[Comparison on biological activities of Lycium barbarum polysaccharides extracted with five methods].

Studying the physicochemical properties and biological activities of Lycium barbarum polysaccharides(LBPs) is of great significance. The previous study had extracted LBPs(LBP-1, LBP-2, LBP-3, LBP-4, and LBP-5) by five different methods(cold water extraction, boiling water reflux extraction of the residue after cold water extraction, ultrasonic extraction with 50% ethanol, ultrasonic extraction with 25% ethanol of the residue after 50% ethanol extraction, and hot water extraction). In this study, the structures of the obtained five LBPs were characterized by UV spectroscopy, thermogravimetric analysis, and scanning electron microscopy. Furthermore, the antioxidant, blood lipid-lowering, nitrosation-inhibting, acetylcholinesterase-inhibiting, and tyrosinase-inhibiting activities of the five LBPs were measured in vitro. The results showed that high-temperature extraction destroyed the polysaccharide structure, while ultrasound-assisted extraction ensured the structural integrity. The thermal stability and degradation behaviors differed among the five LBPs. However, the UV spectroscopic results of the five LBPs did not show significant differences, and all of the five LBPs showed the characteristic absorption peaks of proteins. LBP-3 and LBP-4 exhibited strong antioxidant activity, while LBP-3 had the strongest blood lipid-lowering activity. In addition, LBP-3 outperformed other LBPs in inhibiting nitrosation and acetylcholineste-rase, and LBP-2 showed the strongest inhibitory effect on tyrosinase. This study explored the effects of different extraction methods on the physicochemical properties and biological activities of LBPs, with a view to providing a basis for the selection of suitable extraction methods to obtain LBPs with ideal biological activities.

Mucosal immune responses and protective efficacy elicited by oral administration AMP-ZnONPs-adjuvanted inactivated H9N2 virus in chickens.

The avian influenza virus is infected through the mucosal route, thus mucosal barrier defense is very important. While the inactivated H9N2 vaccine cannot achieve sufficient mucosal immunity, adjuvants are needed to induce mucosal and systemic immunity to prevent poultry from H9N2 influenza virus infection. Our previous study found that polysaccharide from Atractylodes macrocephala Koidz binding with zinc oxide nanoparticles (AMP-ZnONPs) had immune-enhancing effects in vitro. This study aimed to evaluate the mucosal immune responses of oral whole-inactivated H9N2 virus (WIV)+AMP-ZnONPs and its impact on the animal challenge protection, and the corresponding changes of pulmonary metabolomics after the second immunization. The results showed that compared to the WIV, the combined treatment of WIV and AMP-ZnONPs significantly enhanced the HI titer, IgG and specific sIgA levels, the number of goblet cells and intestinal epithelial lymphocytes (iIELs) as well as the expression of J-chain, polymeric immunoglobulin receptor (pIgR), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α) and transforming growth factor-ß (TGF-ß). In viral attack experiments, WIV combing with AMP-ZnONPs effectively reduced lung damage and viral titers in throat swabs. Interestingly, significant changes of both the IgA intestinal immune network and PPAR pathway could also be found in the WIV+AMP-ZnONPs group compared to the non-infected group. Taken together, these findings suggest that AMP-ZnONPs can serve as a potential mucosal vaccine adjuvant, thereby avoiding adverse stress and corresponding costs caused by vaccine injection.

Ultrasound-assisted lumbar puncture with a horizontal and perpendicular paramedian approach based on positioning in patients with abnormal spinal anatomy: a case report and technical description.

BACKGROUND: The use of ultrasound has been reported to be beneficial in challenging neuraxial procedures. The angled probe is responsible for the main limitations of previous ultrasound-assisted techniques. We developed a novel technique for challenging lumbar puncture, aiming to locate the needle entry point which allowed for a horizontal and perpendicular needle trajectory and thereby addressed the drawbacks of earlier ultrasound-assisted techniques. CASE PRESENTATION: Patient 1 was an adult patient with severe scoliosis who underwent a series of intrathecal injections of nusinersen. The preprocedural ultrasound scan revealed a cephalad probe's angulation (relative to the edge of the bed) in the paramedian sagittal oblique view, and then the probe was rotated 90° into a transverse plane and we noted that a rocking maneuver was required to obtain normalized views. Then the shoulders were moved forward to eliminate the need for cephalad angulation of the probe. The degree of rocking was translated to a lateral offset from the midline of the spine through an imaginary lumbar puncture's triangle model, and a needle entry point was marked. The spinal needle was advanced through this marking-point without craniocaudal and lateromedial angulation, and first-pass success was achieved in all eight lumbar punctures. Patient 2 was an elderly patient with ankylosing spondylitis who underwent spinal anesthesia for transurethral resection of the prostate. The patient was positioned anteriorly obliquely to create a vertebral rotation that eliminated medial angulation in the paramedian approach. The procedure succeeded on the first pass. CONCLUSIONS: This ultrasound-assisted paramedian approach with a horizontal and perpendicular needle trajectory may be a promising technique that can help circumvent challenging anatomy. Larger case series and prospective studies are warranted to define its superiority to alternative approaches of lumbar puncture for patients with difficulties.

Correlation between Colour Traits and Intrinsic Quality of Dalbergiae Odoriferae Lignum.

Dalbergia odorifera T. Chen is traditionally referred to as "Dalbergiae Odoriferae Lignum" in traditional Chinese medicine. Its quality is typically assessed subjectively based on colour and texture observations and lacks a universal grading system. Our objective was to establish a relationship between heartwood colour and the content of key constituents, including total flavonoids, six specific flavonoids, alcohol-soluble extracts, and volatile oils, to assess their impact on heartwood quality. Substantial correlations were observed between the colour depth (L*), red-green direction (a*), and yellow-blue direction (b*), as well as the content of the extract, volatile oil, total flavonoids, naringenin, formononetin, pinocembrin, and isoliquiritigenin. Specifically, a* was correlated with the extract, total flavonoids, and isoliquiritigenin, whereas b* was correlated with the extract, volatile oil, total flavonoids, naringenin, formononetin, pinocembrin, and isoliquiritigenin. The results suggested that L*, b*, and chemical composition indices, such as extract, volatile oil, total flavonoids, and naringenin, could serve as primary criteria for classifying the quality of medicinal materials. This is consistent with market classification based on colour and texture, which facilitates material identification and guides the cultivation, harvesting, and processing of D. odorifera. This study provides a scientific foundation for its future development and use.

Exploring the pathogenesis of depression and potential antidepressants through the integration of reverse network pharmacology, molecular docking, and molecular dynamics.

Depression is characterized by a significant and persistent decline in mood and is currently a major threat to physical and mental health. Traditional Chinese medicine can effectively treat depression with few adverse effects. Therefore, this study aimed to examine the use of reverse network pharmacology and computer simulations to identify effective ingredients and herbs for treating depression. Differentially expressed genes associated with depression were obtained from the Gene Expression Omnibus database, after which enrichment analyses were performed. A protein-protein interaction network was constructed using the STRING database to screen core targets. The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform database was used to screen ingredients related to these core targets, and the core ingredients were screened by constructing the "Targets-Ingredients-Herbs" network. Drug evaluation analysis was performed using the SwissADME and ADMETlab platforms, according to Lipinski Rule of 5. The binding between the targets and ingredients was simulated using molecular docking software. The binding stability was determined using molecular dynamics analysis. The "Ingredients-Herbs" network was constructed, and we annotated it for its characteristics and meridians. Finally, the selected herbs were classified to determine the formulation for treating depression in traditional Chinese medicine. The pathogenesis of depression was associated with changes in SPP1, Plasminogen activator inhibitor 1, CCNB1 protein, CCL3, and other genes. Computer simulations have verified the use of quercetin, luteolin, apigenin, and other ingredients as drugs for treating depression. Most of the top 10 herbs containing these ingredients were attributed to the liver meridian, and their taste was symplectic. Perilla Frutescen, Cyperi Rhizoma, and Linderae Radix, the main components of "Tianxiang Zhengqi Powder," can treat depression owing to Qi stagnation. Epimedium and Citicola, the main traditional Chinese herbs in "Wenshen Yiqi Decoction," have a positive effect on depression of the Yang asthenia type. Fructus Ligustri Lucidi and Ecliptae Herba are from the classic prescription "Erzhi Pills" and can treat depression of the Yin deficiency type. This study identified the key targets and effective medicinal herbs for treating depression. It provides herbal blend references for treating different types of depression according to the theory of traditional Chinese medicine.

Characterization of chitosan-gelatin cryogel templates developed by chemical crosslinking and oxidation resistance of camellia oil cryogel-templated oleogels.

In this study, chitosan-gelatin conjugates were prepared by chemical crosslinking of tannic acid. The cryogel templates were developed through freeze-drying and immersed in camellia oil to construct cryogel-templated oleogels. Chemical crosslinking resulted in apparent colour changes and improved emulsion-related/rheological properties on conjugates. The cryogel templates with different formulas exhibited different microstructures with high porosities (over 96 %), and crosslinked samples might have higher hydrogen bonding strength. Tannic acid crosslinking also led to enhanced thermal stabilities and mechanical properties. Cryogel templates could reach a considerable oil absorption capacity of up to 29.26 g/g and prevent oil from leaking effectively. The obtained oleogels with high tannic acid content possessed outstanding antioxidant abilities. After 8 days of rapid oxidation at 40 °C, Oleogels with a high degree of crosslinking owned the lowest POV and TBARS values (39.74 nmol/kg, and 24.40 µg/g, respectively). This study indicates that the involvement of chemical crosslinking would favor the preparation and the application potential of cryogel-templated oleogels, and the tannic acid in the composite biopolymer systems could act as both the crosslinking agent and the antioxidant.

Recent progress and research trend of anti-cataract pharmacology therapy: A bibliometric analysis and literature review.

Cataract is the leading cause of blindness worldwide. Cataract phacoemulsification combined with intraocular lens implantation causes great burden to global healthcare, especially for low- and middle-income countries. Such burden would be significantly relieved if cataracts can effectively be treated or delayed by non-surgical means. Excitingly, novel drugs have been developed to treat cataracts in recent decades. For example, oxysterols are found to be able to innovatively reverse lens clouding, novel nanotechnology-loaded drugs improve anti-cataract pharmacological effect, and traditional Chinese medicine demonstrates promising therapeutic effects against cataracts. In the present review, we performed bibliometric analysis to provide an overview perspective regarding the research status, hot topics, and academic trends in the field of anti-cataract pharmacology therapy. We further reviewed the curative effects and molecular mechanisms of anti-cataract drugs such as lanosterol, metformin, resveratrol and curcumin, and prospected the possibility of their clinical application in future.

The complete chloroplast of Chrysanthemum × morifolium 'Fubaiju'.

Chrysanthemum × morifolium 'Fubaiju,' which is native to Macheng, Hubei, China, has a long cultivation history almost dating back to the early 10th century Song dynasty, and is used as Chrysanthemum tea drink and Chinese traditional medicine. In this study, the complete chloroplast genome sequence of 'Fubaiju' was 151,109 bp, included a large single copy LSC (82,931 bp), a small single copy SSC (18,350 bp), and a pair of inverted repeat regions (24,941 bp). It contained 132 genes with 87 CDS, 8r RNA, and 37 tRNA. The phylogenetic analysis showed that the C. × morifolium 'Fubaiju' was clustered together with C. × morifolium 'Baekma'.

Targeted mutagenesis of CYP76AK2 and CYP76AK3 in Salvia miltiorrhiza reveals their roles in tanshinones biosynthetic pathway.

Salvia miltiorrhiza Bunge, belonging to Lamiaceae family, is one of the most important Chinese medicinal herbs. The dried roots, also called Danshen in Chinese, are usually used in the formula of Chinese traditional medicine due to the bioactive constituents known as phenolic acids and tanshinones, which are a group of abietane nor-diterpenoid quinone natural products. Cytochrome P450 enzymes (CYPs) usually play crucial roles in terpenoids synthesis, especially in hydroxylation processes. Up to now, several important P450 enzymes, such as CYP76AH1, CYP76AH3, CYP76AK1, CYP71D373, and CYP71D375, have been functionally characterized in the tanshinones biosynthetic pathway. Nevertheless, the tanshinones biosynthesis is a so complex network that more P450 enzymes should be identified and characterized. Here, we report two novel P450 enzymes CYP76AK2 and CYP76AK3 that are involved in tanshinones biosynthetic pathway. These two P450 enzymes were highly homologous to previously reported CYP76AK1 and showed the same expression profile as CYP76AK1. Also, CYP76AK2 and CYP76AK3 could be stimulated by MeJA and SA, resulting in increased expression. We used a triple-target CRISPR/Cas9 system to generate targeted mutagenesis of CYP76AK2 and CYP76AK3 in S. miltiorrhiza. The content of five major tanshinones was significantly reduced in both cyp76ak2 and cyp76ak3 mutants, indicating that the two enzymes might be involved in the biosynthesis of tanshinones. This study would provide a foundation for the catalytic function identification of CYP76AK2 and CYP76AK3, and further enrich the understanding of the network of tanshinones secondary metabolism synthesis as well.

Triptolide Inhibits the Proliferation of HaCaT Cells Induced by IL22 via Upregulating miR-181b-5p.

BACKGROUND: Evidence has been shown that triptolide was effective in the treatment of psoriasis; however, the mechanisms remain poorly understood. Thus, this study aimed to investigate the role of triptolide on the proliferation and differentiation of HaCaT cells which are treated with IL22 to mimic abnormal proliferation/differentiation in keratinocyte of psoriasis. MATERIALS AND METHODS: HaCaT cells were transfected with miR-181b-5p antagomir for 24 h, and then exposed to 10 µM Triptolide for 24 h, following by 100 ng/mL of IL22 for 24 h. In addition, the proliferation and cell cycle distribution in HaCaT cells were assessed by immunofluorescence or flow cytometry assays, respectively. RESULTS: Triptolide obviously upregulated the level of miR-181b-5p in HaCaT cells. In addition, triptolide significantly inhibited IL22-induced proliferation of HaCaT cells via inducing cell cycle arrest. Moreover, IL22 markedly inhibited the differentiation of HaCaT cells, and this phenomenon was reversed by triptolide treatment. In contrast, the effects of triptolide on the proliferation and differentiation in IL22-stimulated HaCaT cells were notably reversed by miR-181b-5p antagomir. Moreover, dual-luciferase assay showed that E2F5 was the direct target of miR-181b-5p in HaCaT cells. Meanwhile, upregulation of miR-181b-5p obviously decreased the level of E2F5 in HaCaT cells. CONCLUSION: In this study, we found that triptolide could inhibit the proliferation and promote the differentiation in IL22-stimulated keratinocytes via upregulating miR-181b-5p. These data indicated that triptolide may be a potential agent for the treatment of psoriasis.

Efficacy and safety of Anluohuaxian in the treatment of patients with severe Coronavirus disease 2019- a multicenter, open label, randomized controlled study: a structured summary of a study protocol for a randomised controlled trial.

OBJECTIVES: Patients with severe COVID-19 often suffer from significant pulmonary fibrosis. Although the pathogenesis of pulmonary fibrosis has not been fully explained, the signal pathways and cytokines involved are very similar to hepatic fibrosis. This has been successfully treated with the Anluohuaxian Pill, a proprietary Chinese medicine composed of a variety of Chinese herbal medicines. The aim of this study is to evaluate the efficacy and safety of Anluohuaxian in the treatment of pulmonary fibrosis in patients with severe COVID-19. TRIAL DESIGN: This is a prospective, multicenter, open, randomized controlled trial. The distribution ratio was 2:1, 500 cases in the experimental group and 250 cases in the control group. PARTICIPANTS: Minimum Age: 18 Years Maximum Age: 80 Years Sex: All Gender Based: No Accepts Healthy Volunteers: No Inclusion Criteria: 1.Confirmed COVID-19, and the nucleic acid test of respiratory specimens such as sputum or nasopharyngeal swabs is negative twice after the treatment (sampling interval is at least 24 hours);2.Negative nucleic acid test of respiratory specimens such as sputum or nasopharyngeal swabs during screening visits;3.High-resolution CT of the lung (HRCT) indicates pulmonary fibrosis (thickness of lobular septum, honeycomb-like changes, with or without bronchial / pleural distraction);4.Voluntarily participate in research and sign informed consent. EXCLUSION CRITERIA: 1.Combined with severe heart, lung (diagnosed with interstitial lung disease, bronchial asthma, chronic obstructive pulmonary disease, etc.), liver and kidney disease or with endocrine, rheumatic, neurologic, malignant and other systemic diseases;2.Have been diagnosed with connective tissue disease;3.Pregnant or lactating women;4.History of mental disorders, substance abuse or dependence;5.Have used other anti-pulmonary fibrosis drugs in the past 14 days, such as nintedanib, pirfenidone, penicillamine, colchicine, tumor necrosis factor alpha blocker, imatinib, glucocorticoid hormones, morphomycodyl esters, azathioprine, cyclophosphamide, interferon-γ, and traditional Chinese medicine;6.Researchers consider it inappropriate to participate in research;7.Participating in other clinical research. This mutli-centre RCT will be undertaken in 9 trial centres: The Second People's Hospital of Fuyang, Ezhou Central Hospital, Huoshenshan Hospital of Wuhan, Jinyintan Hospital of Wuhan, Tongji Hospital of Huazhong University of Science and Technology, West Hospital Union Hospital Huazhong University of Science and Technology, Wuhan Pulmonary Hospital, Zhongnan Hospital of Wuhan University, Wenzhou Medical University Affiliated First Hospital. INTERVENTION AND COMPARATOR: The research drug is Anluohuaxian Pill, which is provided by Senlong Pharmaceutical Co., Ltd. The basic therapeutic drugs for COVID-19 involved in the study include antiviral drugs. Brands can be selected according to the treatment routines of each research center to facilitate the improvement of treatment compliance. MAIN OUTCOMES: Primary Outcome Measure: 1.Changes in high-resolution computer tomography of the lung Changes in ground-glass shadows, interstitial or air nodules found on high-resolution computer tomography [Time Frame: 3 months] 2.Change in 6-minute walking distance [Time Frame: 3 months] RANDOMISATION: In this study, the central randomization system (IWRS, an interactive network response system based on network) is used to randomise the groups. The subjects who met the entry criteria were randomly divided into the experimental group and the control group according to the proportion of 2:1. In this study, the block randomized grouping method is used, and the block length is 6. The random grouping program is set up by statistical and computer professionals in the randomization process. BLINDING (MASKING): This is an open label trial. Trial participants, investigators, care givers, outcome assessors, and date analysts are not blinded to group assignment. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): 750 patients are expected to be enrolled and the cases are allocated according to the ratio of 2 (Anluohuaxian combined with regular treatment group):1 (regular treatment group). TRIAL STATUS: Protocol version number 3.0, 10th April 2020. The recruitment has not yet started. Actual Study Start Date: April 1, 2020 Estimated Primary Completion Date: June 1, 2020 Estimated Study Completion Date: December 1, 2020 TRIAL REGISTRATION: ClinicalTrials.gov ID: NCT04334265. Registered on 3 April 2020 FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.

The Mechanism and Clinical Outcome of patients with Corona Virus Disease 2019 Whose Nucleic Acid Test has changed from negative to positive, and the therapeutic efficacy of Favipiravir: A structured summary of a study protocol for a randomised controlled trial.

OBJECTIVES: A variety of possible mechanisms can make the nucleic acid test of patients who meet the discharge conditions positive again, including reinfection, reactivation of the original virus, lack of strict discharge criteria, new infection, and so on. Different reasons will correspond to different prevention and control measures. We will enroll patients who are discharged after treatment, whose nucleic acid test has changed from negative to positive during the screening visit, regardless of the severity of the symptoms, to investigate the mechanism, clinical outcome and therapeutic efficacy with Favipiravir patients with Corona virus Disease 2019. Favipiravir is an anti-viral agent that selectively and potently inhibits the RNA-dependent RNA polymerase, it has been used for treatment of some life-threatening infections such as Ebola virus, Lassa virus and rabies. Its therapeutic efficacy has been proven in these diseases. TRIAL DESIGN: This is a multi-center, two arm, open label, parallel group, randomized controlled trial. PARTICIPANTS: Eligibility criteria: Inclusion criteria: 1.Adults 18 to 80 years, male or female.2.After the first diagnosis and treatment of COVID-19, the nucleic acid test of respiratory specimens such as sputum or nasopharyngeal swabs, has been negative for two consecutive times (sampling time interval of at least 24 hours), in accordance with the COVID-19's diagnosis and treatment Plan (7th Edition), discharged.3.During screening visit (follow-up after discharge), The nucleic acid test of COVID-19 is positive in any one of the following samples: sputum, throat swabs, blood, feces or other specimens. Regardless of whether or not they had symptoms and the severity of symptoms.4.Volunteer to participate in the research and sign the Informed Consent Form. EXCLUSION CRITERIA: 1.Allergic to Favipiravjr;2.Pregnant or lactating women3.Uncontrolled diseases of the blood and cardiovascular system, liver or kidney.4.History of mental disorders, drug abuse or dependence;5.Researchers consider it inappropriate for adults to participate;6.Participating in other clinical studies. Loss to Follow up: Cases that do not complete the clinical trial program will be regarded as lost to follow up. Including the withdrawal of patients by themselves (such as poor compliance, etc.), or the withdrawal of patients ordered by the researcher (those who need other drugs which affect the judgment of the curative effect, and those who need to stop taking drugs for severe adverse events) Study setting: The participating hospitals are some of the designated hospitals that have been or may be admitting patients who meet the eligibility criteria, mainly in Hubei, Shenzhen, Anhui and Beijing. Participants will be recruited from these 15 hospitals: Wuhan Pulmonary Hospital, Hubei; Jinyintan Hospital of Wuhan, Hubei; Ezhou Central Hospital, Hubei; The Second People's Hospital of Fuyang, Anhui; The First Affiliated Hospital of USTC, Anhui; Beijing Youan Hospital, Beijing; Capital Medical University Beijing Institute of Hepatology, Beijing; Ezhou Hospital of Traditional Chinese Medicine, Hubei; Zhongnan Hospital of Wuhan University, Hubei; The Fifth Hospital of ShiJiazhuang, Hebei; Jinan Infectious Diseases Hospital, Shandong; Public Health Clinical Center of Chengdu, Sichuan; Wuxi No.5 People's Hospital, Jiangsu; The Third People's Hospital of Shenzhen, Guangdong; The First Affiliated Hospital of Bengfu Medical College, AnHui. INTERVENTION AND COMPARATOR: Favipiravir group (experimental): Favipiravir 1600mg each dose, twice a day on the 1st day; 600mg each dose, twice a day from the 2nd to the 7th day, Oral administration, the maximum number of days taken will be no more than 14 days plus routine treatment for COVID-19. Regular treatment group (control): Treatments other than Antiviral drugs can be given. Routine treatment for patients with the corona virus will be administered, this includes oxygen therapy, drugs that reduced phlegm and relieve cough, including thymosin, proprietary Chinese medicine, etc. MAIN OUTCOMES: Primary Outcome Measures: Viral nucleic acid test negative [Time Frame: 5 months]: Subjects who tested negative for nucleic acid from sputum or nasopharyngeal swabs for two consecutive times (sampling time interval of at least 24 hours). SECONDARY OUTCOME MEASURES: Clinical cure [Time Frame: 5 months]: 1.Body temperature returned to normal for more than 3 days;2.Lung image improved.3.Clinical manifestation improved;4.The viral nucleic acid test of respiratory specimens was negative for two consecutive times (sampling time interval of at least 24 hours). RANDOMIZATION: The central randomization system (Interactive Web Response Management System), will be used to randomly divide the subjects into the experimental group and the control group according to the ratio of 2:1. In this study, block randomization will be used, in blocks of 6. BLINDING (MASKING): This is an open label trial. Trial participants, investigators, care givers, outcome assessors, and date analysts are not blinded to group assignment. NUMBERS TO BE RANDOMISED: 210 patients are expected to be enrolled and allocated according to the ratio of 2 (Favipiravir group, n=140): 1(regular treatment group, n=70). TRIAL STATUS: Protocol version number 3.0, 10th April 2020 First Patient, first visit 17th March 2020; recruitment end date anticipated June 1, 2020. TRIAL REGISTRATION: ClinicalTrials.gov, NCT04333589, April 3, 2020. Registered April 3, 2020. FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.

Proteomic Analysis of Auricularia auricula-judae Under Freezing Treatment Revealed Proteins and Pathways Associated With Melanin Reduction.

Auricularia auricula-judae is an edible nutrient-rich mushroom, which is a traditional medicinal resource in China. It is known that environment stimuli will affect the production of melanin by A. auricula-judae, but the mechanism of the effects of freezing treatment on melanin accumulation remains unknown. In the present study, the synthesis of melanin in A. auricula-judae was analyzed by physiological assays and a proteomics approach. Our findings showed that a longer freezing treatment causes a lighter color of A. auricula-judae fruiting bodies. The proteomic analysis showed that proteins involved in glycolysis/gluconeogenesis, tyrosine metabolism, ribosome, and arginine biosynthesis might contribute to the color differences in the A. auricula-judae after freezing treatment. This work will be expected to provide valuable information on the physiological and molecular mechanisms of freezing treatment on the color quality of A. auricula-judae.