Catalytic efficiency and thermal stability promotion of the cassava linamarase with multiple mutations for better cyanogenic glycoside degradation.

In our previous study, we found that cassava cyanogenic glycosides had an acute health risk. Therefore, to solve this problem, the improvement of specific degradation of cyanogenic glycosides of cassava linamarase during processing is the key. In this study, the catalytic activity and thermal stability of enzymes were screened before investigating the degradation efficiency of cyanogenic glycosides with a cassava linamarase mutant K263P-T53F-S366R-V335C-F339C (CASmut) -controlled technique. The CASmut was obtained with the optimum temperature of 45 °C, which was improved by 10 °C. The specific activity of CASmut was 85.1 ± 4.6 U/mg, which was 2.02 times higher than that of the wild type. Molecular dynamics simulation analysis and flexible docking showed there were more hydrogen bonding interactions at the pocket, and the aliphatic glycoside of the linamarin was partially surrounded by hydrophobic residues. The optimum conditions of degradation reactions was screened with CASmut addition of 47 mg/L at 45 °C, pH 6.0. The CASmut combined with ultrasonication improved the degradation from 478.2 ± 10.4 mg/kg to 86.7 ± 7.4 mg/kg. Those results indicating the great potential of CASmut in applying in the cassava food or cyanogenic food. However, challenges in terms of the catalytic mechanism research is worthy of being noticed in further studies.

Investigation of the mechanism of different 3D printing performance of starch and whole flour gels from tuber crops.

This study aims to reveal the variation in 3D printing performance of whole flour and starch gels as derived from different varieties of tuber crops including cassava, potato, and yam, along with its mechanism. The whole flour of the same tuber crops showed a higher branching degree, average molecular weight (R¯h), and the proportion of AM chains for 100 < X ≤ 1000 than its starch. Due to the higher degree of branching, the crystallinity of whole flour reached a higher level. In this circumstance, G2' of the dispersion system decreased, which facilitated smooth extrusion of ink from the nozzle, thus improving the precision of printing for the final product. Besides, a higher R¯h and the percentage of AM chains for 100 < X ≤ 1000 made it easier for the material to extrude, thus enhancing the printing accuracy of the product. The higher short-range ordered structure of whole flour also enhanced the printing performance of 3D printed products. This research contributes an effective solution to the selection of starch and whole flour for food 3D printing.

Effects of the structure and interaction force of phytosterol/whey protein isolate self-assembly complex on phytosterol digestion properties.

Phytosterol (PS)/whey protein isolate (WPI) self-assembly complex was formed with different PS:WPI mass ratios (from 1:1 to 1:15) to reveal the relationship of interaction mechanism in PS/WPI complex, WPI secondary structure, and PS digestion properties. The sample with 1:5 mass ratio had the strongest hydrogen bonds and the largest encapsulation efficiency (EE). Circular dichroism analysis indicated that the mass ratio of PS/WPI complex had a vital effect on WPI secondary structure. The content of ß-sheet increased with the increase in PS ratio, which, in turn, inhibited the release rate and bioaccessibility of PS. The ß-turn of WPI was positively correlated with hydrogen bonds in the complex, thus playing a significant role in increasing EE, promoting PS release, and even further enhancing PS bioaccessibility in digestion simulation. These findings appeared to be promising for future applications in enhancing the bioaccessibility of phytosterol through specific structure in high-protein food systems.

Investigation of the mechanism of casein protein to enhance 3D printing accuracy of cassava starch gel.

This work evaluated the potential mechanism of casein protein (CP) in enhancing the 3D printing performance of cassava starch (CS) gel in terms of the multi-scale structure of starch and gel properties. The addition of CP could increase the average molecular weight (-Rh) of starch after thermal processing, which reduced the line width and increased the centre height of the 3D-printed product, despite the reduction of the structural recovery of the gel system. In addition, the increase in CS content caused a decrease in the short-range ordered structure of starch, resulting in a decline in relaxation time and an increase in free water content (A23), which in turn provided the gel system with a greater elastic modulus and ultimately increased the printing precision of 3D printed products. This study presented meaningful information for the application of 3D printing to starch-protein complex foods.

CDK4/6 Inhibition Enhances Oncolytic Virus Efficacy by Potentiating Tumor-Selective Cell Killing and T-cell Activation in Refractory Glioblastoma.

Glioblastoma (GBM) is among the most aggressive human cancers. Although oncolytic virus (OV) therapy has been proposed as a potential approach to treat GBM, it frequently fails because GBM cells are usually nonpermissive to OV. Here, we describe a dual-step drug screen for identifying chemical enhancers of OV in GBM. From a high-throughput screen of 1416 FDA-approved drugs, an inhibitor of CDK4/6 was identified as the top enhancer, selectively increasing potency of two OV strains, VSVΔ51 and Zika virus. Mechanistically, CDK4/6 inhibition promoted autophagic degradation of MAVS, resulting in impaired antiviral responses and enhanced tumor-selective replication of VSVΔ51 in vitro and in vivo. CDK4/6 inhibition cooperated with VSVΔ51 to induce severe DNA damage stress and amplify oncolysis. In GBM xenograft models, combined treatment with CDK4/6 inhibitor and VSVΔ51 significantly inhibited tumor growth and prolonged the survival of tumor-bearing mice. Further investigation revealed that CDK4/6 inhibitor and VSVΔ51 synergistically induced immunogenic cell death and boosted antitumor immunity. Together, this study features a promising approach of treating aggressive GBM through the combination of CDK4/6 inhibitor with OV. SIGNIFICANCE: This study proposes inhibition of cyclin-dependent kinases as a clinically translatable combinatorial strategy to enhance the efficacy of oncolytic virotherapy in GBM.

Atmospheric pressure plasma jet pretreatment to facilitate cassava starch modification with octenyl succinic anhydride.

Cassava starch (CS) was pretreated with atmospheric pressure plasma jet (APPJ), followed by esterification with octenyl succinic anhydride (OSA). This study was the first report investigating the effect of APPJ on CS modification with OSA. Results showed that APPJ pretreatment could change the morphological characteristics and crystallinity of CS. Consequently, the degree of substitution and reaction efficiency significantly improved compared with the unpretreated CS (P < 0.05). In Confocal laser scanning microscopy, the fluorescence intensity of OSA-modified CS pretreated with APPJ for 10 min and 15 min was higher than those pretreated with APPJ for 1, 3, and 5 min. The onset temperature and enthalpy (ΔH) of native starch decreased after APPJ pretreatment and further decreased by OSA modification. APPJ-OSA-CS also showed better emulsion stability and emulsion activity. This study demonstrated that APPJ could be used as a novel approach to facilitate starch modification with OSA.

Tuber flours improve intestinal health and modulate gut microbiota composition.

The different health effects between starch and whole flour from tubers are rarely studied. Here, we investigated the effects of cassava flour (CF), cassava starch (CS), potato flour (PF), and potato starch (PS) on gut health and gut microbiota of normal rats. Feed analysis showed that CF and PF diet provided significantly more slowly digestible and resistant starch, less rapidly digestible starch. Compared with rats fed with PS and CS diets, rats fed with PF and CF diets gained less body weight and have tighter intestinal barrier. Butyric acid contents were increased by tuber flours. CF and PF selectively promoted the relative abundance of Akkermansia and Eubacterium ruminantium in cecal and colonic content. In conclusion, tuber flour has intestinal protection, body weight control, and gut microbiota improving ability compared with starch. The different composition of starch might be the basis for these effects.

Health benefits and phenolic compounds of Moringa oleifera leaves: A comprehensive review.

BACKGROUND: Moringa oleifera Lam (MO) is native to India and is a cash crop widely cultivated in tropical and sub-tropical areas. The health improving properties of MO has been studied from a long time ago for the numerous phenolic compounds, including vitamins, flavonoids, phenolic acids, isothiocyanates, tannins and saponins, which are present in considerable amounts in the plant. A growing spectrum of therapeutic characteristics of MO leaves has been found and used in the remission or treatment of oxidative stress, liver disease, neurological disease, hyperglycemia and cancer. HYPOTHESIS: This review focused on researches applying MO or MO leaf extract as a functional food or cure against various disease and cellular injuries. We believed it would help the discovery of therapeutic application of MO and understanding of MO phytochemistry. METHODS: The data collected in this review were extracted from researches indexed in Web of Science, google scholar, PubMed, Science Direct and Scopus to find out health benefits and biological activities of MO leaves polyphenols. The studies reporting mechanistic route of phenolic compounds of MO leaves were also considered in the present study. RESULTS: It has been reported that polyphenols of MO leaf have protective characteristics against neurodegenerative disorders through reducing DNA damage, activation of AchE activity and inhibition of caspase-3 activity. It has been reported that, they protected the kidney from damage caused by melamine through suppressed the pro-inflammatory cytokine, metallopeptidase inhibitor 1 (TIMP-1), and kidney injury molecule 1 (KIM-1). Similarly, methanol extract of MO leaves has low hypoglycemic attributes and attenuate the risk of diabetes caused by alloxan by enhancing lipid metabolism and stimulating insulin release, glucose uptake, and glycogen synthesis. In addition, MO leaves are becoming the best phytomedicine to reduce hypertension, which are naturally known as angiotensin-1converting enzyme (ACE), acetylcholinesterase, arginase and phosphodiesterase 5 (PDE5) inhibitors. CONCLUSION: MO leaves extract as a health promoting food additives for human and animals due to its great protective effect against many diseases and the widely persistent environmental toxins which disrupted cellular metabolic function. More studies are required to use the phenolic compounds of MO leaves to develop and produce drugs for controlling and treatment of various diseases.

Effect of ultrasonic pretreatment on eliminating cyanogenic glycosides and hydrogen cyanide in cassava.

Traditional soaking method takes days to remove cassava cyanide. Ten minutes of ultrasonic pretreatment (UPT) was found to be a new effective method to eliminate both cyanogenic glycosides and hydrogen cyanide in cassava. Here, the parameters of UPT were optimized and the underlying mechanisms were investigated. 40.36% and 24.95% of hydrogen cyanide and cyanogenic glycosides in cassava juice were eliminated under 10 min of UPT (45℃, 81 W). UPT before boiling enhanced the total cyanide elimination to 41.94%. The degradation patterns of hydrogen cyanide and cyanogenic glycosides were different. Ultrasound directly eliminated hydrogen cyanide and indirectly degraded cyanogenic glycosides through promoting enzymatic hydrolysis. The ß-glucosidase activity was increased by 17.99% induced by ultrasound. This was supported by the movement of hydrophobic residual and the rearrangement of the secondary structure of the molecular as found in fluorescence, CD, FTIR, DSC and TG analysis. This study revealed that UPT acted as a fast and simple technical way in improving cassava safety.

Non-Structural Protein 5 of Zika Virus Interacts with p53 in Human Neural Progenitor Cells and Induces p53-Mediated Apoptosis.

Zika virus (ZIKV) infection could disrupt neurogenesis and cause microcephaly in neonates by targeting neural progenitor cells (NPCs). The tumor suppressor p53-mediated cell cycle arrest and apoptotic cell death have been suggested to be activated upon ZIKV infection, yet the detailed mechanism is not well understood. In the present study, we investigated the effects of ZIKV-encoded proteins in the activation of p53 signaling pathway and found that, among the ten viral proteins, the nonstructural protein 5 (NS5) of ZIKV most significantly activated the transcription of p53 target genes. Using the immunoprecipitation-coupled mass spectrometry approach, we identified that ZIKV-NS5 interacted with p53 protein. The NS5-p53 interaction was further confirmed by co-immunoprecipitation and GST pull-down assays. In addition, the MTase domain of NS5 and the C-terminal domain of p53 were mapped to be responsible for the interaction between these two proteins. We further showed that ZIKV-NS5 was colocalized with p53 and increased its protein level in the nuclei and able to prolong the half-life of p53. Furthermore, lentivirus-mediated expression of ZIKV-NS5 in hNPCs led to an apparent cell death phenotype. ZIKV-NS5 promoted the cleavage of PARP1 and significantly increased the cell apoptosis of hNPCs. Taken together, these findings revealed that ZIKV-NS5 is a previously undiscovered regulator of p53-mediated apoptosis in hNPCs, which may contribute to the ZIKV-caused abnormal neurodevelopment.

Endogenous reverse transcriptase and RNase H-mediated antiviral mechanism in embryonic stem cells.

Nucleic acid-based systems play important roles in antiviral defense, including CRISPR/Cas that adopts RNA-guided DNA cleavage to prevent DNA phage infection and RNA interference (RNAi) that employs RNA-guided RNA cleavage to defend against RNA virus infection. Here, we report a novel type of nucleic acid-based antiviral system that exists in mouse embryonic stem cells (mESCs), which suppresses RNA virus infection by DNA-mediated RNA cleavage. We found that the viral RNA of encephalomyocarditis virus can be reverse transcribed into complementary DNA (vcDNA) by the reverse transcriptase (RTase) encoded by endogenous retrovirus-like elements in mESCs. The vcDNA is negative-sense single-stranded and forms DNA/RNA hybrid with viral RNA. The viral RNA in the heteroduplex is subsequently destroyed by cellular RNase H1, leading to robust suppression of viral growth. Furthermore, either inhibition of the RTase activity or depletion of endogenous RNase H1 results in the promotion of virus proliferation. Altogether, our results provide intriguing insights into the antiviral mechanism of mESCs and the antiviral function of endogenized retroviruses and cellular RNase H. Such a natural nucleic acid-based antiviral mechanism in mESCs is referred to as ERASE (endogenous RTase/RNase H-mediated antiviral system), which is an addition to the previously known nucleic acid-based antiviral mechanisms including CRISPR/Cas in bacteria and RNAi in plants and invertebrates.

Dietary exposure and risk assessment of cyanide via cassava consumption in Chinese population.

The intake of cassava would probably induce adverse health effects since there are toxic cyanide in cassava. However, the risk assessment of cassava consumption has not been reported in China. Therefore, this paper aimed to evaluate the dietary risks of cassava cyanide and proposed a maximum residue limit (MRL) for cyanogenic glycosides (CNGs) in cassava. The retention rate of CNGs and CN- were 61% and 11% after boiling, respectively. The acute dietary exposure of CN- and CNGs were 0.6-fold and 1.7-fold of acute risk reference dose, respectively. There was no chronic health risk across all populations concerning cassava consumption. The MRL of CNGs was proposed as 200 mg/kg in cassava. Risk assessment of cyanide for foods rich in CNGs was suggested to be based on CNGs quantification rather than that of CN-.

The Ubiquitin E3 Ligase Parkin Inhibits Innate Antiviral Immunity Through K48-Linked Polyubiquitination of RIG-I and MDA5.

Innate immunity is the first-line defense against antiviral or antimicrobial infection. RIG-I and MDA5, which mediate the recognition of pathogen-derived nucleic acids, are essential for production of type I interferons (IFN). Here, we identified mitochondrion depolarization inducer carbonyl cyanide 3-chlorophenylhydrazone (CCCP) inhibited the response and antiviral activity of type I IFN during viral infection. Furthermore, we found that the PTEN-induced putative kinase 1 (PINK1) and the E3 ubiquitin-protein ligase Parkin mediated mitophagy, thus negatively regulating the activation of RIG-I and MDA5. Parkin directly interacted with and catalyzed the K48-linked polyubiquitination and subsequent degradation of RIG-I and MDA5. Thus, we demonstrate that Parkin limits RLR-triggered innate immunity activation, suggesting Parkin as a potential therapeutic target for the control of viral infection.

Development and validation of eight cyanogenic glucosides via ultra-high-performance liquid chromatography-tandem mass spectrometry in agri-food.

An ultra-high-performance liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS) method was established and validated for the simultaneous quantification of eight cyanogenic glucosides (CNGs) in agri-food. The eight CNGs were linamarin, lotaustralin, linustatin, neolinustatin, taxiphyllin, amygdalin, dhurrin and prunasin. CNGs were extracted with aqueous methanol and cleaned via solid-phase extraction. Analytes were separated with a C18 column via gradient elution. MS/MS analysis was performed with electrospray ionisation in positive mode. Quantification was performed in multiple reaction monitoring mode. Satisfactory validation results were obtained in terms of linearity, sensitivity, precision and accuracy, matrix effect and stability. The method was applied in typical cyanogenic agri-food. CNGs in cassava, linseed, bamboo, sorghum, apricot, almond and lima bean were analyzed.

Phenolic acid profiles of common food and estimated natural intake with different structures and forms in five regions of China.

Phenolic acids have been reported to have many biological activities, but daily intake information is scarce. In this study, the phenolic acid contents of 116 commonly consumed food in five regions of China (Beijing, Hangzhou, Wuhan, Chongqing and Guangzhou) were analyzed by high performance liquid chromatography-mass spectrometry (HPLC-MS) and dietary intakes estimated. Tea had the highest total phenolic acids (TPA) content in all regions investigated. Phenolic acids were in esterified and bound forms, and hydroxycinnamic acids accounted for more than half of TPA content, except in tea and legumes. Average TPA intake was 193.50 mg/ day at the national level but ranged from 157.09 to 263.01 mg/ day among the regions. Rice, tea, and fruits were the main contributors to dietary intakes of TPA. Also, TPA intake in the period 2009-2013 increased 31.65 mg/ day compared with 2002, largely due to increased intakes of fruits, vegetables, and legumes.

Discovery of Keap1-Nrf2 small-molecule inhibitors from phytochemicals based on molecular docking.

Various phytochemicals have been reported to protect against oxidative stress. However, the mechanism underlying has not been systematically evaluated, which limited their application in disease treatment. Nuclear factor erythroid 2-related factor 2 (Nrf2), a central transcription factor in oxidative stress response related to numerous diseases, is activated after dissociating from the cytoskeleton-anchored Kelch-like ECH-associated protein 1 (Keap1). The Keap1-Nrf2 protein-protein interaction has become an important drug target. This study was designed to clarify whether antioxidantive phytochemicals inhibit the Keap1-Nrf2 protein-protein interaction and activate the Nrf2-ARE signaling pathway efficiently. Molecular docking and 3D-QSAR were applied to evaluate the interaction effects between 178 antioxidant phytochemicals and the Nrf2 binding site in Keap1. The Nrf2 activation effect was tested on a H2O2-induced oxidative-injured cell model. Results showed that the 178 phytochemicals could be divided into high-, medium-, and low-total-score groups depending on their binding affinity with Keap1, and the high-total-score group consisted of 24 compounds with abundant oxygen or glycosides. Meanwhile, these compounds could bind with key amino acids in the structure of the Keap1-Nrf2 interface. Compounds from high-total-score group show effective activation effects on Nrf2. In conclusion, phytochemicals showed high binding affinity with Keap1 are promising new Nrf2 activators.

Bioaccessibility and Absorption Mechanism of Phenylethanoid Glycosides Using Simulated Digestion/Caco-2 Intestinal Cell Models.

Acteoside and salidroside are major phenylethanoid glycosides (PhGs) in Osmanthus fragrans Lour. flowers with extensive pharmacological activities and poor oral bioavailability. The absorption mechanisms of these two compounds remain unclear. This study aimed to investigate the bioaccessibility of these compounds using an in vitro gastrointestinal digestion model and to examine the absorption and transport mechanisms of PhGs using the Caco-2 cell model. The in vitro digestion model revealed that the bioaccessibility of salidroside (98.7 ± 1.35%) was higher than that of acteoside (50.1 ± 3.04%), and the superior bioaccessibility of salidroside can be attributed to its stability. The absorption percentages of total phenylethanoid glycoside, salidroside, and acteoside were 1.42-1.54%, 2.10-2.68%, and 0.461-0.698% in the Caco-2 model, respectively. Salidroside permeated Caco-2 cell monolayers through passive diffusion. At the concentration of 200 µg/mL, the apparent permeability ( Papp) of salidroside in the basolateral (BL)-to-apical (AP) direction was 23.7 ± 1.33 × 10-7 cm/s, which was 1.09-fold of that in the AP-to-BL direction (21.7 ± 1.38 × 10-7 cm/s). Acteoside was poorly absorbed with low Papp (AP to BL) (4.75 ± 0.251 × 10-7 cm/s), and its permeation mechanism was passive diffusion with active efflux mediated by P-glycoprotein (P-gp). This study clarified the bioaccessibility, absorption, and transport mechanisms of PhGs. It also demonstrated that the low bioavailability of acteoside might be attributed to its poor bioaccessibility, low absorption, and P-gp efflux transporter.

Phytosterol Profiles of Common Foods and Estimated Natural Intake of Different Structures and Forms in China.

Phytosterols are well-known for their cholesterol-lowering effects, and the structures and forms of phytosterols affect their bioactivity. We aimed to illustrate the phytosterol profiles in common foods and estimate their natural intake in five geographical regions and among different age groups in China. In total, 12 phytosterols in free and esterified forms of 119 foods from five regions across China were examined using gas chromatography-mass spectrometry. Then, the dietary intake of phytosterols was calculated combined with the dietary foods intake data of Chinese people. The total phytosterol content was highest in vegetable oils (150.4-1230.9 mg/100 g), followed by legumes (129.6-275.6 mg/100 g), nuts (18.9-255.2 mg/100 g), and cereals (11.9-93.8 mg/100 g). Vegetables and fruits contained lower contents of total phytosterols. Phytosterols were mainly esterified in most common foods except in nuts. The predominant phytosterols were ß-sitosterol, campesterol, and stigmasterol, all of which belonged to plant sterols and 4-desmethylsterols. Total phytosterol intake varied across different regions, ranging between 257.7 and 473.7 mg/standard-person (sp)/day, with the highest intake in Beijing, followed by Hangzhou, Wuhan, Chongqing, and Guangzhou. However, phytosterol proportion was similar across regions, with ß-sitosterol accounting for 46.5-50.3% of the natural intake. Phytosterol intake was mainly constituted by plant sterols and 4-desmethylsterols in esterified form (61.9-74.6%). At the age of 2-70 years, phytosterol intake ranged from 154.3 mg/day to 348.0 mg/day in the national scale.