of psoriasis was established by topical imiquimod application on back skin. LncRNA MEG3 was cloned into the pcDNA3.1 vector and transfected in TNF-α-treated HaCaT and NHEK cells to overexpress its expression. Liposome-encapsulated pcDNA3.1-MEG3 was injected into imiquimod-treated mice via tail vein. RT-qPCR and western blot assays were used to examine the expression of lncRNA MEG3, IL-6, IL-8, IFN-γ, IL-1ß, LC3, Beclin 1, p62, p-p65, p65, NLRP3, p-PI3K, PI3K, p-AKT, AKT, p-mTOR, mTOR respectively. The secretion of IL-6, IL-8, IFN-γ and IL-1ß was determined using ELISA assay. Immunofluorescence and immunohistochemistry methods were performed for analyzing the expression of LC3 and NLRP3 in cells and skin tissues respectively. LY294002 was used to block the PI3K/AKT/mTOR signalling. MTT assay was applied to test the toxicity of LY294002 to HaCaT and NHEK cells. RESULTS: LncRNA MEG3 expression levels were downregulated in TNF-α-treated HaCaT and NHEK cells and skin tissues of psoriatic mice model. TNF-α treatment enhanced inflammation and suppressed autophagy in HaCaT and NHEK cells, which were largely reversed by overexpression of lncRNA MEG3. Autophagy puncta and NLRP3 inflammasome assembly showed the same patterns with the expression of inflammation and autophagy markers in TNF-α-treated HaCaT and NHEK cells with or without lncRNA MEG3 overexpression. TNF-α-induced activation of the PI3K/AKT/mTOR signalling was abolished by lncRNA MEG3 overexpression in HaCaT and NHEK cells. Blocking the PI3K/AKT/mTOR signalling inhibited TNF-α-induced inflammation and restored autophagy level in TNF-α-treated HaCaT and NHEK cells. Overexpression of lncRNA MEG3 suppressed inflammation, promoted autophagy and inhibited the activation of the PI3K/AKT/mTOR signalling in a mouse model of psoriasis. CONCLUSION: LncRNA MEG3 facilitates autophagy and suppresses inflammation in TNF-α-treated keratinocytes and psoriatic mice, which is dependent on the PI3K/AKT/mTOR signalling pathway. Our study enhances the understanding of psoriasis and provides potential therapeutic targets for psoriasis.

You are what you eat: Microplastics in the feces of young men living in Beijing.

PURPOSE: Microplastics have been widely detected in the environment and marine organisms. However, few studies have investigated the presence of microplastics in humans. This preliminary study identified and quantified the microplastic content in human feces. METHODS: A total of 26 young male students aged 18-25 years were recruited from Beijing, China. A self-administered 7-day 24-h fluid intake record was used to document fluid intake, and food intake was recorded for 3 days. Feces were collected by participants using a sterile fecal collector. Microplastics in the remaining fecal residues were measured and identified using fourier transform infrared micro-spectroscopy. RESULTS: Eventually, twenty-four participants completed the study. The fecal samples of 23 (95.8%) participants tested positive for microplastics. In these 23 samples, the abundance of microplastics varied from 1 particle/g to 36 particles/g (size 20 to 800 µm). The summed mass of all microplastic particles per participant ranged from 0.01 to 14.6 mg. Qualitative analysis of the microplastics indicated the presence of one to eight types of microplastics in each sample, with polypropylene (PP) being the most abundant; it was found in 95.8% of fecal samples. We examined associations between water intake habits and the abundance of microplastics in their feces. A moderate correlation was observed between packaged water and beverage intake and microplastic abundance in feces (r = 0.445, P = 0.029). CONCLUSION: Various types of microplastics were detected in human feces, with PP being found in the highest proportion. There may be an association between water intake habits and microplastic abundance in feces.

The Influence of Fluid Intake Behavior on Cognition and Mood among College Students in Baoding, China.

INTRODUCTION: Water is a critical nutrient, and it is important for the maintenance of the physiological function of the human body [1-3]. In addition to fluid amounts, fluid intake frequency is also important for hydration status [4, 5]. At present, only few guidelines mention fluid intake behavior that recommend drinking water frequently while in small quantities, however, there is no scientific evidence to support it. Therefore, it is necessary to explore the appropriate fluid intake behavior. OBJECTIVE: The objective of this study is to evaluate the influence of different fluid intake behavior on cognition and mood, to provide scientific basis for proposing the appropriate fluid intake behavior. METHODS: A double-blinded randomized controlled trial was designed and implemented among college students aged 18-23 years in Baoding, China. Subjects were randomly assigned into each of 3 groups using a random number generated by computer software: the subjects consuming plain water 200 mL/2 h, that is, 1,600 mL during whole day (group 1), 100 mL/2 h, that is, 800 mL during whole day (group 2), and 110 mL/1 h, that is, 1,650 mL during whole day (group 3), respectively. Subjects were asked to fast from 11:00 p.m., without consuming any foods or drinks the day before the intervention. From 8:00 a.m. to 10:00 p.m. of the first study day, subjects consumed water according to the instructions and repeated it from 8:00 a.m. to 4:00 p.m. of the second study day. Cognition, mood, and urine osmolality were collected twice at 10:00 a.m. (time 1) and 4:00 p.m. (time 2) of the second study day (shown in Fig. 1). RESULTS: A total of 92 subjects (46 males, 46 females) completed this study. It was found that the increasing fluid intake amounts lead to an increase in urine output and a decrease in urine osmolality (p < 0.05). Use the mixed models to compare measurements for groups 1 and 2, which showed that when compared with those drinking 800 mL per day, people who drank 1,600 mL per day scored higher in vigor (11.8 vs. 9.1, p < 0.05) and portrait memory test (22.6 vs. 20.8, p < 0.05) but lower in total mood disturbance (90.8 vs. 97.8, p < 0.05). By comparing groups 1 and 3, the results indicated that compared with drinking 8 times per day, people who drank 15 times per day scored lower in portrait memory test (21.8 vs. 22.6, p < 0.05) and hunger (3.3 vs. 3.6, p < 0.05). CONCLUSIONS: Reasonable fluid intake behavior may be beneficial to improve the cognition and mood of college students. The fluid intake behavior, which is consuming water 200 mL each time and 8 times per day, is recommended. More studies are needed to advise people to have health-beneficial fluid intake behavior.

LncRNA MEG3 influences the proliferation and apoptosis of psoriasis epidermal cells by targeting miR-21/caspase-8.

BACKGROUND: It was reported that microRNA-21(miR-21) was differentially expressed in the keratinocytes of psoriasis patients, and it may influence the apoptosis and proliferation of cells. The role of lncRNA maternally expressed gene3 (MEG3), a competing endogenous RNAs of miR-21, in the progression of psoriasis remains unclear. We aimed to unfold the influence of MEG3 and miR-21 on the proliferation and apoptosis of psoriasis epidermal cells. METHODS: 50µg/L TNF-α was used to treat HaCaTs and NHEKs cells for 24 h, and then different experiments were conducted. qRT-PCR were applied for measuring the mRNA level of MEG3, miR-2, and caspase-8, and the protein expression of caspase-8 was measured with western blotting. Flow cytometry was used for assessing apoptosis. Cell proliferation was detected using MTT and colony formation assays. Dual luciferase reporter assay was applied for confirming the binding site between MEG3 and miR-21, miR-21 and Caspase-8. RESULTS: A cell model for in vitro studying the role of MEG3 in psoriasis pathophysiology was established using HaCaT and HHEKs. MEG3 was significantly down-regulated in HaCaT, HHEKs, and psoriatic skin samples. MEG3 inhibits proliferation and promotes apoptosis of Activated-HaCaT (Act-HaCaT) and Activated-HHEKs (Act- HHEK) by regulating miR-21, and the binding site between MEG3 and miR-21 was identified. We also found that miR-21 could inhibit the level of caspase-8 and identified the binding site between caspase-8 and miR-21. Some down-stream proteins of caspase-8, Cleaved caspase-8, cytc, and apaf-1 were regulated by miR-21 and MEG3. CONCLUSION: MEG3/miR-21 axis may regulate the expression of caspase-8, and further influence the proliferation and apoptosis of psoriasis keratinocyte, Act-HaCaT and Act- HHEK. Therefore, our findings may provide a new thought for the study of pathogenesis and treatment of psoriasis.

A spectrophotometric screening method for avermectin oxidizing microorganisms.

A spectrophotometric screening method for avermectin oxidizing microbes by determination of 4″-oxo-avermectin was established based on the reaction between 4″-oxo-avermectin and 2,4-dinitrophenylhydrazine. Combined with a gradient HPLC assay, microorganisms capable of regioselectively oxidizing avermectin to 4″-oxo-avermectin were successfully obtained by this method.

Gene cloning and expression of a glucoside 3-dehydrogenase from Sphingobacterium faecium ZJF-D6, and used it to produce N-p-nitrophenyl-3-ketovalidamine.

In this study, we report the cloning and expression of a functional glucoside 3-dehydrogenase (G3DH) gene from Sphingobacterium faecium ZJF-D6. This gene is 1686 bp in length and encodes a peptide of 562 amino acids. The G3DH gene was successfully expressed in E. coli, and the recombinant enzyme could oxidize glucosides, galactosides and analogues at C-3 position. The sequence and multiple alignment analysis showed that the enzyme has highest identity with G3DHs from Paraglaciecola polaris LMG 21857, Aliiglaciecola lipolytica E3 and Halomonas sp. alpha-15. The recombinant G3DH was purified on Ni-NTA column and exhibited the highest activity at pH 7.6 and 30 °C. It was sensitive to acid and alkali, and showed well thermostability. The SfG3DH could oxidize a wild range of sugars. When recombinant E. coli BL21 cells were used as catalyst, a high rate of conversion to N-p-nitrophenyl-3-ketovalidamine was achieved, and no p-nitroaniline was detected. This process offers a promising approach to fulfill substrate of 3-ketovalidoxylamine A C-N lyase production.

[Chemical Constituents from Rhizome of Valeriana jatamansi].

OBJECTIVE: To study the chemical constituents from the rhizome of Valeriana jatamansi. METHODS: The chemical constituents were separated and purified by silica gel, medium pressure column chromatography, and preparative HPLC. Their structures were determined by physicochemical properties and spectral data. RESULTS: Six compounds were isolated from the dibromochloromethane extract in the rhizome of Valeriana jatamansi, and identified as decursidin (1), decursitin B (2), decursitin A (3), 3'(S)-acetoxy-4'(R)-angeloyloxy-3',4'-dihydroxanthyletin (4), 8-acetoxyl-pathchouli alcohol (5) and dibutyl phthalate (6). CONCLUSION: Compounds 1-4 are coumarins which are isolated from this genus for the first time,and compound 6 is isolated from this genus for the first time.

Purification and characterization of the glucoside 3-dehydrogenase produced by a newly isolated Sphingobacterium faecium ZJF-D6 CCTCC M 2013251.

A soluble glucoside 3-dehydrogenase (G3DH) was purified from a newly isolated Sphingobacterium faecium ZJF-D6 CCTCC M 2013251. The enzyme was purified to 35.71-fold with a yield of 41.91 % and was estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with a molecular mass of 62 kDa. The sequences of two peptides of the enzyme were all contained in a GMC family oxidoreductase (EFK55866) by mass spectrometry analysis. The optimal pH of the enzyme was around 6.2. The enzyme was stable within a pH range of 5.0-6.6 and was sensitive to heat. G3DH from S. faecium exhibited extremely broad substrate specificity and well regioselectivity to validoxylamine A. The enzyme was completely inhibited by Hg2Cl2 and partly inhibited by Cu(2+), Fe(2+), Ca(2+), and Cd(2+). The apparent K m values for D-glucose, sucrose, and validoxylamine were calculated to be 1.1, 1.7, and 2.1 mM, respectively. With this purified enzyme, 3-keto sucrose was prepared at pH 5.0, 30 °C for 10 h with a yield of 28.7 %.

Purification and characterization of 3-ketovalidoxylamine A C-N lyase produced by Stenotrophomonas maltrophilia.

A soluble 3-ketovalidoxylamine A C-N lyase from Stenotrophomonas maltrophilia was purified to 367.5-fold from the crude enzyme, with a yield of 16.4% by column chromatography on High S IEX, Methyl HIC, High Q IEX, and Sephadex G 100. The molecular mass of the enzyme was estimated to be 34 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the enzyme was a neutral protein having an isoelectric point value at pH 7.0. The optimal pH of 3-ketovalidoxylamine A C-N lyase was around 7.0. The enzyme was stable within a pH range of 7.0-10.5. The optimal temperature was found to be near 40 degrees C, and the enzyme was sensitive to heat. The enzyme was completely inhibited by ethylenediaminetetraacetic acid, and it was reversed by Ca2+. The product, p-nitroaniline, inhibited the enzyme activity significantly at low concentration. The enzyme has C-N lyase activity and C-O lyase activity, and need 3-keto groups. The apparent K (m) value for p-nitrophenyl-3-ketovalidamine was 0.14 mM.

Preparation of 3-ketovalidoxylamine A C-N lyase substrate: N-p-nitrophenyl-3-ketovalidamine by Stenotrophomonas maltrophilia CCTCC M 204024.

3-Ketovalidoxylamine A C-N lyase is one of three key enzymes in the production of valienamine, which is a potent glucosidase inhibitor from validamycin A. N-p-Nitrophenyl-3-ketovalidamine, used as the substrate of 3-ketovalidoxylamine A C-N lyase, was prepared from N-p-nitrophenylvalidamine with free cells of Stenotrophomonas maltrophilia CCTCC M 204024. The yield and selectivity of N-p-nitrophenyl-3-ketovalidamine from cells were improved by treatment with 10 mM ethylenediaminetetraacetic acid. The optimal pH and temperature for N-p-nitrophenyl-3-ketovalidamine formation was pH 6.0 and 30 degrees C, respectively. N-p-Nitrophenyl-3-ketovalidamine was formed with a yield of 0.68 in the first batch.

Purification and characterization of the glucoside 3-dehydrogenase produced by a newly isolated Stenotrophomonas maltrophilia CCTCC M 204024.

A soluble glucoside 3-dehydrogenase (G3DH) from Stenotrophomonas maltrophilia CCTCC M 204024, recently isolated from wheat soil in our laboratory, was purified to 37.4-fold with a yield of 24.7% and was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 66 kDa. 2,6-Dichlorophenolindophenol (DCPIP) and ferricyanide were able to act as artificial electron acceptors for the enzyme. The optimal pH of G3DH was in the range of 6.0-7.0 in the presence of DCPIP. The enzyme was stable in the pH range of 4.4-10.6 and was sensitive to heat. G3DH exhibited extremely broad substrate specificity by converting many sugars to their corresponding 3-ketoglucosides. They produced a characteristic spectrum by alkaline treatment with a peak at 340 nm. The apparent Km values for validoxylamine A and D: -glucose were 8.3 and 1.1 mM, respectively. Cu2+, Ag2+, and Hg2Cl2 inhibited the activity of G3DH.