RUNX1 targeting AKT3 promotes alveolar hypercoagulation and fibrinolytic inhibition in LPS induced ARDS.

BACKGROUND: Alveolar hypercoagulation and fibrinolytic inhibition are mainly responsible for massive alveolar fibrin deposition, which are closely related with refractory hypoxemia in acute respiratory distress syndrome (ARDS). Our previous study testified runt-related transcription factor (RUNX1) participated in the regulation of this pathophysiology in this syndrome, but the mechanism is unknown. We speculate that screening the downstream genes associated with RUNX1 will presumably help uncover the mechanism of RUNX1. METHODS: Genes associated with RUNX1 were screened by CHIP-seq, among which the target gene was verified by Dual Luciferase experiment. Then the efficacy of the target gene on alveolar hypercoagulation and fibrinolytic inhibition in LPS-induced ARDS was explored in vivo as well as in vitro. Finally, whether the regulatory effects of RUNX1 on alveolar hypercoagulation and fibrinolytic in ARDS would be related with the screened target gene was also sufficiently explored. RESULTS: Among these screened genes, AKT3 was verified to be the direct target gene of RUNX1. Results showed that AKT3 was highly expressed either in lung tissues of LPS-induced rat ARDS or in LPS-treated alveolar epithelia cell type II (AECII). Tissue factor (TF) and plasminogen activator inhibitor 1 (PAI-1) were increasingly expressed both in lung tissues of ARDS and in LPS-induced AECII, which were all significantly attenuated by down-regulation of AKT3. Inhibition of AKT3 gene obviously ameliorated the LPS-induced lung injury as well as the collagen I expression in ARDS. RUNX1 overexpression not only promoted the expressions of TF, PAI-1, but also boosted AKT3 expression in vitro. More importantly, the efficacy of RUNX1 on TF, PAI-1 were all effectively reversed by down-regulation of AKT3 gene. CONCLUSION: AKT3 is an important target gene of RUNX1, through which RUNX1 exerted its regulatory role on alveolar hypercoagulation and fibrinolytic inhibition in LPS-induced ARDS. RUNX1/ATK3 signaling axis is expected to be a new target for the exploration of ARDS genesis and treatment.

Enteral nutrition alleviated lipopolysaccharides-induced hypercatabolism through ghrelin/GHS-R1α-POMC.

Sustained hypercatabolism induced by sepsis contributed to serious complications and mortality in the intensive care unit. Enteral nutrition (EN) was required to maintain the energy balance during sepsis. Ghrelin, which was stimulated secretion by EN, had been shown to regulate energy homeostasis. Therefore, we tested whether EN alleviated hypercatabolism through ghrelin/GH secretagogue receptor 1α (GHS-R1α)-proopiomelanocortin (POMC) in endotoxemic rats. Rats in the Sham and lipopolysaccharide (LPS) groups were free access to rodent chow diet and water. Rats in the EN, EN + Lys and EN + 3-MA groups were intracerebroventricularly injected with saline, D-Lys3-GHRP-6 or 3-MA and then received EN for three days. Hypercatabolism was measured by the change of body weight, insulin resistance, leptin, corticosterone, muscle protein synthesis and atrophy. Serum and hypothalamic total ghrelin, acylated ghrelin, GHS-R1α and AMP-activated protein kinase (AMPK)-autophagy-POMC pathway were also detected. The results showed that EN increased serum and hypothalamic total ghrelin, acylated ghrelin and GHS-R1α, effectively activated the hypothalamic AMPK-autophagy-POMC pathway and alleviated hypercatabolism in endotoxemic rats. The improving effects of EN on hypercatabolism and hypothalamic AMPK-autophagy-POMC pathway were abolished with the central administration of D-Lys3-GHRP-6 to inhibited hypothalamic GHS-R1α. And with the central administration of 3-MA to inhibited hypothalamic autophagy, the improving effect of EN on hypercatabolism was also abolished in endotoxemic rats. In conclusions, EN could significantly alleviate hypercatabolism through ghrelin/GHS-R1α-POMC in endotoxemic rats.

Isolation and identification of Candida tropicalis in sows with fatal infection: a case report.

BACKGROUND: Candida is the common conditionally pathogenic fungus that infected human and animal clinically. C. tropicalis had been isolated from the skin and hair of healthy pigs, but with no report of fatal infection in gastrointestinal diseases. CASE PRESENTATION: In a pig farm in Henan Province of China, about 20 % of pregnant and postpartum sows suffered from severe gastrointestinal diseases, with a mortality rate higher than 60 % in the diseased animals. The sows had gastrointestinal symptoms such as blood in stool and vomiting. Necropsy revealed obvious gastric ulcers, gastrointestinal perforation, and intestinal hemorrhage in the gastrointestinal tract, but no lesions in other organs. The microbial species in gastric samples collected from gastric ulcer of the diseased sows then was initially identified as Candida by using routine systems of microscopic examination, culture characteristics on the medium Sabouraud dextrose agar medium. The fungus was further identified as C. tropicalis by species-specific PCR and sequencing. This study revealed an infection of C. tropicalis in sows through gastrointestinal mucosa could cause fatal digestive system disease and septicemia. CONCLUSIONS: For the first time, a strain of C. tropicalis was isolated and identified from the gastric tissue of sows with severe gastrointestinal diseases. PCR and sequencing of ITS-rDNA combined with morphology and histopathological assay were reliable for the identification of Candida clinically.

Deficient and excess dietary selenium levels affect growth performance, blood cells apoptosis and liver HSP70 expression in juvenile yellow catfish Pelteobagrus fulvidraco.

We investigated the effects of deficient and excess dietary selenium (Se) on growth, blood cells apoptosis and liver heat shock protein 70 (HSP70) expression in juvenile yellow catfish (Pelteobagrus fulvidraco). After 8 weeks, yellow catfish (initial weight: 2.12 ± 0.01 g) fed isonitrogenous and isolipid diets containing <0.05 (deficient dietary Se) or 6.5 (excess dietary Se) mg Se/kg displayed a significantly lower weight gain ratio (WGR) than those fed a diet containing 0.23 (normal dietary Se) mg Se/kg. As dietary Se levels increased, liver Se concentration, glutathione peroxidase activity and the hepatosomatic index increased significantly. Plasma glucose concentration was highest in the normal treatment compared with the excess dietary Se treatment. Both deficient and excess dietary Se lead to increased reactive oxygen species (ROS) production and apoptosis ratio in blood cells, whereas only excess dietary Se increased their cytoplasmic free-Ca(2+) (CF-Ca(2+)) concentration. Excess dietary Se also resulted in the highest level of HSP70 expression, thereby possibly providing a protective mechanism against oxidative stress. These results indicate that both deficient and excess dietary Se restrained the growth of juvenile yellow catfish and caused oxidative stress. The overproduction of ROS may act as a signal molecule mediate apoptosis when dietary Se deficiency. Both ROS and CF-Ca(2+) were recorded when dietary Se excess, suggesting that Ca(2+) may be activated by Se and play a major role during Se-induced oxidative stress and cell apoptosis.

Changes in nitrogen budget and potential risk to the environment over 20years (1990-2010) in the agroecosystems of the Haihe Basin, China.

The nitrogen balance can serve as an indicator of the risk to the environment of nitrogen loss from agricultural land. To investigate the temporal and spatial changes in agricultural nitrogen application and its potential threat to the environment of the Haihe Basin in China, we used a database of county-level agricultural statistics to calculate agricultural nitrogen input, output, surplus intensity, and use efficiency. Chemical fertilizer nitrogen input increased by 51.7% from 1990 to 2000 and by 37.2% from 2000 to 2010, concomitant with increasing crop yields. Simultaneously, the nitrogen surplus intensity increased by 53.5% from 1990 to 2000 and by 16.5% from 2000 to 2010, presenting a continuously increased environmental risk. Nitrogen use efficiency decreased from 0.46 in 1990 to 0.42 in 2000 and remained constant at 0.42 in 2010, partly due to fertilizer composition and type improvement. This level indicates that more than half of nitrogen inputs are lost in agroecosystems. Our results suggest that although the improvement in fertilizer composition and types has partially offset the decrease in nitrogen use efficiency, the environmental risk has still increased gradually over the past 20 years, along with the increase in crop yields and nitrogen application. It is important to achieve a better nitrogen balance through more effective management to significantly reduce the environmental risk, decrease nitrogen surplus intensity, and increase nitrogen use efficiency without sacrificing crop yields.

[Determination of 39 fatty acids in liver of rats by gas chromatography-mass spectrometry].

Fatty acids not only form phospholipids that contribute to the formation of cell membranes but also participate in many metabolic activities, such as energy storage and cell signal transduction. The liver plays a key role in the synthesis and metabolism of fatty acids. The composition and contents of fatty acids in the liver are closely related to body health. Most fatty acid-detection methods require a large sample size and can detect only a small number of fatty acids. Therefore, a sensitive and efficient method to determine fatty acids in the liver is urgently required. Herein, a method based on gas chromatography-mass spectrometry (GC-MS) was established for the simultaneous determination of 39 fatty acids in 1.1 mg of liver tissue. Different extraction methods and derivatization conditions were compared to develop an optimal sample-treatment method. The performance of two different columns in separating the target fatty acids were also compared. A total of 10 mg of liver was added to 450 µL of normal saline and ground at -35 ℃ to obtain a homogenate. Next, 50 µL of the homogenate (equivalent to 1.1 mg of liver) was added with 750 µL of chloroform-methanol (1∶2, v/v) to extract total fatty acids. The fatty acid extracts were dried under nitrogen, and then derivatized at 100 ℃ for 90 min after being added with methanol containing 5% sulfuric acid. The fatty acid methyl esters were extracted with hexane and then separated on an SP-2560 capillary column (100 m×0.25 mm×0.2 µm; Supelco, USA) via GC-MS. The results revealed that all 39 fatty acid methyl esters detected had good linearities in the certain mass concentration ranges with correlation coefficients (R2) greater than 0.9940. The limits of detection (LOD) and quantification (LOQ) of these methyl esters in the liver were 2-272 ng/mg and 7-906 ng/mg, respectively. The accuracy and precision of the method were evaluated by spiking the liver homogenate with tridecylic acid and eicosanoic acid at low (0.09 µg/mg), moderate (0.90 µg/mg), and high (5.40 µg/mg) concentration levels. The recoveries ranged from 82.4% to 101.0% with an intraday relative standard deviations (RSDs) (n=5) of 3.2%-12.0% and interday RSDs (n=3) of 5.4%-13.4%. The method was successfully applied to detect fatty acids in the livers of four healthy male Sprague-Dawley (SD) rats and four male SD rats with abnormal liver function induced by perfluorooctane sulfonate (PFOS). PFOS is a persistent organic pollutant. Twenty-six fatty acids were detected in the livers of both groups. Among the fatty acids investigated, pentadecanoic acid (C15∶0), γ-linolenic acid (C18∶3n6), and elaidic acid (C18∶1n9t) cannot be detected by the methods reported in the literature. By contrast, the method developed in this study could separate the isomers of oleic acid (elaidic acid, C18∶1n9t; oleic acid, C18∶1n9c) and linolenic acid (linolelaidic acid, C18∶2n6t; linoleic acid, C18∶2n6c). In conclusion, the developed method is simple and can detect a large number of fatty acids using small sample amounts and few reagents. More importantly, it could successfully separate fatty acid isomers. These findings indicate that the developed method is suitable for the detection of fatty acid composition and contents in the liver in clinical and experimental research.

Long noncoding RNA Linc00337 functions as an E2F1 co-activator and promotes cell proliferation in pancreatic ductal adenocarcinoma.

BACKGROUND: Long noncoding RNA (lncRNA) Linc00337 has been implicated in lung, gastric, colorectal and esophageal squamous cell carcinoma progression via various mechanisms; however, its clinicopathological significance and role in pancreatic ductal adenocarcinoma (PDAC) progression remains largely unknown. METHODS: Multiple approaches such as bioinformatic analysis, Transfection, quantitative real-time-PCR, Western blotting, animal studies, RNA-immunoprecipitation (RIP), RNA-pulldown and RNA-Fluorescence in situ hybridization (RNA-FISH) and were utilized to explore the role of Linc00337 in PDAC. RESULTS: Here we identified Linc00337 is an oncogenic lncRNA during PDAC progression. We found that the expression of Linc00337 is elevated in PDAC tissues and the higher Linc00337 predicts dismal prognosis. Functionally, Linc00337 promotes PDAC cell proliferation and cell cycle transition both in vitro and in vivo. Mechanistically, Linc00337 binds to E2F1 and functions as an E2F1 coactivator to trigger the targets expression during PDAC progression. CONCLUSION: Our results demonstrate a reciprocal regulation mechanism between Linc00337 and E2F1 in PDAC progression and report the clinical value of Linc00337 for PDAC prognosis and treatment.

Inhibitory effect of yuzu essential oil on the formation of N-nitrosodimethylamine in vegetables.

The inhibitory effect of yuzu (Citrus junos Tanaka) essential oil on the formation of N-nitrosodimethylamine (NDMA) in the presence of vegetables (31 species) or saliva was investigated by HPLC. Most vegetable extracts enhanced the formation of NDMA. However, the formation ratio of NDMA in vegetable extracts was decreased by yuzu oil in the range of 59 to 22%. In the presence of yuzu oil and saliva, its ratio ranged between 62 and 24%. These results indicated that yuzu oil inhibited the formation of NDMA even in vegetables and saliva. The contents of ascorbic acid, nitrate, and nitrite in the 31 vegetable species were 0.3-65 mg/100 g, 3-581 mg/100 g, and 10-750 microg/100 g, respectively. Ascorbic acid and nitrite had little effect on the inhibition or formation of NDMA at their intact levels. Nitrate accelerated the formation of NDMA, and the addition of saliva further enhanced it. The mechanism of inhibition of NDMA formation by alpha-terpinene was studied. It was assumed from the results of LC-MS that a new compound formed by the reaction of alpha-terpinene with nitrite would be a derivative of alpha-terpinene with dinitroso groups. The molecular weight of this compound was 194. It is suggested that terpene hydrocarbons in citrus essential oils would contribute to the decrease of NDMA formation.

Comparative proteomic identification of the hemocyte response to cold stress in white shrimp, Litopenaeus vannamei.

To understand molecular responses of crustacean hemocytes to cold stress, flow cytometer analysis and two-dimensional electrophoresis proteomic approach were used to investigate altered proteins in hemocytes of Litopenaeus vannamei during cold stress treatment. Through flow cytometer analysis, 13°C for 24h post-cold stress was selected as the suitable temperature and condition for cold stress treatment. MALDI-TOF/TOF MS analysis revealed that 6 forms of 6 proteins were significantly up-regulated, including three enzymes (cystathionase, glyceraldehyde 3-phosphate dehydrogenase and glyoxalase 1) and one immune-related protein (oncoprotein nm23), whereas 24 forms of 3 proteins were significantly down-regulated in the treated shrimp (hemocyanin, hemocyte transglutaminase and transketolase). There were 20 spots identified as hemocyanin meaning that it may play important roles in environmental regulation in shrimp. Real-time fluorescence quantitative PCR confirmed that the levels of transcription of the hemocyanin, partial mRNA for hemocyanin, cystathionase, glyoxalase 1 and oncoprotein nm23 genes were found to relate well with that of their translation products after cold stress treated, while only the levels of hemocyte transglutaminase transcripts were not corresponded with that of their translation products. Further investigation of these data may lead to better understanding of the molecular responses of crustacean hemocytes to cold stress.