Valorization and protection of anthocyanins from strawberries (Fragaria×ananassa Duch.) by acidified natural deep eutectic solvent based on intermolecular interaction.

This study introduces an innovative approach for the valorization and protection of anthocyanins from 'Benihoppe' strawberry (Fragaria × ananassa Duch.) based on acidified natural deep eutectic solvent (NADES). Choline chloride-citric acid (ChCl-CA, 1:1) was selected and acidified to enhance the valorization and protection of anthocyanins through hydrogen bond. The optimal conditions (ultrasonic power of 318 W, extraction temperature of 61 °C, liquid-to-solid ratio of 33 mL/g, ultrasonic time of 19 min), yielded the highest anthocyanins of 1428.34 µg CGE/g DW. UPLC-Triple-TOF/MS identified six anthocyanins in acidified ChCl-CA extract. Stability tests indicated that acidified ChCl-CA significantly increased storage stability of anthocyanins in high temperature and light treatments. Molecular dynamics results showed that acidified ChCl-CA system possessed a larger diffusion coefficient (0.05 m2/s), hydrogen bond number (145) and hydrogen bond lifetime (4.38 ps) with a reduced intermolecular interaction energy (-1329.74 kcal/mol), thereby efficiently valorizing and protecting anthocyanins from strawberries.

Nontargeted screening of aldehydes and ketones by chemical isotope labeling combined with ultra-high performance liquid chromatography-high resolution mass spectrometry followed by hybrid filtering of features.

Aldehydes and ketones are important carbonyl compounds that are widely present in foodstuffs, biological organisms and human living environment. However, it is still challenging to comprehensively detect and capture them using liquid chromatography - mass spectrometry. In this work, a chemical isotope labeling (CIL) coupled with ultra-high performance liquid chromatography - high resolution mass spectrometry (UHPLC-HRMS) strategy was developed for the capture and detection of this class of compounds. 2,4-Dinitrophenylhydrazine (DNPH) and isotope-labeled DNPH (DNPH-d3) were utilized to selectively label the target analytes. To address the difficulties in processing UHPLC-HRMS data, a post-acquisition data processing method called MSFilter was proposed to facilitate the screening and identification aldehydes and ketones in complex matrices. The MSFilter consists of four independent filters, namely statistical characteristic-based filtering, mass defect filtering, CIL paired peaks filtering, and diagnostic fragmentation ion filtering. These filters can be used individually or in combination to eliminate unrelated interfering MS features and efficiently detect DNPH-labeled aldehydes and ketones. The results of a mixture containing 48 model compounds showed that although all individual filtering methods could significantly reduce more than 95% of the raw MS features with acceptable recall rates above 85%, but they had relatively high false positive ratios of over 90%. In comparison, the hybrid filtering method combining four filters is able to eliminate massive interfering features (> 99.5%) with a high recall rate of 81.25% and a much lower false positive ratio of 15.22%. By implementing the hybrid filtering method in MSFilter, a total of 154 features were identified as potential signals of CCs from the original 45,961 features of real tobacco samples, of which 70 were annotated. We believe that the proposed strategy is promising to analyze the potential CCs in complex samples by UHPLC-HRMS.

Development of a MOF-based SPE method combined with GC-MS for simultaneous determination of alachlor, acetochlor and pretilachlor in field soil.

In this work, a rapid, highly selective, reusable and effective method was developed for simultaneous determination of alachlor, acetochlor and pretilachlor in field soil by GC-MS coupled with MIL-101 based SPE. Main factors affecting the SPE by using MIL-101 were optimized. Moreover, by comparing with the other commercial materials such as C18, PSA and Florisil, the MIL-101(Cr) exhibited excellent adsorption performance, which aimed at amide herbicides. On the other hand, method validation displayed excellent method performance, achieving good linearities with r2 ≥ 0.9921, limits of detection between 0.25-0.45 µg kg-1, enrichment factors ≥ 89, matrix effect in the range of ± 20%, recoveries between 86.3% and 102.4%, and RSD lower than 4.38%. The developed method was successfully applied to the determination of amide herbicides in soil taken from the wheat, corn and soybean field at different depths, where the concentration of alachlor, acetochlor and pretilachlor were in the range of 0.62-8.04 µg kg-1. It was demonstrated that the more depth of soil, the lower of three amide herbicides. This finding could be proposed a novel method to detect the amide herbicides in the agriculture and food industry.

Azacytidine-induced hypomethylation delays senescence and coloration in harvested strawberries by stimulating antioxidant enzymes and modulating abscisate metabolism to minimize anthocyanin overproduction.

DNA methylation is increasingly known to be essential for fruit ripening and senescence. Currently, 5-azacytidine (AZ) was selected as an effective demethylator and it successfully shaped the genomic hypomethylation in harvested strawberries. This was associated with the reprogramming of global gene expressions, which influenced downstream food traits. The alleviation of decay and softening, as well as the deceleration of soluble solid accumulation, were included. Coloration was also delayed as a result of the AZ-induced hypomethylation. Our examinations of anthocyanin biosynthesis and transport revealed that they were markedly minimized, which was probably involved with the decreased abscisate level and its weakened metabolism. Additionally, under AZ, the retarded postharvest senescence process was observed and it might be induced by the inhibited ROS accumulation accompanying the peroxidase and catalase activities alteration. Overall, these findings underlined the importance of methylation in strawberries and suggested the potential role of epigenetic regulators in the postharvest industry.

Generation and stabilization of CO2 nanobubbles using surfactants for extraction of polyphenols from Camellia oleifera shells.

Camellia oleifera shells are abundant in polyphenolic compounds. Green extraction methods of polyphenolic compounds are essential to ensure product quality, efficiency, process cost, environment, and safety. This study investigated the effect of Tween 80 and Rhamnolipid surfactants on the production and utilization of stabilized carbon dioxide nanobubbles (CO2 -NBs). The results confirmed the presence of the CO2 -NBs in ultra-pure water with a concentration of 8.45 ± 1.05 × 108  ml-1 , among which the stable CO2 -NBs possessed a mean size of 40-90 nm and a negative zeta potential (-41.6 ± 1.3 mV). Further, the efficiency of CO2 -NBs combined with ultrasonication (CO2 -NBs-Rh-UAE) was evaluated to extract polyphenols from Camellia oleifera shells (waste). The CO2 -NBs treatment with ultrasonication showed the highest total phenolic content (TPC) and total flavonoid content (TFC) (36.75 ± 0.22 mg GAE/g DW and 24.06 ± 0.22 mg RE/g DW, respectively). Overall, this study demonstrated an innovative approach for producing, stabilizing, and utilizing biosurfactant stabilized CO2 -NBs to extract polyphenolic compounds from the waste agricultural products. These findings highlighted the potential application of biosurfactant-stabilized CO2 -NBs.

Acidified glycerol as a one-step efficient green extraction and preservation strategy for anthocyanin from blueberry pomace: New insights into extraction and stability protection mechanism with molecular dynamic simulation.

In present work, green and efficient glycerol solvent system was coupled with pulse-probe ultrasonication for one-step extraction and preservation of anthocyanin from blueberry pomace. Under optimal conditions (40 min, 174 W, 18.6 mL/g, 20% of glycerol fraction), extraction yield was 23.07 ± 0.09 mg C3GE/g DW. The extracted anthocyanins were characterized by UPLC-Triple-TOF/MS and 10 anthocyanins compounds were tentatively identified. Stability of anthocyanins influenced by solvents were evaluated in varying temperature, pH and light exposure conditions, demonstrating higher stability of anthocyanins in glycerol solvent system than methanol one. Furthermore, mechanism of high efficiency extraction and stability of anthocyanin using glycerol were investigated by quantum chemical calculation with molecular dynamic simulation. Larger solvent accessible surface area (127.16 nm2), hydrogen bonds number (228.16) and hydrogen bonds lifetime (4.35 ps), and lower intermolecular interaction energy (-1080.48 kJ/mol) between anthocyanin and glycerol were responsible for better extraction and preservation of anthocyanins using glycerol system.

Generation and characterization of nanobubbles in ionic liquid for a green extraction of polyphenols from Carya cathayensis Sarg.

Nanobubbles (NBs) generated-nanojets membrane poration have gained enormous attention. In this study, NBs were fabricated as a novel green approach to assist ionic liquid (IL) [C4C1im][BF4] extraction of polyphenols from Carya cathayensis Sarg. husk. NBs were successfully generated with mean size of 85.47 ± 5 nm, zeta potential of +39 ± 2.24 mV, and concentration of 21.15 ± 0.75 × 108 particles/mL (stable for over 48 h in IL solution). Compared to common solutions extract, IL-NBs extract showed significantly higher (p < 0.05) antioxidant activity and polyphenols yields with a total polyphenol, total flavonoid, and total tannins contents of 85.67 ± 2.05 mg GAE/g DW, 42.44 ± 1.17 mg CE/g DW, and 8.2 ± 0.05 mg TAE/g DW, respectively. The SEM results confirmed that NBs' nanojets caused morphological destruction of the husk powder. Overall, IL-NBs solution showed better extraction efficiency of polyphenols than other solutions, giving insight into a new "green" nanotechnology-based extraction method.

Sphingolipids in foodstuff: Compositions, distribution, digestion, metabolism and health effects - A comprehensive review.

Sphingolipids (SLs) are common in all eukaryotes, prokaryotes, and viruses, and played a vital role in human health. They are involved in physiological processes, including intracellular transport, cell division, and signal transduction. However, there are limited reviews on dietary effects on endogenous SLs metabolism and further on human health. Various dietary conditions, including the SLs-enriched diet, high-fat diet, and vitamins, can change the level of endogenous SLs metabolites and even affect human health. This review systematically summarizes the main known SLs in foods concerning their variety and contents, as well as their isolation and identification approaches. Moreover, the present review discusses the role of dietary (particularly SLs-enriched diet, high-fat diet, and vitamins) in endogenous SLs metabolism, highlighting how exogenous SLs are digested and absorbed. The role of SLs family in the pathogenesis of diseases, including cancers, neurological disorders, infectious and inflammatory diseases, and cardiovascular diseases, and in recently coronavirus disease-19 outbreak was also discussed. In the post-epidemic era, we believe that the concern for health and the need for plant-based products will increase. Therefore, a need for research on the absorption and metabolism pathway of SLs (especially plant-derived SLs) and their bioavailability is necessary. Moreover, the effects of storage treatment and processing on the content and composition of SLs in food are worth exploring. Further studies should also be conducted on the dose-response of SLs on human health to support the development of SLs supplements. More importantly, new approaches, such as, making SLs based hydrogels can effectively achieve sustained release and targeted therapies.

A conserved epitope III on hepatitis C virus E2 protein has alternate conformations facilitating cell binding or virus neutralization.

Epitope III, a highly conserved amino acid motif of 524APTYSW529 on the hepatitis C virus (HCV) E2 glycoprotein, resides in the critical loop that binds to the host receptor CD81, thus making it one of the most important antibody targets for blocking HCV infections. Here, we have determined the X-ray crystal structure of epitope III at a 2.0-Å resolution when it was captured by a site-specific neutralizing antibody, monoclonal antibody 1H8 (mAb1H8). The snapshot of this complex revealed that epitope III has a relatively rigid structure when confined in the binding grooves of mAb1H8, which confers the residue specificity at both ends of the epitope. Such a high shape complementarity is reminiscent of the "lock and key" mode of action, which is reinforced by the incompatibility of an antibody binding with an epitope bearing specific mutations. By subtly positioning the side chains on the three residues of Tyr527, Ser528, and Trp529 while preserving the spatial rigidity of the rest, epitope III in this cocrystal complex adopts a unique conformation that is different from previously described E2 structures. With further analyses of molecular docking and phage display-based peptide interactions, we recognized that it is the arrangements of two separate sets of residues within epitope III that create these discrete conformations for the epitope to interact selectively with either mAb1H8 or CD81. These observations thus raise the possibility that local epitope III conformational dynamics, in conjunction with sequence variations, may act as a regulatory mechanism to coordinate "mAb1H8-like" antibody-mediated immune defenses with CD81-initiated HCV infections.

A novel W/O/W double emulsion co-delivering brassinolide and cinnamon essential oil delayed the senescence of broccoli via regulating chlorophyll degradation and energy metabolism.

The postharvest senescence accompanied by yellowing limited the shelf-life of broccoli. In this study, we developed a novel W/O/W double emulsion co-delivering brassinolide and cinnamon essential oil and applied it to broccoli for preservation. Results showed that double emulsion prepared by whey protein concentrate-high methoxyl pectin (1:3) exhibited best storage stability with largest particle size (581.30 nm), lowest PDI (0.23) and zeta potential (-40.31 mV). This double emulsion also exhibited highest encapsulation efficiency of brassinolide (92%) and cinnamon essential oil (88%). The broccoli coated with double emulsion maintained higher chlorophyll contents and activities of chlorophyllase and magnesium-dechelatase were reduced by 9% and 24%, respectively. The energy metabolic enzymes (SDH, CCO, H+-ATPase, Ca2+-ATPase) were also activated, inducing higher level of ATP and energy charge. These results demonstrated W/O/W double emulsion co-delivering brassinolide and cinnamon essential delayed the senescence of broccoli via regulating chlorophyll degradation and energy metabolism.

Preharvest UV-C treatment affected postharvest senescence and phytochemicals alternation of strawberry fruit with the possible involvement of abscisic acid regulation.

As an environmentally friendly approach for fruit quality improvement, the effect of preharvest UV-C on the physiology of strawberry fruit during postharvest storage remains to be assessed. Strawberry fruit developed with supplementary UV-C were stored at room temperature for 2 weeks. Preharvest UV-C attenuated fruit postharvest senescence and altered phytochemicals composition. Higher ester titer was found in the treated fruit at harvest, whereas higher terpene and furanone contents were detected after 72 h of storage. At harvest, polyphenolics accumulated to a higher level in UV-C group, but the difference disappeared after 24 h of storage. Meanwhile, the intrinsic level of abscisic acid and the expressions of FaPYR1, SnRK2, and FaASR in the UV-C-treated fruit was enhanced at harvest but returned to a lower level as storage proceeded. This study highlights the time-dependent effect of preharvest UV-C on strawberry fruit postharvest biochemical indexes and the possible involvement of abscisic acid signaling factors.

Unveiling the Mechanisms for the Plant Volatile Organic Compound Linalool To Control Gray Mold on Strawberry Fruits.

Fungal infections significantly alter the emissions of volatile organic compounds (VOCs) by plants, but the mechanisms for VOCs affecting fungal infections of plants remain largely unknown. Here, we found that infection by Botrytis cinerea upregulated linalool production by strawberries and fumigation with linalool was able to inhibit the infection of fruits by the fungus. Linalool treatment downregulated the expression of rate-limiting enzymes in the ergosterol biosynthesis pathway, and this reduced the ergosterol content in the fungi cell membrane and impaired membrane integrity. Linalool treatment also caused damage to mitochondrial membranes by collapsing mitochondrial membrane potential and also downregulated genes involved in adenosine triphosphate (ATP) production, resulting in a significant decrease in the ATP content. Linalool treatment increased the levels of reactive oxygen species (ROS), in response to which the treated fungal cells produced more of the ROS scavenger pyruvate. RNA-Seq and proteomic analysis data showed that linalool treatment slowed the rates of transcription and translation.

Preharvest Ultraviolet C Treatment Affected Senescence of Stored Strawberry Fruit with a Potential Role of MicroRNAs in the Activation of the Antioxidant System.

Recent studies presented preharvest ultraviolet C (UV-C) as an environmentally friendly approach for the management of horticultural crop diseases. The effect of this approach on quality preservation during postharvest storage has not yet been investigated. Strawberry fruit harvested from plants grown with supplemental UV-C were stored at room temperature for 72 h, and their postharvest shelf-life biochemical indicators were evaluated. The involvement of microRNAs (miRNAs) in the activation of UV-C-induced antioxidant systems was investigated. Preharvest UV-C contributed to the preservation of sugar and organic acid and reduced overall lipid peroxidation in strawberry fruit during storage. We found that miR159 and miR398 were downregulated by preharvest UV-C and that their respective targets were upregulated at the early stage of storage with enhancement of the activity of antioxidant enzymes. The initial burst of H2O2 and O2• - suggested that preharvest UV-C primed the fruit in an antioxidative activated state via reactive-oxygen-species-mediated feedback control with post-transcriptional involvement of miRNAs.

Preharvest Ultraviolet C Irradiation Increased the Level of Polyphenol Accumulation and Flavonoid Pathway Gene Expression in Strawberry Fruit.

Preharvest ultraviolet C (UV-C) irradiation is an innovative approach for increasing the bioactive phytochemical content of strawberries to increase the disease resistance and nutritional value. This study investigated the changes in individual flavonoids in strawberry developed with three different cumulative doses of preharvest UV-C treatment (low, 9.6 kJ m-2; middle, 15 kJ m-2; and high , 29.4 kJ m-2). Significant accumulation (p < 0.05) of phenolics (25-75% increase), namely, cyanidin 3-glucoside, pelargonidin 3-glucoside/rutinoside, glucoside and glucuronide of quercetin and kaempferol, and ellagic acid, was found in the fruit subjected to low and middle supplemental doses of UV-C radiation. The expression of the flavonoid pathway structural genes, i.e., FaCHS1, FaCHI, FaFHT, FaDFR, FaFLS, and FaFGT, was upregulated in the low- and middle-dose groups, while the early stage genes were not affected by the high dose. FaMYB1 was also relatively enhanced in the low- and middle-dose groups, while FaASR was upregulated in only the low-dose group. Hormetic preharvest UV-C dose ranges for enhancing the polyphenol content of strawberries were established for the first time.

Specificity and function of archaeal DNA replication initiator proteins.

Chromosomes with multiple DNA replication origins are a hallmark of Eukaryotes and some Archaea. All eukaryal nuclear replication origins are defined by the origin recognition complex (ORC) that recruits the replicative helicase MCM(2-7) via Cdc6 and Cdt1. We find that the three origins in the single chromosome of the archaeon Sulfolobus islandicus are specified by distinct initiation factors. While two origins are dependent on archaeal homologs of eukaryal Orc1 and Cdc6, the third origin is instead reliant on an archaeal Cdt1 homolog. We exploit the nonessential nature of the orc1-1 gene to investigate the role of ATP binding and hydrolysis in initiator function in vivo and in vitro. We find that the ATP-bound form of Orc1-1 is proficient for replication and implicates hydrolysis of ATP in downregulation of origin activity. Finally, we reveal that ATP and DNA binding by Orc1-1 remodels the protein's structure rather than that of the DNA template.

Erythroid 5-aminolevulinate synthase mediates the upregulation of membrane band 3 protein expression by iron.

Iron deficiency leads to abnormal expression and function of band 3 protein in erythrocytes, but the underlying mechanisms remain elusive. The mRNA of erythroid-specific 5-aminolevulinate synthase (eALAS) contains an iron response element and the eALAS protein is an important mediator of iron utilization by erythrocytes. In this study, we investigated the effect of short hairpin RNA (shRNA) mediated silencing of eALAS on the expression of band 3 protein induced by iron. By real-time RT-PCR and Western blot we showed that at mRNA and protein level iron-induced expression of band 3 protein was lower in eALAS-shRNA transfected K562 cells than in control cells. Of note, the lowest expression was detected in K562 cells cultured in iron deficiency condition (p < 0.01). Thus either iron deficiency or depletion of eALAS could suppress the expression of erythroid band 3 protein. These results demonstrated for the first time that iron and the iron-regulatory system regulate the expression of the erythrocyte membrane proteins.

[An analysis of prognostic factors in 80 myelodysplastic syndrome].

OBJECTIVE: To investigate the WHO classification, clinical and hematological features and risk group of International Prognostic Scoring System (IPSS) in patients with myelodysplastic syndrome (MDS). METHODS: The diagnosis and classification of MDS patients were defined according to the WHO classification. The clinical manifestations, hemogram, bone marrow biopsy and prognosis were retrospectively analyzed. RESULTS: The median age at diagnosis of MDS was 47 yrs being younger than that in some foreign reports. The frequency of abnormal karyotype was 35.14% and +8 was the most frequent abnormal karyotype in our study. Eleven of 74 patients transformed into leukemia. Univariate analysis showed that age, chromosome abnormality, percentage of bone marrow blast cells and number of cytopenias were significantly related to prognosis. There was a statistical difference in cum survival rate between IPSS subcategories (P < 0.05) except that between low- and intermediate I-risk subcategory (P > 0.05). There were statistical differences for refractory anemia (RA) vs RA with excess blast (RAEB), refractory cytopenias with multilineage dysplasia (RCMD) vs RAEB and RAEB-I vs RAEB-II (P < 0.05). CONCLUSIONS: There were differences in age of disease onset, distribution of WHO, sub-classification and abnormal karyotype in this cohort of MDS patients as compared with those in Europe and Japan. It is helpful in diagnosis, treatment and prognosis to divide RAEB into RAEB-I and RAEB-II. IPSS was well applicable in Chinese MDS patients.

PC12 cell model of inducible expression of mutant DISC1: new evidence for a dominant-negative mechanism of abnormal neuronal differentiation.

A balanced chromosomal translocation, segregating with mental illnesses in a large Scottish family, interrupts the disrupted-in-schizophrenia 1 (DISC1) gene, which would result in loss of DISC1 function via haploinsufficiency or dominant-negative effects (or possibly could cause gain-of-function effects) if a truncated protein is present. To evaluate the effects of a predicted protein, mutant DISC1, we generated stable PC12 cell clones with inducible expression of mutant or full-length human DISC1 (hDISC1). Our study presents new observations that the inhibitory effects of mutant hDISC1 on NGF-induced neurite outgrowth are dependent on the level and timing of expression of mutant DISC1 and the concentrations of NGF, and are associated with altered sub-cellular distribution of endogenous DISC1 and ATF4, and decreased protein levels of LIS1. Thus, inducible expression of DISC1 in PC12 cell clones is a valuable in vitro model for further studying the molecular mechanisms likely due to loss of function of DISC1 relevant to the pathogenesis of major mental illnesses.

[A pilot study of spectrum of gene expression of acute leukemia].

OBJECTIVE: To investigate the gene expression of acute leukemia so as to study the pathogenesis of leukemia. METHODS: Five ml of bone marrow was collected from 22 patients with leukemia, 15 males and 7 females, aged 15-86, 17 with acute myelocytic leukemia, 4 with acute lymphocytic leukemia, and 1 with AHL. Mononuclear cells were isolated. Total RNA was extracted and mRNA was purified. DNA microarray technique with 12 848 genes was used to analyze the gene expression profiles. RESULTS: The predicted 45 genes were enormously expressed in 21 patients with acute leukemia, which were consistent with the ALL and AML classification standards reported by Golub and others. 7 genes which were overexpressed in multiple-resistant cell line K562-n/VCR were also overexpressed in 6 cases of refractory acute leukemia. Further analysis showed that 31 genes were upregulated in AML (M(4)-M(6)) and ALL but downregulated in AML (M(1)-M(3)). CONCLUSION: This results support the standard Golub has put forward. The reason that adult patients with ALL and subtypes of AML (M(4)-M(6)) have poorer prognosis in comparison with AMLM(1)-M(3) is attributed to alterations in gene expression. Primary drug resistance may be the major characteristics of refractory leukemia. Analysis of gene expression profile in acute leukemia is significant for classification and prognosis.

[Clinical and experimental study of two cases of myelodysplastic syndromes with double isochromosome 20q- anomaly].

OBJECTIVE: To explore the clinical and laboratory characteristics of two myelodysplastic syndromes (MDS) patients with double isochromosome 20q- anomaly. METHODS: Bone marrow cell chromosome preparations were made with both direct method and short-term culture. Karyotype analysis was performed by R-banding technique, and dual-color FISH (fluorescence in situ hybridization) by using a 20q telomeric probe and a sequence-specific probe for 20q12. RESULTS: The clinical and hematological findings were comparable with diagnosis of MDS. Karyotype analysis showed that both patients had double isochromosome 20q- anomaly: case 1 is 46, XX, der(20)? i(20q-) [6]/46, idem, der (6) i (6p) [1]/47, idem, +der (20)? i (20q-) [3]/47, idem, der(6)i (6p), +der(20)? i (20q-) [20]; case 2 is 45, XY, -7, der (20)? i (20q-) [17]/46, idem, +der(20) ? i(20q-) [3]. Two derivative chromosomes 20 were proved 20q isochromosomes with interstitial deletions by dual-color FISH in one patient. CONCLUSIONS: Double isochromosome 20q- anomaly is a rare recurrent karyotype abnormality in MDS, and signals a poor prognosis.