Inherited KDM6AA649T facilitates tumor-immune escape and exacerbates colorectal signet-ring cell carcinoma outcomes.

Childhood onset of colorectal signet-ring cell carcinoma (CR-SRCC) is extremely rare and featured as highly malignant with poor prognosis. Here we reported a CR-SRCC case of 11-year-old boy with a novel inherited X-linked KDM6AA694T mutation. The H3K27me3 demethylase KDM6A was frequently mutated in varieties of tumors and acts as a tumor suppressor. In vivo H3K27me3 demethylation assay demonstrated that KDM6AA694T had dampened H3K27me3 demethylase activity. Overexpression of KDM6AA694T in SRCC cell line KATO3 promoted cell proliferation, invasion and migration, which were further confirmed in vivo by constructing orthotopic tumor growth and lung metastasis model. Besides, expression of KDM6AA694T in immune cells suppresses inflammatory macrophage response and effector T cell response. In conclusion, we characterized a novel inherited KDM6AA694T mutant from a childhood-onset SRCC case and demonstrated that the mutant with impaired H3K27me3 demethylase activity could potentiate tumor malignancy and suppress antitumor immunity.

Pretreatment of chitin depolymerization by expansin-like protein to improve hydrolysis efficiency.

Chitin is a polysaccharide similar to cellulose that contains abundant hydrogen bonds. Expansin-like proteins disrupt hydrogen bond networks, causing cellulose to swell and accelerating its degradation. We examined the effects of pretreatment with two expansin-like proteins, CxEXL22 (Arthrobotrys sp. CX1) and HcEXL (Hahella chejuensis), on chitin depolymerisation and enzymatic degradation. The efficiency of chitin degradation increased more than two-fold after pretreatment with expansin-like proteins. Following pretreatment with expansin-like proteins, chitin had a lower crystallinity index, greater d-spacing and crystallite size, and weaker hydrogen bonds, and the loosened porous microfibrils were more exposed than in untreated chitin. The rupture characterisation of crystalline chitin indicated that expansin-like proteins loosened the hydrogen bonds of the chitin polysaccharide chains, causing significant depolymerisation to expose more porous structures and enhance chitin accessibility.

Inhibition of Chitosan with Different Molecular Weights on Barley-Borne Fusarium graminearum during Barley Malting Process for Improving Malt Quality.

There are many Fusarium graminearum contaminations in barley that are often associated with malt and beer quality issues. Thus, it is important to find a biological antifungal agent to prevent the growth of F. graminearum during malting. Minimum inhibition concentration (MIC) of chitosan for mycelial growth and spore germination of F. graminearum was 2.6 g/L and 1.6 g/L, respectively, indicating that the F. graminearum strain was highly sensitive toward chitosan. Chitosan with a molecular weight of 102.7 kDa was added at 0.5 g/kg during the first steeping stage, resulting in the maximum inhibition rate of F. graminearm in barley. The biomass of F. graminearm and deoxynivalenol content in the infected barley at the end of germination with 0.5 g/kg chitosan treatment were decreased by 50.7% and 70.5%, respectively, when compared with the infected barley without chitosan. Chitosan could remove the negative effects of F. graminearm infection on barley germination and malt quality, which makes the application of chitosan during the steeping process as a potential antifungal agent in the malting process to protect from F. graminearum infection.

Enhancement of Vindoline and Catharanthine Accumulation, Antioxidant Enzymes Activities, and Gene Expression Levels in Catharanthus roseus Leaves by Chitooligosaccharides Elicitation.

Catharanthus roseus (L.) G. Don is a plant belonging to the genus Catharanthus of the Apocynaceae family. It contains more than one hundred alkaloids, of which some exhibit significant pharmacological activities. Chitooligosaccharides are the only basic aminooligosaccharides with positively charged cations in nature, which can regulate plant growth and antioxidant properties. In this study, the leaves of Catharanthus roseus were sprayed with chitooligosaccharides of different molecular weights (1 kDa, 2 kDa, 3 kDa) and different concentrations (0.01 µg/mL, 0.1 µg/mL, 1 µg/mL and 10 µg/mL). The fresh weights of its root, stem and leaf were all improved after chitooligosaccharides treatments. More importantly, the chitooligosaccharides elicitor strongly stimulated the accumulation of vindoline and catharanthine in the leaves, especially with the treatment of 0.1 µg/mL 3 kDa chitooligosaccharides, the contents of them were increased by 60.68% and 141.54%, respectively. Furthermore, as the defensive responses, antioxidant enzymes activities (catalase, glutathione reductase, ascorbate peroxidase, peroxidase and superoxide dismutase) were enhanced under chitooligosaccharides treatments. To further elucidate the underlying mechanism, qRT-PCR was used to investigate the genes expression levels of secologanin synthase (SLS), strictosidine synthase (STR), strictosidine glucosidase (SGD), tabersonine 16-hydroxylase (T16H), desacetoxyvindoline-4-hydroxylase (D4H), deacetylvindoline-4-O-acetyltransferase (DAT), peroxidase 1 (PRX1) and octadecanoid-responsive Catharanthus AP2-domain protein 3 (ORCA3). All the genes were significantly up-regulated after chitooligosaccharides treatments, and the transcription abundance of ORCA3, SLS, STR, DAT and PRX1 reached a maximal level with 0.1 µg/mL 3 kDa chitooligosaccharides treatment. All these results suggest that spraying Catharanthus roseus leaves with chitooligosaccharides, especially 0.1 µg/mL of 3 kDa chitooligosaccharides, may effectively improve the pharmaceutical value of Catharanthus roseus.

A highly versatile fungal glucosyltransferase for specific production of quercetin-7-O-ß-D-glucoside and quercetin-3-O-ß-D-glucoside in different hosts.

Glycosylation is an effective way to improve the water solubility of natural products. In this work, a novel glycosyltransferase gene (BbGT) was discovered from Beauveria bassiana ATCC 7159 and heterologously expressed in Escherichia coli. The purified enzyme was functionally characterized through in vitro enzymatic reactions as a UDP-glucosyltransferase, converting quercetin to five monoglucosylated and one diglucosylated products. The optimal pH and temperature for BbGT are 35 ℃ and 8.0, respectively. The activity of BbGT was stimulated by Ca2+, Mg2+, and Mn2+, but inhibited by Zn2+. BbGT enzyme is flexible and can glycosylate a variety of substrates such as curcumin, resveratrol, and zearalenone. The enzyme was also expressed in other microbial hosts including Saccharomyces cerevisiae, Pseudomonas putida, and Pichia pastoris. Interestingly, the major glycosylation product of quercetin in E. coli, P. putida, and P. pastoris was quercetin-7-O-ß-D-glucoside, while the enzyme dominantly produced quercetin-3-O-ß-D-glucoside in S. cerevisiae. The BbGT-harboring E. coli and S. cerevisiae strains were used as whole-cell biocatalysts to specifically produce the two valuable quercetin glucosides, respectively. The titer of quercetin-7-O-ß-D-glucosides was 0.34 ± 0.02 mM from 0.83 mM quercetin in 24 h by BbGT-harboring E. coli. The yield of quercetin-3-O-ß-D-glucoside was 0.22 ± 0.02 mM from 0.41 mM quercetin in 12 h by BbGT-harboring S. cerevisiae. This work thus provides an efficient way to produce two valuable quercetin glucosides through the expression of a versatile glucosyltransferase in different hosts. KEY POINTS: • A highly versatile glucosyltransferase was identified from B. bassiana ATCC 7159. • BbGT converts quercetin to five mono- and one di-glucosylated derivatives in vitro. • Different quercetin glucosides were produced by BbGT in E. coli and S. cerevisiae.

The biogenic amine-producing bacteria from craft beer and their kinetic analysis between growth characteristics and biogenic amine formation in beer.

Craft beer because of its fresh flavor, unique taste, and rich nutrition is becoming more popular to consumers. Compared with industry beer, craft beer is often nonfiltered and nonpasteurized, for this reason, it has a short shelf life and is more susceptible to microbial spoilage, which may cause the quality deterioration of craft beer and the formation of biogenic amine as a harmful factor for consumer's health. In this study, the 23 beer-spoilage bacteria were isolated from craft beer, which were identified as 15 Lactobacillus (L.) brevis, 3 L. plantarum, 1 L. parabuchneri, 2 L. paracasei, and 2 Pediococcus damnosus. Among 23 beer-spoilage isolates, 20 representatives were able to form tyramine, histamine, putrescine, cadaverine, and/or tryptamine in MRS broth. The nine Lactobacillus strains were incubated in beer and produced tyramine, histamine, putrescine, cadaverine, and/or tryptamine during beer storage process. Logistic and Gompertz model could be adopted to respectively describe the kinetics of microorganism growth and biogenic amine formation. The relationship between the biogenic amines and biomass was simulated by Luedeking-Piret model very well, and showed that the formation of biogenic amine was mainly bacteria growth-associated in beer. These findings may be helpful for finding the preventive measures to control biogenic amine formation and for enhancing the safety of craft beer. PRACTICAL APPLICATION: The selection of the biogenic amine-producing spoilage bacteria from craft beer and the investigation their kinetics of the growth and biogenic amines production under beer environmental conditions was very helpful for finding preventive measures to eliminate or reduce biogenic amine formation and for appropriate increase in food safety.

Ophiopogonin D alleviates diabetic myocardial injuries by regulating mitochondrial dynamics.

ETHNOPHARMACOLOGICAL RELEVANCE: Ophiopogonin D (OP-D) is a steroidal saponin extracted from Ophiopogon japonicus (Thunb.) Ker Gawl. (Liliaceae), that has been traditionally used to treat cough, sputum, and thirst in some Asian countries. Recently, various pharmacological roles of OP-D have been identified, including anti-inflammatory, cardioprotective, and anti-cancer effects. However, whether OP-D can prevent diabetic myocardial injury remains unknown. AIM OF THE STUDY: In this study, we aimed to observe the effects of OP-D on the diabetic myocardium. MATERIALS AND METHODS: Leptin receptor-deficient db/db mice were used as an animal model for type 2 diabetes. The effects of OP-D on blood glucose, blood lipids, myocardial ultrastructure, and mitochondrial function in mice were observed after four weeks of intragastric administration. Palmitic acid was used to stimulate cardiomyocytes to establish a myocardial lipotoxicity model. Cell apoptosis, mitochondrial morphology, and function were observed. RESULTS: Blood glucose and blood lipid levels were significantly increased in db/db mice, accompanied by myocardial mitochondrial injury and dysfunction. OP-D treatment reduced blood lipid levels in db/db mice and relieved mitochondrial injury and dysfunction. OP-D inhibited palmitic acid induced-mitochondrial fission and dysfunction, reduced endogenous apoptosis, and improved cell survival rate in H9C2 cardiomyocytes. Both in vivo and in vitro models showed increased phosphorylation of DRP1 at Ser-616, reduced phosphorylation of DRP1 at Ser-637, and reduced expression of fusion proteins MFN1/2 and OPA1. Meanwhile, immunofluorescence co-localization analysis revealed that palmitic acid stimulated the translocation of DRP1 protein from the cytoplasm to the mitochondria in H9C2 cardiomyocytes. The imbalance of mitochondrial dynamics, protein expression, and translocation of DRP1 were effectively reversed by OP-D treatment. In isolated mice ventricular myocytes, palmitic acid enhanced cytoplasmic Ca2+ levels and suppressed contractility in ventricular myocytes, accompanied by activation of calcineurin, a key regulator of DRP1 dephosphorylation at Ser-637. OP-D reversed the changes caused by palmitic acid. CONCLUSIONS: Our findings indicate that OP-D intervention could alleviate lipid accumulation and mitochondrial injury in diabetic mouse hearts and palmitic acid-stimulated cardiomyocytes. The cardioprotective effect of OP-D may be mediated by the regulation of mitochondrial dynamics.

A novel decrystallizing protein CxEXL22 from Arthrobotrys sp. CX1 capable of synergistically hydrolyzing cellulose with cellulases.

A novel expansin-like protein (CxEXL22) has been identified and characterized from newly isolated Arthrobotrys sp. CX1 that can cause cellulose decrystallization. Unlike previously reported expansin-like proteins from microbes, CxEXL22 has a parallel ß-sheet domain at the N terminal, containing many hydrophobic residues to form the hydrophobic surface as part of the groove. The direct phylogenetic relationship implied the genetic transfers occurred from nematode to nematicidal fungal Arthrobotrys sp. CX1. CxEXL22 showed strong activity for the hydrolysis of hydrogen bonds between cellulose molecules, especially when highly crystalline cellulose was used as substrate. The hydrolysis efficiency of Avicel was increased 7.9-fold after pretreating with CxEXL22. The rupture characterization of crystalline region indicated that CxEXL22 strongly binds cellulose and breaks up hydrogen bonds in the crystalline regions of cellulose to split cellulose chains, causing significant depolymerization to expose much more microfibrils and enhances cellulose accessibility.

Manipulation of Purine Metabolic Networks for Riboflavin Production in Bacillus subtilis.

Guanosine monophosphate, the precursor for riboflavin biosynthesis, can be converted to or generated from other purine compounds in purine metabolic networks. In this study, genes in these networks were manipulated in a riboflavin producer, Bacillus subtilis R, to test their contribution to riboflavin biosynthesis. Knocking out adenine phosphoribosyltransferase (apt), xanthine phosphoribosyltransferase (xpt), and adenine deaminase (adeC) increased the riboflavin production by 14.02, 6.78, and 41.50%, respectively, while other deletions in the salvage pathway, interconversion pathway, and nucleoside decomposition genes have no positive effects. The enhancement of riboflavin production in apt and adeC deletion mutants is dependent on the purine biosynthesis regulator PurR. Repression of ribonucleotide reductases (RNRs) led to a 13.12% increase of riboflavin production, which also increased in two RNR regulator mutants PerR and NrdR by 37.52 and 8.09%, respectively. The generation of deoxyribonucleoside competed for precursors with riboflavin biosynthesis, while other pathways do not contribute to the supply of precursors; nevertheless, they have regulatory effects.

Energy Conservation and Carbon Flux Distribution During Fermentation of CO or H2/CO2 by Clostridium ljungdahlii.

Both CO and H2 can be utilized as energy sources during the autotrophic growth of Clostridium ljungdahlii. In principle, CO is a more energetically and thermodynamically favorable energy source for gas fermentation in comparison to H2. Therefore, metabolism may vary during growth under different energy sources. In this study, C. ljungdahlii was fed with CO and/or CO2/H2 at pH 6.0 with a gas pressure of 0.1 MPa. C. ljungdahlii primarily produced acetate in the presence of H2 as an energy source, but produced alcohols with CO as an energy source under the same fermentation conditions. A key enzyme activity assay, metabolic flux analysis, and comparative transcriptomics were performed for investigating the response mechanism of C. ljungdahlii under different energy sources. A CO dehydrogenase and an aldehyde:ferredoxin oxidoreductase were found to play important roles in CO utilization and alcohol production. Based on these findings, novel metabolic schemes are proposed for C. ljungdahlii growing on CO and/or CO2/H2. These schemes indicate that more ATP is produced during CO-fermentation than during H2-fermentation, leading to increased alcohol production.

Improvement of barley (Hordeum vulgare L.) germination by application of biochar leacheate in steeping solution to upgrade malt quality.

OBJECTIVE: To investigate an improvement of barley germination by application of biochar leacheate in the steeping solution for upgrading malt quality. RESULTS: Barley germination was improved when biochar leacheate was used in the steeping water during the first steeping cycle. A clear decrease in the time to reach 50% of final germination percentage was detected due to an addition of biochar leacheate, but no significant difference was observed in the percent germination at the end of germination. Hydrolase activities including α-amylase, proteinase and ß-glucanase in barley grains were maximally increased during the malting process when 10% biochar leacheate was added to the first steeping water. The wort yielding indexes including both glucose and maltose content and the free amino nitrogen content were significantly increased but the ß-glucan content was significantly decreased at a level of p < 0.05 when 10% biochar leacheate was added to the steeping water. CONCLUSIONS: Biochar leacheate could be used as a stimulator in the steeping solution during the first steeping cycle to improve barley germination and so upgrade malt quality.

Shengmai San Alleviates Diabetic Cardiomyopathy Through Improvement of Mitochondrial Lipid Metabolic Disorder.

BACKGROUND/AIMS: Shengmai San (SMS), prepared from Panax ginseng, Ophiopogon japonicus, and Schisandra chinensisin, has been widely used to treat ischemic disease. In this study, we investigated whether SMS may exert a beneficial effect in diabetic cardiomyopathy through improvement of mitochondrial lipid metabolism. METHODS: A leptin receptor-deficient db/db mouse model was utilized, and lean age-matched C57BLKS mice served as non-diabetic controls. Glucose and lipid profiles, myocardial structure, dimension, and function, and heart weight to tibial length ratio were determined. Myocardial ultrastructural morphology was observed with transmission electron microscopy. Protein expression and activity of oxidative phosphorylation (OXPHOS) complex were assessed using western blotting and microplate assay kits. We also observed cellular viability, mitochondrial membrane potential, OXPHOS complex activity, and cellular ATP level in palmitic acid-stimulated H9C2 cardiomyocytes. Changes in the sirtuin (SIRT1)/AMP-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) pathway and mitochondrial uncoupling signaling were assessed using western blotting and quantitative real-time PCR. RESULTS: Leptin receptor-deficient db/db mice exhibit obesity, hyperglycemia, and hyperlipidemia, accompanied by distinct myocardial hypertrophy and diastolic dysfunction. SMS at a dose of 3 g/kg body weight contributed to a recovery of diabetes-induced myocardial hypertrophy and diastolic dysfunction. SMS administration led to an effective restoration of mitochondrial structure and function both in vivo and in vitro. Furthermore, SMS markedly enhanced SIRT1 and p-AMPKα protein levels and decreased the expression of acetylated-PGC-1α and uncoupling protein 2 protein. SMS also restored the depletion of NRF1 and TFAM levels in diabetic hearts and H9C2 cardiomyocytes. CONCLUSION: The results indicate that SMS may alleviate diabetes-induced myocardial hypertrophy and diastolic dysfunction by improving mitochondrial lipid metabolism.

Enhancing the production of phenolic compounds during barley germination by using chitooligosaccharides to improve the antioxidant capacity of malt.

OBJECTIVE: To enhance the production of phenolic compounds during barley germination using chitooligosaccharide as an elicitor to improve the antioxidant capacity of malt. RESULTS: When used as an elicitor for barley germination, chitooligosaccharide with a molecular weight of 3 kDa, added at 10 mg/kg barley kernels during the first steeping cycle, led to the maximum production of phenolic compounds. Compared with the control with no chitooligosaccharide added to the steeping water, the total phenolic content was increased by 54.8%. Increases in the total phenolic content of the barley malt occurred when chitooligosaccharide was applied during the first or both the first and the second steeping cycles. Thus the antioxidant capacity of barley malt was increased significantly by adding chitooligosaccharide during the steeping process. CONCLUSION: Applying chitooligosaccharides during the steeping process increased the content of phenolic compounds thus improving the antioxidant capacity of the barley malt.

Buyanghuanwu Decoction alleviated pressure overload induced cardiac remodeling by suppressing Tgf-ß/Smads and MAPKs signaling activated fibrosis.

Buyanghuanwu Decoction (BHD), a traditional Chinese medicine recipe, is a representative prescription for the treatment of qi-deficiency and blood-stasis syndrome. In this study, the effect of BHD on pressure overload induced cardiac remodeling was investigated and possible mechanism underlying was explored. Rats were randomly divided into four groups: sham, transverse aorta constriction (TAC) with saline, TAC with telmisartan (TAC+Tel), and TAC with BHD (TAC+BHD) for 16 weeks (n=6∼8 in each group). Cardiac morphological and functional changes were evaluated by echocardiography and histological methods, the molecular alterations were detected by western blotting. Our results revealed that pressure overload prominently induced cardiac dysfunction, dilated and atrophied left ventricle, decreased cardiomyocyte cross sectional area, and fibrosis. However, BHD, similar to Tel, greatly reversed cardiac dysfunction, left ventricular dilation, and fibrosis, together with increased left ventricular wall thickness and size of cardiomyocyte. Furthermore, activated classical pro-fibrotic signaling of Tgf-ß/Smads and MAPKs after TAC was dramatically suppressed by BHD or Tel treatment. Taken together, it was demonstrated in this study that BHD exerted a cardioprotective effect against pressure overload induced cardiac remodeling via inactivation of Tgf-ß/Smads and MAPKs signaling triggered fibrosis.

Influence of biochar application on potassium-solubilizing Bacillus mucilaginosus as potential biofertilizer.

Biochar can enhance soil fertility to increase agricultural productivity, whereas its improvement in soil microbial activity is still unclear. In this article, the influence of biochar on the cell growth and the potassium-solubilizing activity of Bacillus mucilaginosus AS1153 was examined. The impact on cell growth is related to the biochar-derived feedstocks and the particle size of biochar. Both intrinsic features and inner component fraction can promote the cell growth of B. mucilaginosus AS1153. The potassium-solubilizing activity was increased by 80% when B. mucilaginosus was incubated in conjunction with the biochar derived from corn stover. The survival time of B. mucilaginosus also was prolonged by adsorption in biochar. The experimental results suggested that the biochar containing B. mucilaginosus could be used as a potential biofertilizer to sustain crop production.

Production of a single cyclic type of fructooligosaccharide structure by inulin-degrading Paenibacillus sp. LX16 newly isolated from Jerusalem artichoke root.

A novel inulin-degrading bacterium was isolated from a soil sample collected on Jerusalem artichoke roots. It is a Gram-positive, aerobic, motile and central endospore-forming straight rod, and exhibits phenotypic properties being consistent with its classification in the genus Paenibacillus. The predominant cellular fatty acids were anteiso-C15:0, C16:0 and anteiso-C17:0. This strain represents a novel species of the genus Paenibacillus on the basis of phenotypic data together with phylogenetic analysis, and it is here designated as LX16 and deposited in China centre for type collection, China (= CCTCC 2015256). Strain LX16 could produce a cyclofructooligosaccharide fructanotransferase catalysing the formation of one type of fructooligosaccharide (FOS) from inulin. The FOS was identified as a cyclofructooligosaccharide with a degree of polymerization of 6. Such homology in inulin degradation products may be beneficial for the functional FOS production.

Invertase Suc2-mediated inulin catabolism is regulated at the transcript level in Saccharomyces cerevisiae.

BACKGROUND: Invertase Suc2 was recently identified as a key hydrolase for inulin catabolism in Saccharomyces cerevisiae, whereas the Suc2 activity degrading inulin varies greatly in different S. cerevisiae strains. The molecular mechanism causing such variation remained obscure. The aim of this study is to investigate how Suc2 activity is regulated in S. cerevisiae. RESULTS: The effect of SUC2 expression level on inulin hydrolysis was investigated by introducing different SUC2 genes or their corresponding promoters in S. cerevisiae strain BY4741 that can only weakly catabolize inulin. Both inulinase and invertase activities were increased with the rising SUC2 expression level. Variation in the promoter sequence has an obvious effect on the transcript level of the SUC2 gene. It was also found that the high expression level of SUC2 was beneficial to inulin degradation and ethanol yield. CONCLUSIONS: Suc2-mediated inulin catabolism is regulated at transcript level in S. cerevisiae. Our work should be valuable for engineering advanced yeast strains in application of inulin for ethanol production.

Isolation and characterization of xanthan-degrading Enterobacter sp. nov. LB37 for reducing the viscosity of xanthan in petroleum industry.

A Gram-negative, straight rod and facultative anaerobic bacterium was isolated from soil sample. It exhibits the phenotypic characteristics consistent with its classification in the genus Enterobacter. The isolate ferment glucose to acid and gas. Arginine dihydrolase, ornithin decarboxylase and gelatinase but not deoxyribonuclease was produced by this isolate. There was no hydrogen sulfide production. On the basis of the phenotypic data, together with phylogenetic analysis based on 16S rDNA gene sequences, this strain should represent a novel species of the genus Enterobacter and was designated as LB37. The strain LB37 could degrade xanthan molecules resulting in the rapid decrease of the viscosity of xanthan solution used in oil drilling process. Endoxanthanase activity was also detected in the culture supernatant. To our knowledge, it is the first report on the microbes being involved in the xanthan degradation for oil industry. The isolate LB37 would be useful for potential application in enhanced oil recovery and oil drilling field.