Laboratory Evolution of Metalloid Reductase Substrate Recognition and Nanoparticle Product Size.

Glutathione reductase-like metalloid reductase (GRLMR) is an enzyme that reduces selenodiglutathione (GS-Se-SG), forming zerovalent Se nanoparticles (SeNPs). Error-prone polymerase chain reaction was used to create a library of ∼10,000 GRLMR variants. The library was expressed in BL21Escherichia coli in liquid culture with 50 mM of SeO32- present, under the hypothesis that the enzyme variants with improved GS-Se-SG reduction kinetics would emerge. The selection resulted in a GRLMR variant with two mutations. One of the mutations (D-E) lacks an obvious functional role, whereas the other mutation is L-H within 5 Šof the enzyme active site. This mutation places a second H residue within 5 Šof an active site dicysteine. This GRLMR variant was characterized for NADPH-dependent reduction of GS-Se-SG, GSSG, SeO32-, SeO42-, GS-Te-SG, and TeO32-. The evolved enzyme demonstrated enhanced reduction of SeO32- and gained the ability to reduce SeO42-. This variant is named selenium reductase (SeR) because of its emergent broad activity for a wide variety of Se substrates, whereas the parent enzyme was specific for GS-Se-SG. This study overall suggests that new biosynthetic routes are possible for inorganic nanomaterials using laboratory-directed evolution methods.

Mutational Analysis of TYR, OCA2, SLC45A2, and TYRP1 Genes Identifies Novel and Reported Mutations in Chinese Families with Oculocutaneous Albinism.

Objective: This study aims to investigate the main types of oculocutaneous albinism (OCA) and the distribution characteristics of mutations in the Chinese population. Additionally, genetic diagnosis and prenatal diagnosis were conducted for Chinese OCA families. Methods: A total of 116 blood DNA samples were collected from 40 unrelated families with suspected albinism. OCA gene testing and mutation screening were performed to identify mutated genes and genotypes. The prenatal genetic diagnosis was conducted on 20 fetal DNA samples (17 amniotic fluid DNA samples, 2 villus DNA samples, and 1 umbilical cord blood DNA sample). Follow-up was conducted on the born fetuses, and the feasibility and accuracy of prenatal genetic diagnosis were assessed based on the clinical phenotype of the fetuses. Results: Analysis of 40 pedigrees led to a molecular diagnosis for the patients or their parents: 24 (60%) had OCA1, 12 (30%) had OCA2, 1 (2.5%) had OCA3, and 2 (5%) had OCA4. Furthermore, 2.5% of the patients harbored only one heterozygous mutation in OCA2. The most common form of albinism observed was OCA1, followed by OCA2, OCA4, and OCA3. Prenatal diagnosis was performed on 20 fetuses, and the clinical phenotype of the fetuses aligned with the predictions of prenatal genetic diagnosis after follow-up. Conclusions: The results of gene mutation analysis in 40 families with oculocutaneous albinism indicate that OCA1 is the predominant type of albinism in the Chinese population, with all four types of OCA identified. Further research is needed to expand the understanding of pathogenic mutations associated with different types of OCA. Prenatal genetic testing, based on determining the albinism type and genotype of the proband and their parents, proves to be the most accurate and least traumatic method in eugenics. This study provides valuable insights into identifying novel therapeutic targets.

Overexpression and RNAi-mediated Knockdown of Two 3ß-hydroxy-Δ5-steroid dehydrogenase Genes in Digitalis lanata Shoot Cultures Reveal Their Role in Cardenolide Biosynthesis.

3ß-hydroxy-Δ5-steroid dehydrogenases (3ßHSDs) are supposed to be involved in 5ß-cardenolide biosynthesis. Here, a novel 3ßHSD (Dl3ßHSD2) was isolated from Digitalis lanata shoot cultures and expressed in E. coli. Recombinant Dl3ßHSD1 and Dl3ßHSD2 shared 70% amino acid identity, reduced various 3-oxopregnanes and oxidised 3-hydroxypregnanes, but only rDl3ßHSD2 converted small ketones and secondary alcohols efficiently. To explain these differences in substrate specificity, we established homology models using borneol dehydrogenase of Salvia rosmarinus (6zyz) as the template. Hydrophobicity and amino acid residues in the binding pocket may explain the difference in enzyme activities and substrate preferences. Compared to Dl3ßHSD1, Dl3ßHSD2 is weakly expressed in D. lanata shoots. High constitutive expression of Dl3ßHSDs was realised by Agrobacterium-mediated transfer of Dl3ßHSD genes fused to the CaMV-35S promotor into the genome of D. lanata wild type shoot cultures. Transformed shoots (35S:Dl3ßHSD1 and 35S:Dl3ßHSD2) accumulated less cardenolides than controls. The levels of reduced glutathione (GSH), which is known to inhibit cardenolide formation, were higher in the 35S:Dl3ßHSD1 lines than in the controls. In the 35S:Dl3ßHSD1 lines cardenolide levels were restored after adding of the substrate pregnane-3,20-dione in combination with buthionine-sulfoximine (BSO), an inhibitor of GSH formation. RNAi-mediated knockdown of the Dl3ßHSD1 yielded several shoot culture lines with strongly reduced cardenolide levels. In these lines, cardenolide biosynthesis was fully restored after addition of the downstream precursor pregnan-3ß-ol-20-one, whereas upstream precursors such as progesterone had no effect, indicating that no shunt pathway could overcome the Dl3ßHSD1 knockdown. These results can be taken as the first direct proof that Dl3ßHSD1 is indeed involved in 5ß-cardenolide biosynthesis.

Multiomics analysis of the mechanisms behind flavonoid differences between purple and green tender shoots of Camellia sinensis var. assamica.

Flavonoids are rich in tea plants (Camellia sinensis), and responsible for the flavor and healthful benefits of tea beverage. The anthocyanin levels in the purple tender shoots are higher than in the general green leaves of tea plant, which provide special materials to search metabolic mechanisms of flavonoid enrichment in plant. In this work, flavonoid differences between purple and green shoots from tea cultivars "Zijuan" (ZJ) and "Yunkang10" (YK-10) were investigated through metabolomic analysis, and mechanisms for their difference were surveyed by comparative transcriptomic and proteomic analysis. Levels of 34 flavonoids were different between ZJ and YK-10 shoots. Among them, 8 and 6 were marker metabolites in ZJ and YK-10, respectively. The differentially expressed genes (DEGs), differentially expressed proteins (DEPs), and different-level metabolites (DLMs) between ZJ and YK-10 were researched, respectively; and interactions including DEG-DLM, DEP-DLM, DEG-DEP, and DEG-DEP-DLM were analyzed; the contents of 18 characteristic flavonoids in tea leaves and expressions of 34 flavonoid metabolic genes were measured to verify the omics results. Integrated above analyses, a proposed model of flavonoids biosynthesis in tea shoots were established. The differential expression of the leucoanthocyanidin reductase (LAR), anthocyanidin synthase (ANS), anthocyanidin reductase (ANR), UDPG-flavonoid glucosyltransferase (UGT) 75L12 and 94P1 at gene level, and the ANS, ANR, and UGT78A15 at protein level, were closely associated with differences in flavonoids between ZJ and YK-10 shoot. Together, this study provides new information on the flavonoid accumulation mechanism in tea plant.

StLTO1, a lumen thiol oxidoreductase in Solanum tuberosum L., enhances the cold resistance of potato plants.

Cold stress reduces plant photosynthesis and increases the accumulation of reactive oxygen species (ROS) in plants, thereby dramatically affecting plant growth, crop productivity and quality. Here, we report that lumen thiol oxidoreductase 1 (StLTO1), a vitamin K epoxide reductase (VKOR)-like protein in the thylakoid membrane of Solanum tuberosum L., enhances the cold tolerance of potato plants. Under normal conditions, overexpression of StLTO1 in plants promoted plant growth. In addition, potato plants overexpressing StLTO1 displayed enhanced photosynthetic capacity and increased capacity for scavenging ROS compared to StLTO1 knockdown and wild-type potato plants under cold conditions. Overexpression of StLTO1 in potato plants also improved cold-regulated (COR) gene expression after cold stress. Our results suggest that StLTO1 acts as a positive regulator of cold resistance in potato plants.

Enhancement of TRP Gene Expression and UV Absorption by Bioconverted Chestnut Inner Shell Extracts Using Lactiplantibacillus plantarum.

In this work, the suppression of tyrosinase-related genes, including an improvement in UV absorption effects of bioconverted CS extracts (BCS), was investigated to improve the skin-whitening effect. Total polyphenols and total flavonoids, which are bioactive components, increased 2.6- and 5.4-times in bioconversion using Lactiplantibacillus plantarum SM4, respectively, as compared to ultrasound-assisted extracts (UCS). The effect of BCS on radical scavenging activity, UV-A absorption, and tyrosinase activity inhibition, contributing to skin-whitening, were 1.3-, 1.2-, and 1.2-times higher than those of UCS, respectively. The main component identified in high-performance liquid chromatography (HPLC) was gallic acid in both UCS and BCS, which increased by 2.9-times following bioconversion. The gene expression of tyrosinase-related proteins, including TRP-1 and TRP-2 genes, was studied to confirm the suppression of melanin synthesis by BCS in order to identify the skin-whitening mechanism, and BCS decreased both genes' expression by 1.7- and 1.6-times, demonstrating that BCS effectively suppressed melanin synthesis. These findings imply that the chestnut inner shell can be employed as a cosmetic material by simultaneously inhibiting melanogenesis and enhancing UV-A absorption through bioconversion using L. plantarum SM4.

Mutation of glucose-methanol-choline oxidoreductase leads to thermosensitive genic male sterility in rice and Arabidopsis.

Thermosensitive genic male sterility (TGMS) lines serve as the major genetic resource for two-line hybrid breeding in rice. However, their unstable sterility under occasional low temperatures in summer highly limits their application. In this study, we identified a novel rice TGMS line, ostms18, of cultivar ZH11 (Oryza sativa ssp. japonica). ostms18 sterility is more stable in summer than the TGMS line carrying the widely used locus tms5 in the ZH11 genetic background, suggesting its potential application for rice breeding. The ostms18 TGMS trait is caused by the point mutation from Gly to Ser in a glucose-methanol-choline (GMC) oxidoreductase; knockout of the oxidoreductase was previously reported to cause complete male sterility. Cellular analysis revealed the pollen wall of ostms18 to be defective, leading to aborted pollen under high temperature. Further analysis showed that the tapetal transcription factor OsMS188 directly regulates OsTMS18 for pollen wall formation. Under low temperature, the flawed pollen wall in ostms18 is sufficient to protect its microspore, allowing for development of functional pollen and restoring fertility. We identified the orthologous gene in Arabidopsis. Although mutants for the gene were fertile under normal conditions (24°C), fertility was significantly reduced under high temperature (28°C), exhibiting a TGMS trait. A cellular mechanism integrated with genetic mutations and different plant species for fertility restoration of TGMS lines is proposed.

Comparative transcriptomic analysis reveals the regulatory mechanisms of catechins synthesis in different cultivars of Camellia sinensis.

Camellia sinensis (L.) O. Kuntze is used to produce tea, a beverage consumed worldwide. Catechins are major medically active components of C. sinensis and can be used clinically to treat hyperglycaemia, hypertension, and cancer. In this study, we aimed to identify the genes involved in catechins biosynthesis. To this end, we analysed transcriptome data from two different cultivars of C. sinensis using DNBSEQ technology. In total,47,717 unigenes were obtained from two cultivars of C. sinensis, of which 9429 were predicted as new unigenes. In our analyses of the Kyoto Encyclopedia of Genes and Genomes database, 212 unigenes encoding 13 key enzymes involved in catechins biosynthesis were identified; the structures of leucoanthocyanidin reductase and anthocyanidin reductase were spatially modelled. Some of these key enzymes were verified by real-time quantitative polymerase chain reaction, and multiple genes encoding plant resistance proteins or transcription factors were identified and analysed. Furthermore, two microRNAs involved in the regulation of catechins biosynthesis were explored. Differentially expressed genes involved in the flavonoid biosynthesis pathway were identified from pairwise comparisons of genes from different cultivars of tea plants. Overall, our findings expanded the number of publicly available transcript datasets for this valuable plant species and identified candidate genes related to the biosynthesis of C. sinensis catechins, thereby establishing a foundation for further in-depth studies of catechins biosynthesis in varieties or cultivars of C. sinensis.

Iron oxide nanoparticles and selenium supplementation improve growth and photosynthesis by modulating antioxidant system and gene expression of chlorophyll synthase (CHLG) and protochlorophyllide oxidoreductase (POR) in arsenic-stressed Cucumis melo.

Current research reveals the positive role of iron oxide nanoparticles (IONPs) and selenium (Se) in extenuation of arsenic (As) induced toxicity in Cucumis melo. C. melo plants grown in As spiked soil (20 mg kg-1 As) showed reduced growth, chlorophyll (Chl) content, photosynthetic rate, stomatal conductivity and transpiration. On the other hand, the alone applications of IONPs or Se improved growth and physiochemical parameters of C. melo plants. Additionally, exogenous application IONPs and Se synergistically improved the activity of antioxidative enzymes and glyoxalase system in C. melo plants. In addition, the collective treatment of IONPs and Se reduced As uptake, enhanced rate of photosynthesis and increased gas exchange attributes of C. melo plants under As stress. Interactive effect of IONPs and Se regulated reduced glutathione (GSH), oxidized glutathione (GSSG) and ascorbate (AsA) content in C. melo plants exposed to As-contaminated Soil. IONPs and Se treatment also regulated expression of respiratory burst oxidase homologue D (RBOHD) gene, chlorophyll synthase (CHLG) and protochlorophyllide oxidoreductase (POR). Therefore, the combined treatment of IONPs and Se may enhance the growth of crop plants by alleviating As stress.

[Drought and Heat Stress-Mediated Modulation of Alternative Splicing in the Genes Involved in Biosynthesis of Metabolites Related to Tea Quality].

Alternative splicing (AS) regulates mRNAs at the post-transcriptional level to affect both their amounts and the protein function. However, little is known about the roles of AS in regulation of biosynthesis of amino acids, flavonoids, and volatile compounds in tea plants. In this study, we used Iso-seq and transcriptome deep sequencing (RNA-seq) to identify AS events, and analyzed the expression of respective mRNAs in tea plants under drought (DS), heat stress (HS), and their combination (HD). By RT-PCR, we validated the AS events in nine genes involved in the biosynthesis of amino acids and flavonoids. The genes accumulating AS transcripts under DS, HS, and HD conditions included those encoding for anthocyanidin reductase (ANR), dihydrofavonol-4-reductase-like (DFRA), and chalcone isomerase (CHI). Similarly, genes directly or indirectly involved in the biosynthesis of volatile compounds such as lipoxygenase (LOX), terpenoid/terpene synthase (TPS), and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) also had AS events. Our study revealed that AS might specifically regulate the biosynthesis of amino acids in tea plants under stressful conditions. Moreover, we suggest that the AS events within the ANR and DFRA transcripts might play an important role in the regulation of flavonoid biosynthesis under DS, HS, and HD conditions. This study improved our understanding of the genetic drivers of the changes in the content of bioactive ingredients of tea plants subjected to abiotic stresses.

Analysis of the application of a gene chip method for detecting Mycobacterium tuberculosis drug resistance in clinical specimens: a retrospective study.

Most Mycobacterium tuberculosis (Mtb) resistant to rifampicin (RIF) has mutations in the rpoB gene, while most Mtb resistant to isoniazid (INH) has mutations in the katG gene or inhA promoter. We used gene chip technology to detect mutations in these genes to determine the resistance of Mtb to RIF and INH. A total of 4148 clinical specimens with sputum smear positivity for acid-fast bacilli (AFB) were detected. Then, taking the results of the drug sensitivity test (DST) as the reference standard, the detection efficiency of sputum samples from different grades of positive smears was compared in detail. We found that the sensitivity of the gene chip method for detecting sputum samples with a grade ≥ AFB 2 + was higher than that of sputum samples with a grade ≤ AFB 1 + (P < 0.05). When the grade of the sample was ≤ AFB 1 +, the sensitivity of the gene chip method was 72.6% for RIF, 67.3% for INH, and 60.0% for MDR-TB. When the grade of the sample was ≥ AFB 2 +, the sensitivity of the gene chip method was 84.5% for RIF, 78.2% for INH, and 73.9% for MDR-TB. The results show that gene chip technology can be directly used to diagnose drug-resistant tuberculosis in clinical specimens, and the diagnostic efficiency for the detection of sputum specimens with a grade ≥ AFB 2 + is better than that of other sputum specimens.

Efficient CRISPR/Cas9-mediated genome editing in Rehmannia glutinosa.

KEY MESSAGE: Here, we cloned a phytoene desaturase (PDS) gene from Rehmannia glutinosa, and realized RgPDS1 knock out in R. glutinosa resulted in the generation of albino plants. Rehmannia glutinosa is a highly important traditional Chinese medicine (TCM) with specific pharmacology and economic value. R. glutinosa is a tetraploid plant, to date, no report has been published on gene editing of R. glutinosa. In this study, we combined the transcriptome database of R. glutinosa and the reported phytoene desaturase (PDS) gene sequences to obtain the PDS gene of R. glutinosa. Then, the PDS gene was used as a marker gene to verify the applicability and gene editing efficiency of the CRISPR/Cas9 system in R. glutinosa. The constructed CRISPR/Cas9 system was mediated by Agrobacterium to genetically transform into R. glutinosa, and successfully regenerated fully albino and chimeric albino plants. The next-generation sequencing (NGS) confirmed that the albino phenotype was indeed caused by RgPDS gene target site editing, and it was found that base deletion was more common than insertion or replacement. Our results revealed that zCas9 has a high editing efficiency on the R. glutinosa genome. This research lays a foundation for further use of gene editing technology to study the molecular functions of genes, create excellent germplasm, accelerate domestication, and improve the yield and quality of R. glutinosa.

Effects of N-acetyl-l-cysteine on heat stress-induced oxidative stress and inflammation in the hypothalamus of hens.

The purpose of this study was to discuss the effects of N-acetyl-l-cysteine (NAC) on heat stress-induced oxidative stress and inflammation in the hypothalamus of hens in different periods. A total of 120 Hy-Line variety brown laying hens (12 weeks old) were randomly assigned to 4 groups with 6 replicates. The control group (C group) (22 ± 1 °C) received a basal diet, the NAC-treated group (N group) (22 ± 1 °C) received a basal diet with 1000 mg/kg NAC, and 2 heat-stressed groups (36 ± 1 °C for 10 h per day and 22 ± 1 °C for the remaining time) were fed a basal diet (HS group) or a basal diet with 1000 mg/kg NAC (HS + N group) for 21 consecutive days. The influence of NAC on histologic changes, oxidative stress and proinflammatory cytokine production was measured and analysed in hens with heat stress-induced hypothalamic changes. NAC effectively alleviated the hypothalamic morphological changes induced by heat stress. In addition, NAC attenuated the activity of the Nf-κB pathway activated by heat stress and decreased the expression of the proinflammatory cytokines IL-6, IL-18, TNF-α, IKK, and IFN-γ. In addition, NAC treatment regulated the expression of HO-1, GSH, SOD2 and PRDX3 by regulating the activity of Nrf2 at different time points to resist oxidative stress caused by heat exposure. In summary, dietary NAC may be an effective candidate for the treatment and prevention of heat stress-induced hypothalamus injury by preventing Nf-κB activation and controlling the Nrf2 pathway.

Skin-Whitening and Anti-Wrinkle Effects of Bioactive Compounds Isolated from Peanut Shell Using Ultrasound-Assisted Extraction.

Response surface methodology was employed to optimize the ultrasound-assisted extraction (UAE) conditions for simultaneous optimization of dependent variables, including DPPH radical scavenging activity (RSA), tyrosinase activity inhibition (TAI), and collagenase activity inhibition (CAI) of peanut shell extracts. The effects of the main variables including extraction time (5.0~55.0 min, X1), extraction temperature (26.0~94.0 °C, X2), and ethanol concentration (0.0%~99.5%, X3) were optimized. Based on experimental values from each condition, quadratic regression models were derived for the prediction of optimum conditions. The coefficient of determination (R2) of the independent variable was in the range of 0.89~0.96, which demonstrates that the regression model is suitable for the prediction. In predicting optimal UAE conditions based on the superimposing method, extraction time of 31.2 min, extraction temperature of 36.6 °C, and ethanol concentration of 93.2% were identified. Under these conditions, RSA of 74.9%, TAI of 50.6%, and CAI of 86.8% were predicted, showing good agreement with the experimental values. A reverse transcription polymerase chain reaction showed that peanut shell extract decreased mRNA levels of tyrosinase-related protein-1 and matrix metalloproteinase-3 genes in B16-F0 cell. Therefore, we identified the skin-whitening and anti-wrinkle effects of peanut shell extracts at protein as well as gene expression levels, and the results show that peanut shell is an effective cosmetic material for skin-whitening and anti-wrinkle effects. Based on this study, peanut shell, which was considered a byproduct, can be used for the development of healthy foods, medicines, and cosmetics.

Cloning and characterization of steroid 5ß-reductase from the venom gland of Bufo bufo gargarizans.

Bufadienolides are the main active ingredients of Venenum Bufonis, which is a widely used traditional Chinese medicine secreted from parotoid gland and skin glands of Bufo bufo gargarizans. According to the transcriptome analysis, "cholesterol-bile acid-bufadienolidies pathway" was proposed as animal-derived bufadienolides biosynthesis pathway by us previously. In this pathway 3ß-hydroxysteroid dehydrogenase (3ßHSD) and steroid 5ß-reductase (SRD5ß) might be the key enzymes to convert the A/B ring to cis-configuration. Therefore, as the second report of our group, here we report the cloning of the full length of SRD5ß cDNA of B. bufo gargarizans (Bbg-SRD5ß) from the parotoid gland of B. bufo gargarizans for the first time, and site-directed mutagenesis was used to explored the character of Bbg-SRD5ß. Bbg-SRD5ß had an open reading frame of 981 bp and encoded 326 amino acids residues. The expression conditions of the recombinant Bbg-SRD5ß in E. coli BL21 (DE3) harbored with pCold-Bbg-SRD5ß was optimized as induction for 10 h at 15 °C with 0.1 mM IPTG. With NADPH as a cofactor, Bbg-SRD5ß can reduce the Δ4,5 double bonds of progesterone to generate dihydroprogesterone õwithout substrate inhibition effect. The catalytic rate of mutant type Bbg-SRD5ß-Y132G was 1.8 times higher than that of wild type Bbg-SRD5ß. Although Bbg-SRD5ß was almost unable to reduce the progesterone to dihydroprogesterone after mutation of V309, the affinity of enzyme with NADPH changed significantly. Bbg-SRD5ß is the key enzymes to convert the A/B ring of steroid to cis-configuration, and V309 is a key site affecting the binding affinity of enzyme with NADPH, and the mutation of Y132 can adjust the catalytic rate of Bbg-SRD5ß.

Methionine dependence in tumor cells: The potential role of cobalamin and MMACHC.

Methionine dependence of tumor cell lines, the inability to grow in tissue culture media lacking methionine but supplemented with homocysteine, has been known for decades, but an understanding of the mechanism underlying this phenomenon remains incomplete. Methionine dependence of certain glioma and melanoma cell lines has been linked to alterations in the metabolism of cobalamin (vitamin B12). In the MeWo LC1 melanoma line, complementation analysis demonstrated that the genetic defect affected the same locus mutated in the cblC inborn error of cobalamin metabolism; hypermethylation of the MMACHC promoter was subsequently demonstrated. Analysis of data in the Cancer Cell Line Encyclopedia showed increased MMACHC methylation levels in melanoma lines compared to other types of cancer. RNA sequencing data from isolated tumors, tabulated at the cBioPortal for Cancer Genomics website, showed decreased MMACHC expression compared to other tumors; and methylation data tabulated at the TGGA Wanderer website demonstrated increased MMACHC methylation. These data suggest that disruptions in cobalamin metabolism might play a more general role in methionine dependence, and potentially in the pathogenesis of melanoma cell lines and primary tumors.

Physiological and metabolic analysis of winter jujube after postharvest treatment with calcium chloride and a composite film.

BACKGROUND: Ziziphus jujuba Miller cv. Dongzao is extremely susceptible to reddening, browning, nutritional loss, and perishability after harvest. In this study, we evaluated the mechanisms of calcium chloride and chitosan/nano-silica composite film treatments on the quality, especially in reddening, by physiological and metabolomic assays. RESULTS: The treatment delayed the decline of phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), and chalcone isomerase (CHI) activities. Meanwhile, the treated groups retarded the increases in anthocyanin and quercetin contents by inhibiting the gene expressions of flavonol synthase (ZjFLS), dihydroflavonol 4-reductase (ZjDFR), and anthocyanidin synthase (ZjANS), while promoting leucoanthocyanidin reductase (ZjLAR) expression, which leads to retardation of fruit reddening. Anthocyanins were found to be responsible for post-harvest winter jujube reddening through principal component analysis. Results from the technique for order preference by similarity to an ideal solution indicated that the treated group delayed the decline of the quality of 'Dongzao' and extended its shelf life. CONCLUSION: The treatment induced the heightening of flavonoids metabolism. They enhanced the nutritional value and the ability to resist stress by delaying the decline of PAL, CHS, and CHI activities. Meanwhile, the treated groups retarded the increase in anthocyanin and quercetin contents by inhibiting the gene expressions of ZjFLS, ZjDFR, and ZjANS and promoting ZjLAR expression, which leads to retardation of fruit reddening. Anthocyanins are responsible for post-harvest winter jujube reddening. Coating treatment effectively delayed the decline of winter jujube quality. © 2020 Society of Chemical Industry.

The blood and mRNA levels of antioxidant-related factors in common carp (Cyprinus carpio) fed p-Coumaric acid.

The natural antioxidants are well known for their antioxidative activity without side effects when compared to antibiotics. Hence, the present study aimed at evaluating p-Coumaric acid as an antioxidant additive on the blood and mRNA levels of antioxidant-related factors in common carp (Cyprinus carpio). Fish fed the basal diet supplemented with p-Coumaric at 0, 0.5, 1, and 1.5 g/kg for 56 days, then the serum, intestine, and liver samples were collected. The growth performance of fish fed with CA showed significantly (P < 0.05) improved FW, WG, and SGR compared to those of the control one. However, the feed conversion ratio was significantly (P < 0.05) reduced in fish fed 1 and 1.5 g/kg diet levels. SOD was not significantly differed among the groups fed with varied p-Coumaric acid (P > 0.05). Serum GPX and TAC were enhanced considerably by p-Coumaric acid regarding the control with the highest being in fish fed 1.5 g/kg diet (P < 0.05). Serum CAT was more elevated in fish provided p-Coumaric acid at 1 or 1.5 g/kg than the control while fish fed 0.5 g/kg did not display significant changes. MDA level significantly decreased by all p-Coumaric acid groups compared to the control one, and the lowest level was observed in 1.5 g/kg (P < 0.05). The mRNA level of CAT was significantly upregulated in the liver by p-Coumaric acid at 1 or 1.5 g/kg (P < 0.05), while the intestine CAT did not influence by p-Coumaric acid (P > 0.05). The measured SOD in the liver and intestine samples revealed no changes in common carp fed p-Coumaric acid (P > 0.05). GPX was significantly upregulated in the intestine by p-Coumaric acid at 1 or 1.5 g/kg (P < 0.05), whereas the liver GPX was upregulated by p-Coumaric acid at 1.5 g/kg. The mRNA level of the GST gene in the intestine of common carp was upregulated by p-Coumaric acid at 1.5 g/kg, whereas the liver displayed upregulated GST in fish fed 1 g/kg diet. The present study approved the application of p-Coumaric acid as a natural antioxidant for friendly, sustainable aquaculture.

Thiolatocobalamins repair the activity of pathogenic variants of the human cobalamin processing enzyme CblC.

Thiolatocobalamins are a class of cobalamins comprised of naturally occurring and synthetic ligands. Glutathionylcobalamin (GSCbl) occurs naturally in mammalian cells, and also as an intermediate in the glutathione-dependent dealkylation of methylcobalamin (MeCbl) to form cob(I)alamin by pure recombinant CblC from C. elegans. Glutathione-driven deglutathionylation of GSCbl was demonstrated both in mammalian as well as in C. elegans CblC. Dethiolation is orders of magnitude faster than dealkylation of Co-C bonded cobalamins, which motivated us to investigate two synthetic thiolatocobalamins as substrates to repair the enzymatic activity of pathogenic CblC variants in humans. We report the synthesis and kinetic characterization of cysteaminylcobalamin (CyaCbl) and 2-mercaptopropionylglycinocobalamin (MpgCbl). Both CyaCbl and MpgCbl were obtained in high purity (90-95%) and yield (78-85%). UV-visible spectral properties agreed with those reported for other thiolatocobalamins with absorbance maxima observed at 372 nm and 532 nm. Both CyaCbl and MpgCbl bound to wild type human recombinant CblC inducing spectral blue-shifts characteristic of the respective base-on to base-off transitions. Addition of excess glutathione (GSH) resulted in rapid elimination of the ß-ligand to give aquacobalamin (H2OCbl) as the reaction product under aerobic conditions. Further, CyaCbl and MpgCbl underwent spontaneous dethiolation thereby repairing the loss of activity of pathogenic variants of human CblC, namely R161G and R161Q. We posit that thiolatocobalamins could be exploited therapeutically for the treatment of inborn errors of metabolism that impair processing of dietary and supplemental cobalamin forms. While these disorders are targets for newborn screening in some countries, there is currently no effective treatment available to patients.

Evaluation of the effect of narrow band-ultraviolet B on the expression of tyrosinase, TYRP-1, and TYRP-2 mRNA in vitiligo skin and their correlations with clinical improvement: A retrospective study.

Narrowband-ultraviolet B (NB-UVB) is considered one of the main therapeutic tools in vitiligo, which is able to induce repigmentation and halt depigmentation. However, little remains known about the effect of NB-UVB on TYR gene family, the main pigmentary genes, in vitiligo patients. To assess the effect of NB-UVB on expression of some genes related to the pigmentary problem of vitiligo; tyrosinase (TYR), tyrosinase related protein 1 (TYRP1) and tyrosinase related protein 2 (TYRP2), mRNA levels of those genes were quantitatively evaluated by Real-Time quantitative Polymerase Chain Reaction (RT-qPCR) in skin biopsies obtained from 30 patients with nonsegmental vitiligo and five healthy controls. Vitiligo patients were classified into two groups; group 1, involving 12 untreated vitiligo patients and group 2, including 18 vitiligo patients treated by NB-UVB. The levels of TYR, TYRP-1, and TYRP-2 mRNAs in untreated group were significantly lower than in control subjects (P < .001). In NB-UVB treated group, the three genes were significantly higher than in group 1 (P < .001), however, they were still significantly lower than in the control subjects (P < .001). A significant positive correlation was detected between TYR and TYRP-2 genes in group 2 (P = .03). This study demonstrated that mRNA level of TYR, TYRP-1, and TYRP-2, which decreased in vitiligo, was significantly increased upon treatment with NB-UVB. Accordingly, the mechanism of depigmentation in vitiligo disease and repigmentation by NB-UVB treatment may be related to the changes in the expression of these genes.