Integrated physio-biochemistry and RNA-seq revealed the mechanism underlying biochar-mediated alleviation of compound heavy metals (Cd, Pb, As) toxicity in cotton.

Biochar has been recognised as an efficacious amendment for the remediation of compound heavy metal contamination in soil. However, the molecular mechanism of biochar-mediated tolerance to compound heavy metal toxicity in cotton is unknown. The objective of this research was to investigate the positive impact of biochar (10 g·kg-1) on reducing damage caused by compound heavy metals (Cd, Pb, and As) in cotton (Gossypium hirsutum L.). The results revealed that biochar reduced Cd concentrations by 24.9 % (roots), and decreased Pb concentrations by 37.1 % (roots) and 59.53 % (stems). Biochar maintained ionic homoeostasis by regulating the expression of metal transporter proteins such as ABC, HIPP, NRAMP3, PCR, and ZIP, and genes related to the carbon skeleton and plasma membrane. Biochar also downregulated genes related to photosynthesis, thereby increasing photosynthesis. Biochar re-established redox homoeostasis in cotton by activating signal transduction, which regulated the activity of the enzymes POD, SOD, and CAT activity; and the expression of related genes. This research revealed the molecular mechanism by which biochar confers resistance to the harmful effects of compound heavy metal toxicity in cotton. The application of biochar as a soil amendment to neutralise the toxicity of compound heavy metals is recommended for cash crop production.

3D-QSAR studies on the structure-bitterness analysis of citrus flavonoids.

Despite their important bioactivities, the unpleasant bitter taste of citrus derived flavonoids limits their applications in the food industry, and the structure-bitterness relationship of flavonoids is still far from clear. In this study, 26 flavonoids were characterized by their bitterness threshold and their common skeleton using sensory evaluation and molecular superposition, respectively. The quantitative conformational relationship of the structure-bitterness of flavonoids was explored using 3D-QSAR based on comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA). The results showed that increases of a hydrogen bond donor at A-5 or B-3', a bulky group at A-8, or an electron-withdrawing group at B-4' would enhance the bitterness of flavonoids. The bitterness of some flavonoids was predicted and evaluated, and the results were similar to the bitter intensity of the counterparts from the 3D-QSAR and contour plots, confirming the validation of 3D-QSAR. This study explains the theory of the structure-bitterness relationship of flavonoids, by showing potential information for understanding the bitterness in citrus flavonoids and developing a debittering process.

Lactobacillus rhamnosus GG Promotes Recovery of the Colon Barrier in Septic Mice through Accelerating ISCs Regeneration.

Disruption of the intestinal barrier is both the cause and result of sepsis. The proliferation and differentiation of intestinal stem cells (ISCs) promote the regenerative nature of intestinal epithelial cells, repairing the injured intestinal mucosal barrier; however, it is uncertain whether the recovery effects mediated by the ISCs are related to the gut microbiota. This research found that the survival rate of septic mice was improved with a Lactobacillus rhamnosus GG (LGG) treatment. Furthermore, an increased proliferation and decreased apoptosis in colon epithelial cells were observed in the LGG-treated septic mice. In vitro, we found that a LGG supernatant was effective in maintaining the colonoid morphology and proliferation under the damage of TNF-α. Both in the mice colon and the colonoid, the LGG-induced barrier repair process was accompanied by an increased expression of Lgr5+ and lysozyme+ cells. This may be attributed to the upregulation of the IL-17, retinol metabolism, NF-kappa B and the MAPK signaling pathways, among which, Tnfaip3 and Nfkbia could be used as two potential biomarkers for LGG in intestinal inflammation therapy. In conclusion, our finding suggests that LGG protects a sepsis-injured intestinal barrier by promoting ISCs regeneration, highlighting the protective mechanism of oral probiotic consumption in sepsis.

Fructose oxidase-like activity of CuO nanoparticles supported by phosphate for a tandem catalysis-based fructose sensor.

A surge of nanozymes with oxidase-like activities is emerging in various fields, whereas nanozymes with the ability to catalyze the oxidation of saccharides have less been explored. Herein, CuO nanoparticles (NPs) with phosphate-supported fructose oxidase-like activity have been reported. Notably, reactive oxygen species (ROS) have been confirmed as the products during the process. By coupling the fructose oxidase-like activity with the peroxidase-like activity of CuO NPs, a tandem catalysis-based fructose sensor can be fabricated. In detail, CuO NPs can catalyze the fructose oxidation under O2 to yield ROS (e.g., H2O2, •OH, and O2·-) and effectively decompose H2O2 into ·OH. After that, terephthalic acid can be oxidized by •OH produced from the tandem catalysis to generate a fluorescent product. This sensor shows a linear range toward fructose (0.625-275 µÐœ) with a low limit of detection (0.5 µÐœ), which can be successfully conducted to detect fructose from real samples. Overall, this work aims to expand the catalytic types of nanozymes and provide a desirable fructose sensor.

Effects of WeChat platform-based nursing intervention on disease severity and maternal and infant outcomes of patients with gestational diabetes mellitus.

OBJECTIVE: To determine the effects of WeChat platform-based nursing intervention on the disease control and pregnancy outcomes of patients with gestational diabetes mellitus (GDM). METHODS: A total of 112 patients with GDM treated in our hospital from December 2018 to December 2020 were enrolled, and their clinical data were retrospectively analysed. Among them, 61 pregnant women were given routine nursing as the control group (Con group), and the other 51 were given WeChat platform-based interactive continuous nursing intervention as the observation group (Obs group). The blood glucose (BG) of the two groups before and after nursing was compared, and their self-management level and nursing satisfaction were evaluated. The maternal and infant outcomes of the two groups were also compared. RESULTS: Before nursing, BG and glycosylated hemoglobin (HbA1c) levels in the two groups were comparatively high, without notable difference between the two groups (P>0.05); after nursing, the levels of fasting blood glucose, 2 hour postprandial blood glucose (2h-PG), and HbA1c in the Obs group decreased significantly, and were significantly lower than those in the Con group (P<0.05). Additionally, the two groups were similar in self-management level scores before nursing (P>0.05), while after nursing, the scores of diet management, exercise management, BG monitoring management and foot care management in the Obs group increased and were significantly higher than those in the Con group (P<0.05). The Obs group expressed significantly higher nursing satisfaction than the Con group (χ2=6.078, P<0.05). The total incidence of adverse pregnancy outcome in Obs group was lower than that in Con group (χ2-5.566, P<0.05). According to the analysis of risk factors, older age, pre-pregnancy BMI ≥24 kg/m2, and history of diabetes mellitus were independent risk factors for adverse pregnancy outcomes of pregnant women, while WeChat platform-based interactive continuous nursing was a protective factor against adverse pregnancy outcome (P<0.05). CONCLUSION: WeChat platform-based interactive continuous nursing intervention can help patients master comprehensive self-management skills to achieve good control of GDM, improve their satisfaction toward nursing and lower the risk of adverse outcome.

Parishin From Gastrodia Elata Ameliorates Aging Phenotype in Mice in a Gut Microbiota-Related Manner.

The physiological and pathological processes that accompany aging can seriously affect the quality of life of the elderly population. Therefore, delaying aging and developing antiaging products have become popular areas of inquiry. Gut microbiota plays an important role in age-related phenotypes. The present study aimed to investigate the antiaging effects and underlying mechanism of parishin, a phenolic glucoside isolated from traditional Chinese medicine Gastrodia elata. Samples from adult (12 weeks), low-dose (10 mg/kg/d) or high-dose (20 mg/kg/d) parishin-treated and untreated aged (19 months) mice were collected to determine blood indicators, gut microbiota and metabolome, and cardiopulmonary histopathological features. The results showed that parishin treatment ameliorates aging-induced cardiopulmonary fibrosis and increase in serum p16 Ink4a , GDF15, and IL-6 levels. Furthermore, parishin treatment alleviated dysbiosis in gut microbiota, including altered microbial diversity and the aberrant abundance of opportunistic pathogenic bacteria such as Turicibacter and Erysipelatoclostridium. Gene function prediction and gut metabolome analysis results indicated that the parishin treatment-altered gut microbiota played important roles in sugar, lipid, amino acid and nucleic acid metabolism, and improved gut metabolic disorders in aged mice. In conclusion, the present study provides an experimental basis of potential applications of parishin against aging.

Estimation of pollutant sources in multi-zone buildings through different deconvolution algorithms.

Effective identification of pollution sources is particularly important for indoor air quality. Accurate estimation of source strength is the basis for source effective identification. This paper proposes an optimization method for the deconvolution process in the source strength inverse calculation. In the scheme, the concept of time resolution was defined, and combined with different filtering positions and filtering algorithms. The measures to reduce effects of measurement noise were quantitatively analyzed. Additionally, the performances of nine deconvolution inverse algorithms under experimental and simulated conditions were evaluated and scored. The hybrid algorithms were proposed and compared with single algorithms including Tikhonov regularization and iterative methods. Results showed that for the filtering position and algorithm, Butterworth filtering performed better, and different filtering positions had little effect on the inverse calculation. For the calculation time step, the optimal Tr (time resolution) was 0.667% and 1.33% in the simulation and experiment, respectively. The hybrid algorithms were found to not perform better than the single algorithms, and the SART (simultaneous algebraic reconstruction technique) algorithm from CAT (computer assisted tomography) yielded better performances in the accuracy and stability of source strength identification. The relative errors of the inverse calculation for source strength were typically below 25% using the optimization scheme.

Periodontitis in elderly patients with type 2 diabetes mellitus: impact on gut microbiota and systemic inflammation.

Elderly patients with type 2 diabetes mellitus (T2DM) exhibit considerable periodontitis frequency, which causes tooth loss and poor quality of life. To investigate the impact of periodontitis on gut microbiota, we used 16S rRNA amplicon sequencing to characterize the composition and structure of gut microbiota among elderly patients with T2DM and periodontitis (T2DM_P), elderly patients with T2DM alone (T2DM_NP), and healthy volunteers. We identified 34 key gut microbiota markers that distinguished participants with different periodontal conditions and investigated their connections to other gut bacteria, as well as their clinical correlates. The most striking differences in co-occurrence networks between the T2DM_P and T2DM_NP groups comprised interactions involving dominant genera in the oral cavity (i.e., Streptococcus and Veillonella). Of the 34 identified key gut microbiota markers that distinguished participants with different periodontal conditions, 25 taxa were correlated with duration of diabetes, dry mouth or the peripheral levels of pro-inflammatory cytokines (e.g., tumor necrosis factor-α, interferon-γ, prostaglandin E2, interleukin-17, and interleukin-6) and metabolic parameters (e.g., hemoglobin A1c), respectively. Our findings suggest that gut microbial shifts driven by periodontitis may contribute to systemic inflammation and metabolic dysfunction during the progression of T2DM.

Probiotic Lactobacillus rhamnosus GG reduces mortality of septic mice by modulating gut microbiota composition and metabolic profiles.

OBJECTIVE: The use of probiotics to reduce mortality of sepsis was supported by a series of clinical research subjects. However, the exact mechanisms underlying protective effects of probiotic in sepsis has not been elucidated clearly. The aim of this study was to explore the therapeutic effects of Lactobacillus rhamnosus GG (LGG) prophylaxis on the host co-microbiota and metabolism in mice with sepsis-induced colon microbiota dysbiosis. METHODS: Mice were fed either probiotic LGG or saline 4 wk before cecum ligation and puncture (CLP) operation. Fecal samples were collected and analyzed by 16S rDNA sequencing and ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS)-based metabolomics. RESULTS: LGG treatment could noticeably reduce the mortality of sepsis and reverse gut microbiota dysbiosis caused by sepsis. Specifically, LGG reduced conditional pathogenic bacteria, such as Proteobactria and Deferribacteres; lipopolysaccharide producers like Enterobacteriaceae, facultative anaerobes, including Bacteroidaceae and Erysipelotrichaceae, and increased the abundance of bacteria related to energy harvest, such as Firmicutes; colon barrier restorers like Akkermansia; and liver function regulators like Coprococcus and Sutterella. Furthermore, the changes in fecal metabolites were prevented by LGG. These changes were found mainly to be correlated with the bile acid and metabolism pathways, lysophosphatidylcholines metabolism, and eicosatetraenoic acid metabolism. Finally, correlation analysis shown that microbiota dysbiosis was closely related to metabolic imbalance. CONCLUSIONS: Probiotic LGG may has a positive effect on reducing mortality of sepsis through rebalancing the metabolic profiles and gut microbiota.

The association between gut butyrate-producing bacteria and non-small-cell lung cancer.

BACKGROUND: Recently, it has been found that the gut microbiota may affect the development of lung cancer through the "gut-lung axis." To investigate this relationship, we performed this study to determine whether the gut microbiota in non-small-cell lung cancer (NSCLC) patients is different from that in healthy adults. METHODS: Quantitative PCR (qPCR) was used to detect the expression levels of eight gut butyrate-producing bacteria in healthy adults and NSCLC patients. We enrolled 30 patients with NSCLC and 30 subjects from 100 healthy adults after matching for age and sex. RESULTS: Compared to healthy adults, most of the gut butyrate-producing bacteria in NSCLC patients were significantly decreased; these included Faecalibacterium prausnitzii, Clostridium leptum, Clostridial cluster I, Ruminococcus spp., Clostridial Cluster XIVa, and Roseburia spp. Among the gut butyrate-producing bacteria, we analyzed Clostridial cluster IV and Eubacterium rectale were not decreased in NSCLC patients. CONCLUSIONS: We conclude that NSCLC patients had gut butyrate-producing bacteria dysbiosis. Further studies should be performed to investigate the underlying mechanisms of how these specific bacteria affect lung cancer progression and prognosis.

Lactobacillus rhamnosus GG treatment improves intestinal permeability and modulates microbiota dysbiosis in an experimental model of sepsis.

Decrease of 'health­benefiting' microbes and increase of pathogenic bacteria (a condition termed dysbiosis) in intensive care unit patients is considered to induce or aggravate sepsis (gut­origin sepsis). Orally administered probiotics have been effective in the prevention of nosocomial infections. However, the mechanisms of probiotic­induced anti­infection and anti­sepsis remain to be explored. In the present study, 4­week­old C57BL6 mice were orally administrated with Lactobacillus rhamnosus GG (LGG) or normal saline (control) 4 weeks prior to cecal ligation and puncture (CLP). A subset of the mice were sacrificed at 24 h post­CLP, and the others were used for survival studies. Ileum tissues, blood and fecal samples were collected. The survival rate of septic mice pretreated with LGG was significantly improved compared with untreated mice. The levels of inflammatory cytokines were reduced in LGG­pretreated septic mice. A decrease of colonic proliferation and epithelial tight junctions and an increase of colonic apoptosis were observed in control septic CLP+saline mice. LGG pretreatment reversed the colonic proliferation, apoptosis and expression of tight junction proteins to the levels of the sham group. LGG pretreatment improved the richness and diversity of intestinal microbiota in septic mice. The principal coordinates analysis clustering plots revealed a significant separate clustering in microbiota structure between three groups. Bacteria associated with energy consumption, including Bacteroidetes, with opportunistic infection, including Proteobacteria, Staphylococcaceae and Enterococcaceae, lipopolysaccharide producers, including Enterobacteriaceae, and facultative anaerobes, such as Bacteroidaceae and Erysipelotrichaceae, increased in septic mice. By contrast, bacteria associated with energy harvest, including Firmicutes, intestinal barrier function regulators, including Akkermansia, hepatic function regulators, including Coprococcus and Oscillospira, and obligate anaerobes, including Prevotellaceae, decreased in septic mice. With LGG pretreatment, the sepsis­induced microbiota dysbiosis was reversed. The present results elucidated the potential mechanism of LGG treatment in sepsis, by improving intestinal permeability and modulating microbiota dysbiosis.

Associations between dietary protein and vitamin intake and the physical functioning of older adults with sarcopenia.

PURPOSE: To investigate associations between dietary protein and vitamin intake and physical function status in older adults with sarcopenia. METHODS: Data of 707 participants with sarcopenia aged > 60 years from the National Health and Nutrition Examination Survey (NHANES) 1999-2004 were analyzed. Body composition, body mass index (BMI), physical function status, demographics, dietary intake (protein and vitamins A, C, E), lifestyle factors and comorbidities were measured, stratified by gender. RESULTS: Dietary levels of carbohydrate, fat and vitamin E differed significantly between genders (P < 0.05). Physical function limitations (48.5 vs. 36%; P < 0.001), basic activities of daily living (ADL) limitations (37 vs. 24.4%; P < 0.001), and instrumental ADL limitations (25.6 vs. 17.8%) were higher in women than in men. Multivariate logistic regression analysis revealed that, in males, intake of optimal amounts of vitamin C (Q3: ≥ 60.71 mg/day) was associated with basic ADL limitations. In females, protein intake of more than 1.11 g/kg/day was associated with both basic and instrumental ADL limitations. CONCLUSIONS: Only dietary or supplemental intake of vitamin C and E, but not protein, was associated with physical functioning in older males with sarcopenia. In contrast, only intake of higher amounts of protein, but not vitamins, was associated with physical functioning in older females with sarcopenia.

Characteristics of hyperuricemia in older adults in China and possible associations with sarcopenia.

OBJECTIVE: This cross-sectional study aimed to investigate the characteristics and epidemiology of hyperuricemia in older adults in China and evaluate possible associations between hyperuricemia and sarcopenia. METHODS: Three hundred and eighty-eight study subjects (>60 years old) meeting the inclusion criteria received blood tests and standardized examinations for bone mineral density, muscle mass, muscle strength, and physical performance. Data including demographic and clinical characteristic and comorbidity were also collected. All data were analyzed retrospectively. RESULTS: In the study population, higher uric acid levels were significantly correlated with higher muscle mass, grip strength, and bone density, but were unrelated to physical performance. When uric acid levels were separated into quartiles and the population was divided by sex, the correlation of uric acid to muscle mass was retained in some quartiles for both men and women, and the correlation to handgrip was only retained for one quartile for men. The correlation to bone density was retained in women in all analyses. CONCLUSION: In the population as a whole, higher uric acid levels were significantly correlated with higher muscle mass, grip strength, and bone density, but had no relationship to physical performance. Differences between men and women in these relationships need to be studied further.

Probiotic pre-administration reduces mortality in a mouse model of cecal ligation and puncture-induced sepsis.

A number of clinical trials have demonstrated that the use of probiotics has the potential to prevent nosocomial infections. However, the mechanism underlying probiotic-induced anti-infection and sepsis remains to be investigated. In the present study, 200 µl/day of Lactobacillus rhamnosus GG (LGG) or normal saline (control) was orally administrated to 4-week-old C57BL6 mice 4 weeks prior to cecal ligation and puncture (CLP). A number of mice were sacrificed 24 h after CLP, and the remaining mice were used for survival studies. Ileum tissues were collected to evaluate the injury on the intestine. Blood samples were also obtained to investigate the changed metabolic pattern in mice that underwent different treatments using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). In the survival studies, the mortality of CLP-induced septic mice pretreated with LGG was significantly lower compared with untreated mice (P=0.029). Ileum mucosal damage was evident in the control septic mice. Based on the data of UPLC-QTOF-MS, phosphatidylcholines were increased and lysophosphatidylcholines (LPCs) that contained polyunsaturated fatty acids were decreased in septic mice, whereas saturated fatty acid LPCs reveal no significant difference between septic and sham mice. In addition, the metabolic profile in the septic mice pretreated with LGG was much closer to that of sham mice compared with control septic mice. The results of the present study suggest that probiotic pre-administration reduces the mortality in septic mice by decreasing ileum mucosal damage, increasing the gut barrier integrity and altering global serum metabolic profiles.

Bafilomycin A1 inhibits the growth and metastatic potential of the BEL-7402 liver cancer and HO-8910 ovarian cancer cell lines and induces alterations in their microRNA expression.

The vacuolar H+-ATPase (V-ATPase) is commonly highly activated in cancer cells and is a potential target of anti-cancer therapy. Bafilomycin A1 is a specific inhibitor of the c subunit of V-ATPase. In the present study, the effects of bafilomycin A1 on the BEL-7402 hepatocellular carcinoma and HO-8910 ovarian cancer cell lines were respectively studied. In addition, the bafilomycin A1-induced alterations in the mRNAs and microRNAs (miRNAs) in the cells were detected using microarray methods. The results demonstrated that the growth of the two cell lines was retarded and the metastatic potential was inhibited by bafilomycin A1. Transmission electron microscopy and assays of capsase-3 and -9 suggested that bafilomycin A1 induced apoptosis. Gene Ontology analysis of the microarrays of mRNA-miRNA integrity showed altered pathways following bafilomycin A1 treatment, including pathways regulating glucose or lipid metabolism, DNA repair or duplication and lysosomes. Quantitative polymerase chain reaction analysis confirmed that miR-923, miR-1246, miR-149*, miR-638 and miR-210 were upregulated and miR-99a, miR-181a-2* and miR-339-5p were downregulated following bafilomycin A1 treatment. The overlapped altered miRs may be effective targets for the two types of solid tumor, and may have potential for application to the treatment of other types of solid tumor.

Ultra­performance liquid chromatography coupled with quadrupole time­of­flight mass spectrometry­based metabolic profiling of human serum prior to and following radical resection of colorectal carcinoma.

Nearly one quarter of patients with colorectal carcinoma (CRC) were diagnosed at an advanced stage. Under these circumstances, radical resection of the tumor is the best strategy to enhance the five-year survival rate. However, up to 50% of post-operative patients experience cancer recurrence within the first few years. Therefore, post­operative surveillance is important. However, currently performed post­operative monitoring relies on relatively dated methods with insufficient sensitivity and specificity. The present study applied an advanced technology of ultra­performance liquid chromatography coupled with quadrupole time­of­flight mass spectrometry in order to examine changes in metabolite patterns in serum with the aim of identifying reliable biomarkers in patients with CRC at various time-points. Serum samples were collected from and 20 CRC patients prior to radical resection (group 1) and one month following radical resection (group 2) as well as from 20 healthy volunteers (group 3). Multivariate pattern recognition was used to identify potential biomarkers of CRC. Compared with healthy volunteers, three groups of biomarkers were identified in patients with CRC (P<0.05), namely phosphatidylcholines (PCs), lysophosphatidylcholines (LPCs) and diacylglycerols (DAGs). However, no statistical difference in the levels of these biomarkers between pre­operative and post­operative CRC patients was identified (P>0.05). PCs and LPCs, which contain polyunsaturated fatty acids, were decreased, whereas LPCs and DAGs, which contain saturated fatty acids, were increased in CRC patients. The present study demonstrated that obvious metabolic disturbances occur during the development of CRC and provided a novel analytic method, which is likely to be used as a diagnostic tool for CRC and may help to improve the patients' prognosis.

Effects of a low-fat diet on the hepatic expression of adiponectin and its receptors in rats with NAFLD.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is correlated with obesity, but specific therapeutic interventions are lacking. Adiponectin is an adipokine with anti-inflammatory activity and is considered a hepatic protector. We aimed to investigate effects of a low-fat diet on the hepatic expression of adiponectin and its receptors in rats with NAFLD. MATERIALS AND METHODS: Sixteen male SD rats were fed a high-fat diet for 8 weeks (HFD1 group) or 16 weeks (HFD2 group) to induce NAFLD, and these rats were compared with rats on a normal diet for 8 weeks (NC1 group) or 16 weeks (NC2 group). Another group of 8 rats was fed an HFD for 8 weeks and then switched to a low-fat diet (DIET group) until the 16th week. The expression of hepatic adiponectin and its receptors was detected by western blotting, immunohistochemistry and RT-qPCR. RESULTS: The NAFLD activity score (NAS) in the HFD groups increased from 3.2 ± 0.45 (8th week) to 6.2 ± 0.84 (16th week) (P < 0.001), reflecting the progression in the NAFLD histology. In contrast to the HFD2 group, the low-fat diet ameliorated the steatosis, ballooning degeneration and inflammation. Dietary intervention augmented the expression of adiponectin and its receptors, which was down-regulated in the HFD2 group. CONCLUSIONS: The NAFLD rat model was successfully developed by feeding the animals a high-fat diet. Adiponectin may play a role in the pathogenesis of NAFLD, especially in the progression from steatosis to NASH. The low-fat diet alleviated the histological lesions associated with NAFLD by up-regulating the expression of adiponectin and its receptors.

Knockout of the HCC suppressor gene Lass2 downregulates the expression level of miR-694.

Homo sapiens longevity assurance homolog 2 of yeast LAG (Lass2) catalyzes the synthesis of long-chain ceramide which is an essential element of membranous structures. Deletion of Lass2 is associated with a high risk of spontaneous or DEN-induced hepatocellular carcinoma (HCC), yet the mechanism remains unclear. In the present study, we found extensive vesicles in hepatocytes of one-month-old Lass2-knockout (KO) mice. Hepatic biochemical indices were increased and expression of albumin was attenuated in the one­month Lass2-KO liver. The results indicate that the injuries of the hepatocytes in young Lass2-KO mice, based on the results of Gene Ontology analysis of mRNA microarray of Lass2-KO liver vs. wild-type liver showed 'wounding response' was the mostly possible altered pathway in the Lass2-KO mice. miR-mRNA integrated analysis revealed that miR-694 was downregulated while its target gene tumor necrosis factor α-induced protein 3 (Tnfaip3) was upregulated, as confirmed by qPCR. The expression of NF-κB which is negatively controlled by Tnfaip3 was detected by qPCR and was found to be downregulated. Herein, we first report that Lass2 deficiency caused the downregulation of miR-694 and the upregulation of its target gene Tnfaip3 in vivo in mice, which may be related to a high risk of occurrence of HCC.

Enhancement of DEN-induced liver tumourigenesis in hepatocyte-specific Lass2-knockout mice coincident with upregulation of the TGF-ß1-Smad4-PAI-1 axis.

Longevity assurance homolog 2 of yeast LAG1 (Lass2) gene is capable of suppressing the proliferation and metastasis of several types of tumours including liver cancer. In the present study, hepatocyte-specific Lass2-knockout (Lass2 KO) and wild-type (WT) mice were exposed to the carcinogen, diethylnitrosamine (DEN), to induced liver tumours. At week 23 following DEN injection, tumours were produced in 100% of the Lass2 KO mice and 21.4% of the WT mice. At week 40, 100% of the Lass2 KO mice and 78.6% of the WT mice developed tumours, with no distinct significant difference in tumour occurrences between the two genotypes; yet, tumours in the Lass2 KO mouse livers were more numerous and larger in size. Hepatocellular carcinoma (HCC) was confirmed by α-fetoprotein (AFP). PCNA and EdU assays indicated more active proliferation whereas TUNEL assay revealed decreased apoptosis in Lass2 KO livers, when compared with the WT control. The expression of plasminogen activator inhibitor type-1 (PAI-1), a tumour-promoting gene, in the liver tissues of the 2 genotypes was detected using qPCR and western blotting, showing that PAI-1 levels were significantly elevated in Lass2 KO livers at week 40 following DEN introduction. Moreover, the expression of PAI-1-related TGF-ß1, Smad-4 and -7 was detected, displaying an elevation in TGF-ß1 and Smad-4 (not including Smad-7) in the Lass2 KO livers. Our data demonstrates that i) Lass2 is a protective gene against DEN-induced liver tumourigenesis; and ii) upregulation of the TGF-ß1-Smad4-PAI-1 axis may contribute to the vulnerability of Lass2-knockout mice to DEN.