Preparation of isoquercitrin and rhamnose from readily accessible rutin by a highly specific recombinant α-L-rhamnosidase (r-Rha1).

Isoquercitrin has superior in vivo bioactivities with respect to its primary glycoside rutin. Its conventional preparation was ineffective, with large chemical consumption and many by-products. Rhamnose, a high value-added monosaccharide, is usually separated from acid hydrolytes of rutin. This study aimed to establish a novel enzymatic hydrolysis-based approach for their preparation. α-L-rhamnosidase was expressed in Pichia pastoris GS115 and applied to enzymolysis of rutin. Then, one-factor-at-a-time optimisation of hydrolysis conditions was performed. Two compounds were produced in 0.02 M HAc-NaAc buffer (pH4.50) containing α-L-rhamnosidase/rutin (1:4, w/w) at 60 °C. Consequently, 20.0 g/L rutin was completely hydrolysed in 2 hrs, and isoquercitrin was obtained after purification by HPD-100 resin. Additionally, rhamnose was enriched by decolorisation and crystallisation. MD simulation analysis suggested that rutin was catalysed on the hydrophobic surface of r-Rha1 with van-der-Waals force being main driving force. This strategy is an efficient approach for preparation of isoquercitrin and rhamnose.

Biotransformation of ginsenoside Rb1 and Rd to four rare ginsenosides and evaluation of their anti-melanogenic effects.

Improving physiological activity of primary ginsenosides through biotransformation is of great significance for food applications. In this study, gynostapenoside XVII, gynostapenoside LXXV, ginsenoside F2, and ginsenoside CK were obtained by enzymolysis of an accessible extract composed of ginsenoside Rb1 and Rd. Their effects on melanin content and tyrosinase activity were compared in vitro, and molecular docking simulation was employed to elucidate the interaction between tyrosinase and individual saponin. The results indicated that four rare ginsenosides decreased tyrosinase activity, melanin content and microphthalmia-associated transcription factor (MITF) expression level, more greatly than their primary ginsenosides, and they were more readily to bind with ASP10 and GLY68 at active site of tyrosinase to inhibit tyrosinase activity as well. These findings suggested that the rare ginsenosides obtained by enzymolysis had excellent anti-melanogenic effect, which could expand the application of ginsenosides in the field of functional foods and health supplements.

A pH-sensitive intelligent packaging film harnessing Dioscorea zingiberensis starch and anthocyanin for meat freshness monitoring.

Abundant starch was isolated from Dioscorea zingiberensis C.H. Wright, a novel and underutilized industrial crop resource. In this study, an intelligent packaging film able to indicate food freshness was developed and characterized. D. zingiberensis starch (DZS) was bleached first, and its particle size, total starch content, amylose content, and gelatinization temperature were then measured. Butterfly pea (Clitoria ternatea Linn.) flowers were selected as the source of polyphenols, which rendered the prepared film intelligent and progressively blue-violet. SEM and FT-IR analyses showed the homogeneous dispersion of butterfly pea flower extract (BPE) in the film. The BPE-loaded film showed improved flexibility and resistance to UV and oxidation while maintaining sufficient mechanical strength and physical properties. Moreover, the film underwent a distinguishable color change from red to blue-violet and finally to green-yellow with increasing pH from 2 to 13. Similar color alteration also occurred when the film was exposed to ammonia. When the film was used to monitor the freshness of chicken stored at room temperature, it exhibited an obvious color change, implying its deterioration. Therefore, the newly developed BPE-DZS film, which was produced from readily accessible natural substances, can serve as an intelligent packaging material, indicating food freshness and prolonging shelf life.

Conversion of Dioscorea zingiberensis saponins to prosapogenin A by enzymatic hydrolysis.

Prosapogenin A is a secondary saponin in Dioscorea zingiberensis, and it showed remarkable pharmacological effects. Due to very low content and lack of well-developed biotransformation, its preparation was not efficient and clean. This study aims to establish an eco-friendly strategy for preparation of Prosapogenin A from plant material. Physical separation was employed to recycle starch and cellulose, and then D101 resin and polyamide packed-bed column was incorporated for purification of total steroidal saponins (TSS). After these pretreatments, purity of TSS was largely increased to 83.2% with recovery at 87.6%, which was subjected to enzymatic hydrolysis. Optimized reaction system was constructed in 0.20 M HAc-NaAc buffer (pH4.2) containing cellulase/TSS (3:1, w/w), and the hydrolysis was performed at 53 °C for 6 h. Consequently, TSS was almost completely hydrolyzed to Prosapogenin A, while the highest yield reached 5.62%. The newly proposed approach is promising for efficient preparation of Prosapogenin A in industrial applications.

Comparative plastomes of Carya species provide new insights into the plastomes evolution and maternal phylogeny of the genus.

Carya, in the Juglandiodeae subfamily, is to a typical temperate-subtropical forest-tree genus for studying the phylogenetic evolution and intercontinental disjunction between eastern Asia (EA) and North America (NA). Species of the genus have high economic values worldwide for their high-quality wood and the rich healthy factors of their nuts. Although previous efforts based on multiple molecular markers or genome-wide SNPs supported the monophyly of Carya and its two EA and NA major subclades, the maternal phylogeny of Carya still need to be comprehensively evaluated. The variation of Carya plastome has never been thoroughly characterized. Here, we novelly present 19 newly generated plastomes of congeneric Carya species, including the recently rediscovered critically endangered C. poilanei. The overall assessment of plastomes revealed highly conservative in the general structures. Our results indicated that remarkable differences in several plastome features are highly consistent with the EA-NA disjunction and showed the relatively diverse matrilineal sources among EA Carya compared to NA Carya. The maternal phylogenies were conducted with different plastome regions and full-length plastome datasets from 30 plastomes, representing 26 species in six genera of Juglandoideae and Myrica rubra (as root). Six out of seven phylogenetic topologies strongly supported the previously reported relationships among genera of Juglandoideae and the two subclades of EA and NA Carya, but displayed significant incongruencies between species within the EA and NA subclades. The phylogenetic tree generated from full-length plastomes demonstrated the optimal topology and revealed significant geographical maternal relationships among Carya species, especially for EA Carya within overlapping distribution areas. The full-length plastome-based phylogenetic topology also strongly supported the taxonomic status of five controversial species as separate species of Carya. Historical and recent introgressive hybridization and plastid captures might contribute to plastome geographic patterns and inconsistencies between topologies built from different datasets, while incomplete lineage sorting could account for the discordance between maternal topology and the previous nuclear genome data-based phylogeny. Our findings highlight full-length plastomes as an ideal tool for exploring maternal relationships among the subclades of Carya, and potentially in other outcrossing perennial woody plants, for resolving plastome phylogenetic relationships.

Preparation and characterization of oregano essential oil-loaded Dioscorea zingiberensis starch film with antioxidant and antibacterial activity and its application in chicken preservation.

In this study, abundant starch was separated from the industrial crop Dioscorea zingiberensis C.H. Wright (DZW), and a novel bioactive packaging film loaded with oregano essential oil (OEO) was prepared and characterized. NaClO solution worked as a bleacher to prepare uniform starch powder from DZW tubers. OEO was selected from among three essential oils of Labiatae family plants for its strongest antibacterial activity. After the addition of OEO into the starch-based film, the UV-vis shielding property and antioxidant activity were enhanced. Meanwhile, the films still have a considerable performance in transparency, mechanical strength and water vapor permeability after incorporated with OEO. Furthermore, the 3% OEO-loaded starch film exhibited the strongest antibacterial activity against Bacillus subtilis, Escherichia coli and Staphylococcus aureus. It effectively lowered the total viable count of fresh chicken under 4 °C preservation conditions. These results revealed that the OEO-loaded DZW starch film can exert a positive effect on maintaining the quality and extending the shelf life of fresh meat. Therefore, readily accessible DZW tubers and oregano are very promising resources for application in degradable bioactive packaging film.

Effect of vacuum impregnation assisted probiotics fermentation suspension on shelf life quality of freshly cut lotus root.

Probiotic fermentation suspension was used to extend the shelf life of freshly cut lotus root for the first time, which played a dual role of biological protection and quality maintenance. Fermentation suspension contained lactic acid bacteria (8-9 log CFU/mL) was prepared from juice of lotus root and used to immerse samples under atmospheric pressure and vacuum. Probiotic fermentation suspension inhibited microorganism and the activity of polyphenol oxidase (PPO), peroxidase (POD) and phenylalanine ammonia lyase (PAL), which slowed down the physiological reaction and was beneficial to maintain the color and hardness of tissues. Lactic acid bacteria antagonized other microorganisms, and metabolic acid production played a continuous role in preservation during storage. The vacuum was helpful for the fermentation suspension to be fully impregnated into samples. The probiotic fermentation suspension had a significant inhibitory effect on E.coli O157:H7, and extended lotus root shelf life from 3 to 9 days.

Analysis of lipidomics profile of Carya cathayensis nuts and lipid dynamic changes during embryonic development.

Hickory (Carya cathayensis) nuts contain higher amount of lipids, and possess high nutritional value and substantial health benefits. However, their lipid composition and dynamic changes during embryogenesis have not been thoroughly investigated. Therefore, lipidomics profile and lipid dynamic changes during embryonic development were analyzed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Totally, 544 kinds of lipids were identified in mature hickory nuts with higher proportions of glycerolipids (59.94%) and glycerophospholipids (38.66%). Notably, diacylglycerols showed gradual uptrends, which corresponded with total glycerolipid and glycerophospholipid at middle and late stage of embryogenesis, suggesting the pivotal role of diacylglycerols in the accumulation of glycerolipids and glycerophospholipids. Moreover, triacylglycerols, diacylglycerols, phosphatidylethanolamines and phosphatidylcholines had high relative content with abundance of unsaturated fatty acids, specifically oleic acid, linoleic acid and linolenic acid, localized mainly at sn-2 lipid position. Together, our study provides innovative perspectives for studying the nutritional benefits of hickory nut lipids.

Integrated transcriptome and proteome analysis of developing embryo reveals the mechanisms underlying the high levels of oil accumulation in Carya cathayensis Sarg.

Hickory (Carya cathayensis Sarg.) is an extraordinary nut-bearing deciduous arbor with high content of oil in its embryo. However, the molecular mechanism underlying high oil accumulation is mostly unknown. Here, we reported that the lipid droplets and oil accumulation gradually increased with the embryo development and the oil content was up to ~76% at maturity. Furthermore, transcriptome and proteome analysis of developing hickory embryo identified 32,907 genes and 9857 proteins. Time-series analysis of gene expressions showed that these genes were divided into 12 clusters and lipid metabolism-related genes were enriched in Cluster 3, with the highest expression levels at 95 days after pollination (S2). Differentially expressed genes and proteins indicated high correlation, and both were enriched in the lipid metabolism. Notably, the genes involved in biosynthesis, transport of fatty acid/lipid and lipid droplets formation had high expression levels at S2, while the expression levels of other genes required for suberin/wax/cutin biosynthesis and lipid degradation were very low at all the sampling time points, ultimately promoting the accumulation of oil. Quantitative reverse-transcription PCR analysis also verified the results of RNA-seq. The co-regulatory networks of lipid metabolism were further constructed and WRINKLED1 (WRI1) was a core transcriptional factor located in the nucleus. Of note, CcWRI1A/B could directly activate the expression of some genes (CcBCCP2A, CcBCCP2B, CcFATA and CcFAD3) required for fatty acid synthesis. These results provided in-depth evidence for revealing the molecular mechanism of high oil accumulation in hickory embryo.

A species-specific triplex PCR assay for authentication of Galli Gigerii Endothelium Corneum.

A triplex PCR assay was developed to identify animal species and adulteration of a natural medicine Galli Gigerii Endothelium Corneum (GGEC). Three species-specific primer sets were designed according to the difference in mitochondrial genome of Gallus gallus domesticus, Anas platyrhynchos and Anser anse. The PCR conditions were optimized and the assay was well validated for high specificity and sensitivity (1 mg/µL). Especially, when artificial adulterants made from the mixture of three species were analyzed, the assay has still exhibited strong capability of differentiation. By using this developed method, two batches out of fourteen commercial GGEC products were identified to be adulterated by Anser anse. The newly proposed assay showed sufficient merits as a regular tool for the identification of counterfeits or adulterants of GGEC product for their pulverized and processed form, and even Chinese patent medicines composed of these species.

Efficient and clean preparation of rare prosaikogenin D by enzymatic hydrolysis of saikosaponin B2 and response surface methodology optimization.

Prosaikogenin D, a rare secondary saponin in Radix Bupleuri, has much higher in vivo bioactivities than its original glycoside saikosaponin B2. Its preparation methods, such as conventional acid hydrolysis and column chromatograph, are unfriendly to environment with serious pollution and undesired products. The aim of this study was to establish an efficient and clean approach for convenient preparation of this rare steroid saponin based on the enzymatic hydrolysis. Cellulase was selected from four commercial enzymes due to its higher hydrolysis performance. Then the hydrolysis conditions were optimized by response surface methodology after preliminary investigation on affecting factors by single-factor experiments. The reaction system was constructed by 100 µg/mL of saikosaponin B2 and 8.00 mg/mL of cellulase, which was incubated in HAc-NaAc buffer (pH 4.7) at 60 °C for 33 h. Consequently, a high conversion ratio of the substrate has been achieved at 95.04 %. The newly developed strategy is an efficient and clean approach for the preparation of prosaikogenin D and it is a promising technology in industrial application.

Construction of Fe3O4@α-glucosidase magnetic nanoparticles for ligand fishing of α-glucosidase inhibitors from a natural tonic Epimedii Folium.

Inhibition of α-glucosidase activity is an effective way for treatment of type 2 diabetes mellitus. Epimedii Folium is an important source of α-glucosidase inhibitors (AGIs), however bioactive compounds and pharmacological mechanisms remained unclear. In this study, a novel strategy was established, which harnessed α-glucosidase functionalized magnetic beads to fish out potential AGIs, followed by UPLC-MS/MS analysis for their identification. Furthermore, molecular docking was employed to predict binding patterns between the AGIs and the enzyme, and IC50 values was estimated as well. After response surface methodology optimization, the highest activity of Fe3O4@α-glucosidase has been achieved when 1.17 mg/mL of α-glucosidase was immobilized in phosphate buffer (pH 6.81) for 4.22 h. Moreover, eight flavonoids were fished out from the extract of Epimedii Folium, and then identified to be epimedin A, epimedin B, epimedin C, icariin, sagittatoside A, sagittatoside B, 2"-O-rhamnosyl icariside II and baohuoside I. All of them were further confirmed to be AGIs through in vitro inhibitory assay and molecular docking. Among those, baohuoside I and sagittatoside B possessed stronger inhibitory activity than acarbose. The approach has a significant prospect in conveniently screening bioactive compounds that target various receptors, which provided an efficient platform for new drug development from natural products.

A Narrow Endemic or a Species Showing Disjunct Distribution? Studies on Meehania montis-koyae Ohwi (Lamiaceae).

Meehania montis-koyae Ohwi (Lamiaceae), which has been considered a narrow endemic and endangered species in Japan, was found in eastern China in 2011. China and Japan belong to the same floristic region and share many plant species, but it is very rare that Japanese narrow endemic species are newly found outside of the country. We examined herbarium specimens of both countries, and conducted analyses of molecular phylogenetics, population genetics, and divergence time estimation using two nuclear (ITS and ETS) gene regions and MIG-seq data. Chinese plants tend to become larger than Japanese, and they are different in leaf shape and floral features. Molecular phylogenetic analysis shows Chinese and Japanese M. montis-koyae are the closest relatives to each other. Population genetic analysis indicates no current gene flow between the Chinese and Japanese populations, and divergence time analysis shows they were separated during the late Miocene. We reach the conclusion that Chinese and Japanese M. montis-koyae have already become distinct biological entities, and a new taxon name Meehania zheminensis A. Takano, Pan Li, G.-H.Xia is proposed for the Chinese plants. A key to Asian Meehania species is provided.

Paired associated magnetic stimulation promotes neural repair in the rat middle cerebral artery occlusion model of stroke.

Paired associative stimulation has been used in stroke patients as an innovative recovery treatment. However, the mechanisms underlying the therapeutic effectiveness of paired associative stimulation on neurological function remain unclear. In this study, rats were randomly divided into middle cerebral occlusion model (MCAO) and paired associated magnetic stimulation (PAMS) groups. The MCAO rat model was produced by middle cerebral artery embolization. The PAMS group received PAMS on days 3 to 20 post MCAO. The MCAO group received sham stimulation, three times every week. Within 18 days after ischemia, rats were subjected to behavioral experiments-the foot-fault test, the balance beam walking test, and the ladder walking test. Balance ability was improved on days 15 and 17, and the foot-fault rate was less in their affected limb on day 15 in the PAMS group compared with the MCAO group. Western blot assay showed that the expression levels of brain derived neurotrophic factor, glutamate receptor 2/3, postsynaptic density protein 95 and synapsin-1 were significantly increased in the PAMS group compared with the MCAO group in the ipsilateral sensorimotor cortex on day 21. Resting-state functional magnetic resonance imaging revealed that regional brain activities in the sensorimotor cortex were increased in the ipsilateral hemisphere, but decreased in the contralateral hemisphere on day 20. By finite element simulation, the electric field distribution showed a higher intensity, of approximately 0.4 A/m2, in the ischemic cortex compared with the contralateral cortex in the template. Together, our findings show that PAMS upregulates neuroplasticity-related proteins, increases regional brain activity, and promotes functional recovery in the affected sensorimotor cortex in the rat MCAO model. The experiments were approved by the Institutional Animal Care and Use Committee of Fudan University, China (approval No. 201802173S) on March 3, 2018.

COVID-19 Epidemic Peer Support and Crisis Intervention Via Social Media.

This article describes a peer support project developed and carried out by a group of experienced mental health professionals, organized to offer peer psychological support from overseas to healthcare professionals on the frontline of the COVID-19 outbreak in Wuhan, China. This pandemic extremely challenged the existing health care systems and caused severe mental distress to frontline healthcare workers. The authors describe the infrastructure of the team and a novel model of peer support and crisis intervention that utilized a popular social media application on smartphone. Such a model for intervention that can be used elsewhere in the face of current global pandemic, or future disaster response.

[Clinical Analysis of Autologous Peripheral Blood Hematopoietic Stem Cell Mobilization Regimen in Patients with Malignant Hematological Diseases].

OBJECTIVE: To analyze the factors influencing the mobilization and collection of peripheral blood hematopoietic stem cells (HSC) in the patients with malignant hematological diseases. METHODS: The peripheral blood stem cells in 50 patients with hematological malignancies had been mobilized in the Hematology Department Zhongda Hospital. The factors, such as age, sex, mobilization regimen, disease status, cell separator were analyzed, and the effect of above-mentioned factors on stem cell mobilization was evaluated. And the correlation between WBC, Hb, Plt counts and CD34+ cell collection was anzlyzed. RESULTS: There was significant effect at mobilization regimen on the success rate and collection counts of CD34+ cells, while sex, age, interval between diagnosis and mobilization, previous chemotherapy regimen and bone marrow involvement showed no significant effect on stem cell collection. There was positively correlation of the WBC count, hemoglobin level of peripheral blood before apheresis with the collection of CD34+ cells . The WBC and MNC counts in peripheral blood before apheresis closely related with the success rate of mobilization. CONCLUSION: Chemotherapy plus combined with G-CSF mobilization regimens is superior to mobilization regimen of cytokine alone for collection of HSC. The WBC and MNC count in peripheral blood before apheresis can predict the optimal time for the PBSC collection and increase the success rate of mobilization and collection.

Identification of signature proteins of processed Bombyx batryticatus by comparative proteomic analysis.

Bombyx batryticatus is the dried dead larva of silkworms after infection by a fatal fungi Beauveria bassiana. It's a challenging task to monitor intrinsic biological macromolecules in this natural medicine. In this study, a novel strategy based on comparative proteomic analysis was developed to disclose inherent differences between processed and highly processed Bombyx batryticatus. First, protein and amino acid contents were determined, and the changes in proteins after highly processing the medicine were profiled through electrophoresis-based technologies. Then, the peptides that originated from the differentiated proteins were eluted under reversed-phase NanoLC and sequenced by orbitrap MSD. It was found that both contents had been significantly decreased after processed Bombyx batryticatus was stir-baked with wheat bran. Additionally, protein bands and spots visualized in SDS-PAGE and 2-DE profiles of the processed product were much brighter than those of the highly processed product, allowing them to be distinguished by electrophoresis profiling readily. Moreover, thirty-two signature proteins were identified as potential biomarkers by NanoLC-orbitrap MSD after in-gel tryptic digestion of specific bands and spots. This work provides a solid scientific basis for quality assessment and control of commercial products of this natural medicine, and the developed novel strategy could ensure its safety and efficacy.

Right Temporoparietal Junction Involvement in Autonomic Responses to the Suffering of Others: A Preliminary Transcranial Magnetic Stimulation Study.

Functional neuroimaging studies have emphasized distinct networks for social cognition and affective aspects of empathy. However, studies have not considered whether substrates of social cognition, such as the right temporoparietal junction (TPJ), play a role in affective responses to complex empathy-related stimuli. Here, we used repetitive transcranial magnetic stimulation (TMS) to test whether the right TPJ contributes to psychophysiological responses to another person's emotional suffering. We used a theory of mind functional localizer and image-guided TMS to target the sub-region of the right TPJ implicated in social cognition, and measured autonomic and subjective responses to an empathy induction video. We found evidence that TMS applied at 1 Hz over the right TPJ increased withdrawal of parasympathetic nervous system activity during the empathy induction (n = 32), but did not affect sympathetic nervous system activity (n = 27). Participants who received TMS over the right TPJ also reported feeling more irritation and annoyance, and were less likely to report feeling compassion over and above empathic sadness, than participants who received TMS over the vertex (N = 34). This study provides preliminary evidence for the role of right TPJ functioning in empathy-related psychophysiological and affective responding, potentially blurring the distinction between neural regions specific to social cognition vs. affective aspects of empathy.

Eco-efficient biphasic enzymatic hydrolysis for the green production of rare baohuoside I.

Baohuoside I, a rare secondary glycoside from Epimedii Folium, has anti-osteoporosis and other pharmacological effects. In addition, it exhibits improved bioactivities compared with its original glycoside icariin. Conventional methods for the production of baohuoside I, such as acid hydrolysis, exhibit very low efficiency with serious pollution and substantial byproducts. The aim of this study was to develop an eco-efficient biphasic enzymatic hydrolysis system for the green production of rare baohuoside I. ß-glucanase was selected to hydrolyze icariin and showed better performance than ß-glucosidase. The biphasic system was constructed using HAc-NaAc buffer (pH 4.5) containing ß-glucanase/icariin (Cicariin = 100 mg/mL; 1:1, w/w) and propyl acetate (1:2, v/v), and the hydrolysis was performed at 60℃ for 6 h. The hydrolysis capacity of icariin in the system was largely increased from 0.5 mg/mL to 100 mg/mL. The conversion ratio of icariin was greater than 99% in a 100-fold scale-up pilot test, demonstrating its strong potential for industrial applications. Furthermore, a process flow and production plant were designed to produce 1 ton of baohuoside I annually, and the profit would be approximately 4.5-fold its total cost. Biphasic enzymatic hydrolysis is an eco-efficient technology to produce baohuoside I and represents a promising method in the pharmaceutical industry.

Physiologic Electrical Fields Direct Retinal Ganglion Cell Axon Growth In Vitro.

Purpose: The purpose of this study was to characterize the ability of applied electrical fields (EFs) to direct retinal ganglion cell (RGC) axon growth as well as to assess whether Rho GTPases play a role in translating electrical cues to directional cues. Methods: Full-thickness, early postnatal mouse retina was cultured in electrotaxis chambers and exposed to EFs of varying strengths (50-200 mV/mm). The direction of RGC axon growth was quantified from time-lapsed videos. The rate of axon growth and responsiveness to changes in EF polarity were also assessed. The effect of toxin B, a broad-spectrum inhibitor of Rho GTPase signaling, and Z62954982, a selective inhibitor of Rac1, on EF-directed growth was determined. Results: In the absence of an EF, RGC axons demonstrated indiscriminate directional growth from the explant edge. Retinal cultures exposed to an EF of 100 and 200 mV/mm showed markedly asymmetric growth, with 74.2% and 81.2% of axons oriented toward the cathode, respectively (P < 0.001). RGC axons responded to acute changes in EF polarity by redirecting their growth toward the "new" cathode. This galvanotropic effect was partially neutralized by toxin B and Rac1 inhibitor Z62954982. Conclusions: RGC axons exhibit cathode-directed growth in the presence of an EF. This effect is mediated in part by the Rho GTPase signaling cascade.