Integrated meta-omics approaches reveal Saccharopolyspora as the core functional genus in huangjiu fermentations.

Identification of the core functional microorganisms in food fermentations is necessary to understand the ecological and functional processes for making those foods. Wheat qu, which provides liquefaction and saccharifying power, and affects the flavor quality, is a key ingredient in ancient alcoholic huangjiu fermentation, while core microbiota of them still remains indistinct. In this study, metagenomics, metabolomics, microbial isolation and co-fermentation were used to investigate huangjiu. Although Aspergillus is usually regarded as core microorganism in wheat qu to initiate huangjiu fermentations, our metagenomic analysis showed that bacteria Saccharopolyspora are predominant in wheat qu and responsible for breakdown of starch and cellulose. Metabolic network and correlation analysis showed that Saccharopolyspora rectivirgula, Saccharopolyspora erythraea, and Saccharopolyspora hirsuta made the greatest contributions to huangjiu's metabolites, consisting of alcohols (phenylethanol, isoamylol and isobutanol), esters, amino acids (Pro, Arg, Glu and Ala) and organic acids (lactate, tartrate, acetate and citrate). S. hirsuta J2 isolated from wheat qu had the highest amylase, glucoamylase and protease activities. Co-fermentations of S. hirsuta J2 with S. cerevisiae HJ resulted in a higher fermentation rate and alcohol content, and huangjiu flavors were more similar to that of traditional huangjiu compared to co-fermentations of Aspergillus or Lactiplantibacillus with S. cerevisiae HJ. Genome of S. hirsuta J2 contained genes encoding biogenic amine degradation enzymes. By S. hirsuta J2 inoculation, biogenic amine content was reduced by 45%, 43% and 62% in huangjiu, sausage and soy sauce, respectively. These findings show the utility of Saccharopolyspora as a key functional organism in fermented food products.

Microbial succession and its effect on the formation of umami peptides during sufu fermentation.

Sufu, a traditional Chinese fermented food, is famous for its unique flavor, especially umami. However, the formation mechanism of its umami peptides is still unclear. Here, we investigated the dynamic change of both umami peptides and microbial communities during sufu production. Based on peptidomic analysis, 9081 key differential peptides were identified, which mainly involved in amino acid transport and metabolism, peptidase activity and hydrolase activity. Twenty-six high-quality umami peptides with ascending trend were recognized by machine learning methods and Fuzzy c-means clustering. Then, through correlation analysis, five bacterial species (Enterococcus italicus, Leuconostoc citreum, L. mesenteroides, L. pseudomesenteroides, Tetragenococcus halophilus) and two fungi species (Cladosporium colombiae, Hannaella oryzae) were identified to be the core functional microorganisms for umami peptides formation. Functional annotation of five lactic acid bacteria indicated their important functions to be carbohydrate metabolism, amino acid metabolism and nucleotide metabolism, which proved their umami peptides production ability. Overall, our results enhanced the understanding of microbial communities and the formation mechanism of umami peptides in sufu, providing novel insights for quality control and flavor improvement of tofu products.

Safety evaluation and comparative genomics analysis of the industrial strain Aspergillus flavus SU-16 used for huangjiu brewing.

Huangjiu is a popular Chinese traditional alcoholic beverage, while its brewing processes have rarely been explored. We herein report the first gapless, near-finished genome assembly of the industrial strain Aspergillus flavus SU-16 for huangjiu brewing. This work provides insights and supports for the further industrial applications of A. flavus isolates by comprehensively studying of the safety and genomic variations of SU-16. We demonstrated that SU-16 is a non-aflatoxigenic A. flavus at both molecular and metabolic levels. Using of nanopore sequencing technology resulted in a complete genome sequence for all 8 A. flavus chromosomes, as well as the mitochondrion. Genome comparisons of SU-16 with reference strains identified the chromosomal rearrangements, revealed the adaption mechanism of SU-16 to huangjiu ecological niche, and found that SU-16 is a good repository for CAZymes and some bioactive secondary metabolites. The results will help to develop more scientific huangjiu fermentation processes, and explore metabolism pathways of desired or harmful components in huangjiu to improve its quality.

Therapeutic or prophylactic anticoagulation in acute isolated distal deep vein thrombosis: protocol for a prospective, multicentre, single-blind, randomised controlled trial (TOP-IDDVT).

INTRODUCTION: The efficacy and safety of anticoagulant treatment is not established for patients with acute symptomatic isolated distal deep vein thrombosis (IDDVT). In real-world clinical practice, both therapeutic and prophylactic anticoagulation are used for acute IDDVT. However, therapeutic anticoagulation is associated with higher risk of bleeding than prophylactic anticoagulation. Thus, this study aims to assess the efficacy and safety in patients with first acute symptomatic IDDVT treated with therapeutic or prophylactic anticoagulation using rivaroxaban. METHODS AND ANALYSIS: This study is a prospective, multicentre, single-blind, randomised controlled trial. Outpatients with a first, acute, symptomatic, objectively confirmed IDDVT in four centres from 1 August 2021 are recruited. Eligible patients are randomised in a 1:1 ratio to receive prophylactic anticoagulation (rivaroxaban 10 mg once a day for 3 months) or therapeutic anticoagulation (rivaroxaban 20 mg once a day for 3 months). All patients are followed for 6 months. The primary efficacy outcome is radiographically confirmed recurrent venous thromboembolism. The primary safety outcome is the incidence of major or clinically relevant non-major bleeding events. ETHICS AND DISSEMINATION: This study has been approved by the Ethics Committee of Zhongshan Hospital Fudan University (B2021-175R). Study results will be disseminated through peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT04967573.

Metagenomics-based insights into the microbial community profiling and flavor development potentiality of baijiu Daqu and huangjiu wheat Qu.

Daqu and wheat Qu are saccharification and fermenting agents in Chinese huangjiu and baijiu production. This study aimed to investigate the difference between Daqu and wheat Qu in physicochemical indices, microbial communities, functional genes, and the metabolic network of key microbes responsible for flavor synthesis by whole-metagenome sequencing and metabolite analysis. Herein, physicochemical indices indicated that compared with wheat Qu, Daqu exhibited higher protease and cellulase activity and acidity, and lower glucoamylase and amylase enzyme activity. Metagenomic sequencing reveals that although Daqu and wheat Qu community composition have significant differences at species level, they have similar functional genes. Daqu were enriched in Pediococcus pentosaceus, Weissella paramesenteroides, Rasamsonia emersonii and Byssochlamys spectabilis (22.48% of the total abundance), while wheat Qu harbored greater abundances of Saccharopolyspora (54.78%, Saccharopolyspora rectivirgula, Saccharopolyspora shandongensis, Saccharopolyspora hirsuta, Saccharopolyspora spinose, and Saccharopolyspora erythraea). From a functional perspective, the important functions of Daqu and wheat Qu are both amino acid metabolism and carbohydrate metabolism. Meanwhile, a combined analysis among microbiota, functional genes, and dominant flavors indicated S. shandongensis, S. rectivirgula, and S. spinose might be the main contributor to the synthesis of flavor compounds in wheat Qu, while R. emersonii, W. paramesenteroides, Leuconostoc citreum, Leuconostoc mesenteroides, Weissella cibaria and P. pentosaceus may make the greatest contribution to flavor compounds synthesis in Daqu. This study reveals the microbial and functional dissimilarities of Daqu and wheat Qu, and helps elucidating different metabolic roles of microbes during flavor formation.

Reducing biogenic amine in seriflux and huangjiu by recycling of seriflux inoculated with Lactobacillus plantarum JN01.

High content of biogenic amine (BA) in huangjiu could pose serious quality concerns. More than 71% of BA in huangjiu were carried over from seriflux (rice soaking wastewater), which were produced by some BA producing bacteria during rice soaking process. A BA non-producing strain, Lactobacillus plantarum JN01, was introduced to rice soaking process, which decreased BA content in seriflux by 93.8% by niche competition at bench scale. Recycling of seriflux inoculated with L. plantarum JN01 at pilot run scale for ten batches demonstrated that BA in seriflux and huangjiu were reduced by 78.4% and 87.7%, respectively. The safety of huangjiu was enormously improved without affecting on the profiles of flavor compounds. Our results demostrated that seriflux recycling technology could reduce 50% of water consumption and achieve "zero effluents" in rice soaking process, which might potentially be a "green technology" not only for huangjiu brewing industry, but also for other related traditional fermented food industries.

hsa-miR-15b-5p regulates the proliferation and apoptosis of human vascular smooth muscle cells by targeting the ACSS2/PTGS2 axis.

A previous bioinformatic analysis from our group predicted that the interaction of microRNA (miRNA/miR)-15b with the acyl-CoA synthetase short chain family member 2 (ACSS2) gene was important for the development of abdominal aortic aneurysm (AAA). Apoptosis of aortic vascular smooth muscle cells (VSMCs) is a pathological feature of AAA. The present study aimed to explain the roles of miR-15b/ACSS2 in AAA by exploring their effects on the proliferation and apoptosis of aortic VSMCs. Human aortic VSMCs (T/G HA-VSMC cell line) were divided into six groups and were transfected with miR-15b-5p mimics, mimic negative control (NC), miR-15b-5p inhibitors, inhibitor NC, miR-15b-5p mimics+pcDNA3.1 and miR-15b-5p mimics+ACSS2-overexpessing vector. CCK-8 assay was used to determine cell proliferation. Annexin V-FITC/PI staining and flow cytometry assays were used to measure cell apoptosis. Dual-luciferase reporter assays were used to confirm the targeted relationship between miR-15b-5p and ACSS2. Reverse transcription-quantitative PCR and/or western blotting were used to examine the expression levels of miR-15b-5p, ACSS2 and prostaglandin-endoperoxide synthase 2 (PTGS2). Following transfection of T/G HA-VSMCs with mimics and inhibitors to respectively upregulate and downregulate miR-15b-5p, the results demonstrated that overexpression of miR-15b-5p inhibited cell proliferation and promoted cell apoptosis; silencing of miR-15b-5p obtained the opposite results. ACSS2 may be a direct target of miR-15b-5p, since the luciferase activity of a ACSS2 wild-type vector, but not that of a ACSS2 mutant reporter, was significantly inhibited by miR-15b-5p mimics compared with controls. Additionally, the expression levels of ACSS2 and its downstream gene PTGS2 were significantly reduced or increased following transfection with miR-15b-5p mimics or inhibitors, respectively. Furthermore, overexpression of ACSS2 reversed the antiproliferative and proapoptotic effects of miR-15b-5p mimics by blocking the production of PTGS2 protein. In conclusion, miR-15b-5p may promote the apoptosis and inhibit the proliferation of aortic VSMCs via targeting the ACSS2/PTGS2 axis. The present study provided preliminary evidence indicating that the miR-15b-5p/ACSS2/PTGS2 axis may be a potential target for the treatment of AAA.

Structural Characterization of Peptides From Huangjiu and Their Regulation of Hepatic Steatosis and Gut Microbiota Dysbiosis in Hyperlipidemia Mice.

Hyperlipidemia is a chronic disorder that is difficult to cure and usually treated with long-term lipid-reducing drugs. Recent trends have led to the use of diet therapies or food-derived strategies in the treatment of such long-term diseases. The Chinese rice wine (huangjiu) contains a wide range of bioactive peptides that are produced during the multi-species fermentation process. To clarify the regulation effects of lipid metabolism and gut microbiota by huangjiu bioactive peptides, three huangjiu peptides were isolated, purified and characterized by hyper-filtration, macroporous resin, gel filtration separation and structural identification. Meanwhile, a mouse model of high-fat diet-induced hyperlipidemia was established to study the effects of huangjiu peptides on serum biomarker, hepatic metabolism and gut microbiota dysbiosis. Experimental results showed that huangjiu peptides T1 and T2 (HpT1, HpT2) treatment alleviated the increase in serum total cholesterol, triglyceride, low-density lipoprotein cholesterol levels and aberrant hepatic lipid accumulation in the high-fat diet-induced hyperlipidemia mice. Furthermore, HpT2 and HpT1 restored the α-diversity and structure of gut microbial community after hyperlipidemia-induced microbiota disturbance compared with simvastatin and HpT3. The administration of HpT2 and HpT1 regulated the microbiota-mediated gut ecology through alterations of characteristic taxa including Lactobacillus, Ileibacterium, Faecalibaculum and Alloprevotella by linear discriminant analysis effect size analysis. Collectively, our results offer new insights into the abilities of food-derived peptides on alleviation of high-fat diet-induced hyperlipidemia, hepatic steatosis and gut dysbiosis in mice.

Microbiota stratification and succession of amylase-producing Bacillus in traditional Chinese Jiuqu (fermentation starters).

BACKGROUND: Jiuqu are vital saccharifying and fermenting agents for Chinese fermented foods. Natural ventilation during Jiuqu fermentation causes changes in temperature, oxygen and moisture content, resulting in mass and heat gradients from the outer to inner areas of Jiuqu blocks. In the present study, microbiota stratification in Jiuqu was investigated by single molecule real-time sequencing and culture isolation. The contributors of Bacillus to amylase activity of Jiuqu and the dynamics of their biomass during Jiuqu fermentation were also analyzed. RESULTS: The dominant orders, genera and species between the inner and outer layers of Huangjiu qu (HJQ) were similar, although they displayed greater variance in two layers of Baijiu qu (BJQ). Bacillus possessed the highest diversity (including 27 species) in Jiuqu. Bacillus licheniformis, Bacillus altitudinis, Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus megaterium were most prevalent in HJQ, whereas B. licheniformis, B. amyloliquefaciens and Bacillus cereus were dominant in BJQ. Isolates of B. amyloliquefaciens, B. subtilis and B. cereus exhibited high activities of amylase and glucoamylase. Quantification of Bacillus members possessing genes of α-amylase revealed that B. cereus and B. licheniformis were the most dominant microbes to secret α-amylase in Jiuqu and their biomass were increasing during Jiuqu fermentation. CONCLUSION: The present study demonstrates the microbial distribution in different layers of Jiuqu and clarifies the Bacillus species processing the activity of α-amylase. These results will help industries control the quality of Jiuqu by rationally selecting starters and optimizing their microbiota. © 2020 Society of Chemical Industry.

New insights into the impacts of huangjiu compontents on intoxication.

Although huangjiu is a popular alcoholic beverage in China, the occurrence of quick-intoxication suppresses huangjiu consumption and impedes development of the huangjiu industry. In this study, the Cryprinus carpio intoxication model was used to compare the differences in intoxication effect of alcoholic beverages and to assess the impacts of huangjiu components on intoxication for the first time. Exposure to huangjiu led to the most rapid physical imbalance of C. carpio, followed by red wine and Western liquor. Higher alcohols, biogenic amines and aldehydes could cause physical imbalance of fish by themselves, and synergistic effects were observed when combined with ethanol. 2-Phenylethanoland and isopentanol had the greatest positive effect on huangjiu intoxication, followed by histamine and phenethylamine. No synergistic effect was observed between individual aldehydes and ethanol. Identification of these impactful huangjiu components provides a new perspective on the establishment of more rigorous control on the quality and flavor of huangjiu.

SOX12 expression is associated with progression and poor prognosis in human breast cancer.

The sex-determining region Y-box 12 (SOX12) is implicated in several oncogenic signaling pathways of multiple types of cancer; however, the biological effects of SOX12 on breast cancer has yet to be elucidated. Here, we assessed SOX12 expression using real-time quantitative PCR in 142 pairs of breast cancer and adjacent normal tissues (ANTs) and immunohistochemistry in 524 breast cancer and 147 ANTs. The effects of SOX12 on breast cancer progression, clinicopathological variables, and prognostic value were then investigated. SOX12 expression was markedly elevated in breast cancer tissues relative to that in ANTs at both mRNA and protein levels. Positive SOX12 expression was correlated to tumor size (P = 0.005), estrogen receptor (ER) (P = 0.018) and human epidermal growth factor receptor (HER2) (P = 0.004) status, lymph node metastasis (P < 0.001), and the tumor-node-metastasis (TNM) stage (P < 0.001). Notably, the positive rate of SOX12 expression gradually increased with breast cancer progression. Multivariate analysis indicated that SOX12 was an independent prognostic factor for overall survival (OS, P = 0.023) and distant metastasis-free survival (DMFS, P = 0.012). Subgroup analysis revealed that luminal and HER2 patients with positive SOX12 expression had a shorter OS period than those with negative SOX12 expression. Moreover, SOX12 expression was associated with a high risk of distant metastasis in invasive carcinoma with the lymph node metastasis subgroup. In summary, SOX12 correlates with progression and poor prognosis in human breast cancer, suggesting that SOX12 is a potential target for breast cancer treatment and warrants further functional research.

miR-30a-GNG2 and miR-15b-ACSS2 Interaction Pairs May Be Potentially Crucial for Development of Abdominal Aortic Aneurysm by Influencing Inflammation.

Abdominal aortic aneurysm (AAA) is a lethal vascular degenerative disease for the elderly, but current therapeutic options are limited. This study was to explore the molecular mechanisms of AAA to screen underlying treatment targets for AAA. The gene and microRNA (miRNA) expression profiles of human AAA were downloaded from Gene Expression Omnibus database under accession number GSE57691, GSE62179, and GSE63541. Differentially expressed genes (DEGs) and microRNAs (miRNAs; DEMs) were identified using the Linear Models for Microarray data method. Protein-protein interaction (PPI) network, module analysis, and miRNA-mRNA regulatory network analyses were performed to screen hub genes and miRNAs that regulated the hub genes. The Database for Annotation, Visualization and Integrated Discovery was used to predict the functions of genes. GEPIA and Tumor-miRNA-Pathway online software were used to validate the expressions of crucial DEMs and DEGs in other cancers, respectively. As a result, in the GSE57691 dataset, a total of 584 DEGs were found to be specific for AAA, 521 of which were used for constructing the PPI network. ACSS2 (acyl-CoA synthetase short-chain family member 2), GNG2 (G protein subunit gamma 2), and CXCL1 (C-X-C motif chemokine ligand 1) and CCR7 (C-C motif chemokine receptor 7) were believed to be hub genes by calculating their topological features in the PPI network. Upregulated GNG2 could interact with CXCL1 and CCR7 to involve in chemokine signaling pathway, while downregulated ACSS2 was associated with lipid biosynthetic process. In the miRNA-mRNA regulatory network, ACSS2 was found to be regulated by hsa-miR-15b; hsa-miR-30a could modulate the expression of GNG2. In line with our analysis in AAA, GNG2, ACSS2, hsa-miR-30a, and hsa-miR-15b were also confirmed to be significantly upregulated or downregulated in several cancer types. In conclusion, hsa-miR-30a-GNG2 and hsa-miR-15b-ACSS2 interaction pairs may represent novel mechanisms for explaining the pathogenesis of AAA. Targeted regulation of them may be potential strategies for treatment of AAA.

Combined subfascial endoscopic perforator surgery and endovenous laser treatment without impact on the great saphenous vein for management of lower-extremity varicose veins.

BACKGROUND: Conventional high ligation and stripping of the great saphenous vein (GSV) has a good curative effect but is highly traumatic with a considerable relapse rate. Subfascial endoscopic perforator surgery (SEPS) plus endovenous laser treatment (EVLT) could be applied as individual therapy. This study aimed to evaluate the feasibility of performing combined SEPS and EVLT without impacting GSV in the management of valvular insufficiency of the lower-limb venous perforators. METHODS: Placement of lower-limb venous perforator insufficiency was marked by ascending phlebography in 83 affected limbs from September 2010 to June 2011. After randomization, SEPS was performed on 41 limbs to address the insufficiency of the venous perforators under the deep fascia, in combination with EVLT to close the superficial varicose veins without impacting the GSV. The remaining 42 limbs were treated using traditional GSV phlebectomy as controls. RESULTS: Postoperatively, all varicose veins were resolved, with lightening of the pigmentation and healing of the ulcer. Within a follow-up period of 5 - 11 months, no symptoms had recurred. Compared with the control group, the operation time, the number of incisions sutured, and the in-hospital time decreased on average by 1.5 hours, 4.7, and 6.8 days, respectively (P < 0.01 in all cases). CONCLUSION: Combined SEPS and EVLT for treatment of valvular insufficiency of the lower-limb venous perforators offer the advantages of microtrauma and rapid cure.

A retrospective study comparing endovenous laser ablation and microwave ablation for great saphenous varicose veins.

BACKGROUND: Endo-venous laser or microwave ablation is a minimally invasive surgery for treating varicose veins of lower limbs. AIM: The aim of our study was to determine whether endovenous microwave ablation of the greater saphenous vein was associated with better effectiveness and less complications than the endovenous laser ablation. MATERIALS AND METHODS: From July 2008 to June 2011, 259 cases (306 limbs) of varicose veins were assigned to endovenous laser ablation (n=138, 163 limbs) or endovenous microwave ablation (n=121, 143 limbs). RESULTS: Through analysis there was no significant difference of the operating time, length of hospital stay and Aberdeen score in the two groups. The recanalization rate was statistically higher in the laser group than that in the microwave group. The ecchymosis complication was significantly lower in microwave ablation than that of laser ablation group. However, the skin burn and paralysis complications were significantly lower in the laser ablation than that of microwave ablation group. CONCLUSIONS: Endo-venous microwave ablation is an effective alternative to laser ablation for treatment of varicose veins, associated with higher occlusion rate and without serious complications.