[Acupoint application for Hashimoto's thyroiditis with liver-qi stagnation: a randomized controlled trial].

OBJECTIVE: To observe the clinical efficacy and safety of acupoint application for Hashimoto's thyroiditis (HT) with liver-qi stagnation. METHODS: One hundred and fifty patients of HT with liver-qi stagnation were randomly divided into an acupoint application group (75 cases, 11 cases were excluded, 5 cases dropped out) and a control group (75 cases, 12 cases excluded, 3 cases dropped out). Based on the health education combined with conventional western medicine treatment, the patients in the acupoint application group were treated with acupoint application, while the patients in the control group were treated with placebo acupoint application. Shenque (CV 8), bilateral Yongquan (KI 1), Yeshi, and ashi point were selected in both groups, with Yeshi treated once a week and the remaining acupoints treated every other day, for a total of 4 weeks. The serum levels of thyroglobulin antibody (TgAb), thyroid peroxidase antibody (TPOAb), free triiodothyronine (FT3), free thyroxine (FT4), and thyroid-stimulating hormone (TSH), as well as the thickness of thyroid left lobe, right lobe, and isthmus, TCM symptom score, hospital anxiety and depression scale (HADS) score, and MOS 36-item short form health survey (SF-36) score were compared between the two groups before and after treatment. Adverse reactions in both groups were observed. RESULTS: Compared with before treatment, in the acupoint application group, the serum levels of TgAb and TPOAb were reduced after treatment (P<0.05), and the scores of role physical (RP), body pain (BP), vitality (VT), role emotional (RE), and mental health (MH) in SF-36 were increased after treatment (P<0.01, P<0.001). The thickness of the thyroid isthmus after treatment was smaller than that before treatment (P<0.05), and the TCM symptom scores and HADS anxiety (HADS-A) scores after treatment were lower than those before treatment (P<0.001, P<0.01) in both groups. In the control group, the scores of physical function (PF), RP, BP, VT, and RE in SF-36 after treatment were higher than those before treatment (P<0.05, P<0.01, P<0.001). There was no statistically significant difference in serum FT3, FT4, and TSH levels within the groups (P>0.05). There was no statistically significant difference in the above indexes between the two groups (P>0.05). The incidence of adverse reactions in the acupoint application group and the control group was 20.0% (15/75) and 10.7% (8/75) respectively, with skin allergy being the main adverse reaction. CONCLUSION: Acupoint application could reduce the serum levels of TgAb and TPOAb in patients of HT with liver-qi stagnation, alleviate thyroid enlargement, improve TCM symptoms and anxiety, and improve quality of life, with safe and reliable clinical efficacy.

Discovery of indole-2-one derivatives as BRD4 (BD1) selective inhibitors.

Bromodomain protein 4 (BRD4) is a member of the BET family, and its overexpression is closely associated with the development of many tumors. Inhibition of BRD4 shows great therapeutic potential in anti-tumor, and pan-BRD4 inhibitors show adverse effects of dose limiting toxicity and thrombocytopenia in clinical trials. To improve clinical effects and reduce side effects, more efforts have focused on seeking selective inhibitors of BD1 or BD2. Herein, a series of indole-2-one derivatives were designed and synthesized through docking-guided optimization to find BRD4-BD1 selective inhibitors, and their BRD4 inhibitory and antiproliferation activities were evaluated. Among them, compound 21r had potent BRD4 inhibitory activity (the IC50 values of 41 nM and 313 nM in BD1 and BD2 domain), excellent anti-proliferation (the IC50 values of 4.64 ± 0.30 µM, 0.78 ± 0.03 µM, 5.57 ± 1.03 µM against HL-60, MV-4-11 and HT-29 cells), and displayed low toxicity against normal cell GES-1 cells. Further studies revealed that 21r inhibited proliferation by decreasing the expression of proto-oncogene c-Myc, blocking cell cycle in G0/G1 phase, and inducing apoptosis in MV-4-11 cells in a dose-dependent manner. All the results showed that compound 21r was a potent BRD4 inhibitor with BD1 selectivity, which had potential in treatment of leukemia.

Construct Phenylethanoid Glycosides Harnessing Biosynthetic Networks, Protein Engineering and One-Pot Multienzyme Cascades.

Phenylethanoid glycosides (PhGs) exhibit a multitude of structural variations linked to diverse pharmacological activities. Assembling various PhGs via multienzyme cascades represents a concise strategy over traditional synthetic methods. However, the challenge lies in identifying a comprehensive set of catalytic enzymes. This study explores biosynthetic PhG reconstruction from natural precursors, aiming to replicate and amplify their structural diversity. We discovered 12 catalytic enzymes, including four novel 6'-OH glycosyltransferases and three new polyphenol oxidases, revealing the intricate network in PhG biosynthesis. Subsequently, the crystal structure of CmGT3 (2.62 Å) was obtained, guiding the identification of conserved residue 144# as a critical determinant for sugar donor specificity. Engineering this residue in PhG glycosyltransferases (FsGT61, CmGT3, and FsGT6) altered their sugar donor recognition. Finally, a one-pot multienzyme cascade was established, where the combined action of glycosyltransferases and acyltransferases boosted conversion rates by up to 12.6-fold. This cascade facilitated the reconstruction of 26 PhGs with conversion rates ranging from 5-100 %, and 20 additional PhGs detectable by mass spectrometry. PhGs with extra glycosyl and hydroxyl modules demonstrated notable liver cell protection. This work not only provides catalytic tools for PhG biosynthesis, but also serves as a proof-of-concept for cell-free enzymatic construction of diverse natural products.

A trace amine associated receptor mediates antimicrobial immune response in the oyster Crassostrea gigas.

Trace amine-associated receptors (TAARs) are a class of G protein-coupled receptors, playing an immunomodulatory function in the neuroinflammatory responses. In the present study, a TAAR homologue with a 7tm_classA_rhodopsin-like domain (designated as CgTAAR1L) was identified in oyster Crassostrea gigas. The abundant CgTAAR1L transcripts were detected in visceral ganglia and haemocytes compared to other tissues, which were 55.35-fold and 32.95-fold (p < 0.01) of those in adductor muscle, respectively. The mRNA expression level of CgTAAR1L in haemocytes significantly increased and reached the peak level at 3 h after LPS or Poly (I:C) stimulation, which was 4.55-fold and 12.35-fold of that in control group, respectively (p < 0.01). After the expression of CgTAAR1L was inhibited by the injection of its targeted siRNA, the mRNA expression levels of interleukin17s (CgIL17-1, CgIL17-5 and CgIL17-6), and defensin (Cgdefh1) significantly decreased at 3 h after LPS stimulation, which was 0.51-fold (p < 0.001), 0.39-fold (p < 0.01), 0.48-fold (p < 0.05) and 0.41-fold (p < 0.05) of that in the control group, respectively. The nuclear translocation of Cgp65 protein was suppressed in the CgTAAR1L-RNAi oysters. Furthermore, the number of Vibrio splendidus in the haemolymph of CgTAAR1L-RNAi oysters significantly increased (4.11-fold, p < 0.001) compared with that in the control group. In contrast, there was no significant difference in phagocytic rate of haemocytes to V. splendidus in the CgTAAR1L-RNAi oysters. These results indicated that CgTAAR1L played an important role in the immune defense against bacterial infection by inducing the expressions of interleukin and defensin.

The involvement of interferon regulatory factor 8 in regulating the proliferation of haemocytes in oyster Crassostrea gigas.

Interferon regulatory factor 8 (IRF8) is an important transcriptional regulatory factor involving in multiple biological process, such as the antiviral immune response, immune cell proliferation and differentiation. In the present study, the involvement of a previously identified IRF8 homologue (CgIRF8) in regulating haemocyte proliferation of oyster were further investigated. CgIRF8 mRNA transcripts were detectable in all the stages of C. gigas larvae with the highest level in D-veliger (1.76-fold of that in zygote, p < 0.05). Its mRNA transcripts were also detected in all the three haemocyte subpopulations of adult oysters with the highest expression in granulocytes (2.79-fold of that in agranulocytes, p < 0.01). After LPS stimulation, the mRNA transcripts of CgIRF8 in haemocytes significantly increased at 12 h and 48 h, which were 2.04-fold and 1.65-fold (p < 0.05) of that in control group, respectively. Meanwhile, the abundance of CgIRF8 protein in the haemocytes increased significantly at 12 h after LPS stimulation (1.71-fold of that in seawater, p < 0.05). The immunofluorescence assay and Western blot showed that LPS stimulation induced an obvious nucleus translocation of CgIRF8 protein in haemocytes. After the expression of CgIRF8 was inhibited by the injection of CgIRF8 siRNA, the percentage of EdU positive haemocytes, the proportion of granulocytes, and the mRNA expression levels of CgGATA and CgSCL all declined significantly at 12 h after LPS stimulation, which was 0.64-fold (p < 0.05), 0.7-fold (p < 0.05), 0.31-fold and 0.54-fold (p < 0.001) of that in the NC group, respectively. While the expression level of cell proliferation-related protein CgCDK2, CgCDC6, CgCDC45 and CgPCNA were significantly increased (1.99-fold, and 2.41-fold, 3.76-fold and 4.79-fold compared to that in the NC group respectively, p < 0.001). Dual luciferase reporter assay demonstrated that CgIRF8 was able to activate the CgGATA promoter in HEK293T cells after transfection of CgGATA and CgIRF8. These results collectively indicated that CgIRF8 promoted haemocyte proliferation by regulating the expression of CgGATA and other related genes in the immune response of oyster.

Parotid metastasis of rare lung adenocarcinoma: A case report.

BACKGROUND: Lung cancer (LC) is the leading cause of malignancy-related deaths worldwide. The most common sites of metastasis include the nervous system, bone, liver, respiratory system, and adrenal glands. LC metastasis in the parotid gland is very rare, and its diagnosis presents a challenge. Here, we report a case of parotid metastasis in primary LC. CASE SUMMARY: The patient was a 74-year-old male who was discovered to have bilateral facial asymmetry inadvertently two years ago. The right earlobe was slightly swollen and without pain or numbness. Computed tomography (CT) examination showed bilateral lung space-occupying lesions. Pulmonary biopsy was performed and revealed adenocarcinoma (right-upper-lung nodule tissue). Positron emission tomography-CT examination showed: (1) Two hypermetabolic nodules in the right upper lobe of the lung, enlarged hypermetabolic lymph nodes in the right hilar and mediastinum, and malignant space-occupying lesion in the right upper lobe of the lung and possible metastasis to the right hilar and mediastinal lymph nodes; and (2) multiple hypermetabolic nodules in bilateral parotid glands. Parotid puncture biopsy was performed considering lung adenocarcinoma metastasis. Gene detection of lung biopsy specimens revealed an EGFR gene 21 exon L858R mutation. CONCLUSION: This case report highlights the challenging diagnosis of parotid metastasis in LC given its rare nature. Such lesions should be differentiated from primary tumors of the parotid gland. Simple radiological imaging is unreliable, and puncture biopsy is needed for final diagnosis of this condition.

An LPS-induced TNF-α factor involved in immune response of oyster Crassostrea gigas by regulating haemocytes apoptosis.

LPS induced TNF-α Factor (LITAF) is a transcription factor widely involving in activation of Tumor Necrosis Factor (TNF) and other cytokines in the inflammatory response. In the present study, a homologue of LITAF with a conserved LITAF domain was identified from the Pacific oyster Crassostrea gigas. The transcripts of CgLITAF were detected in all examined tissues with highest expression in hepatopancrease. The immunofluorescence assay and Western blot showed that LPS stimulation induced an obvious nucleus translocation of CgLITAF protein in haemocytes. While the mRNA level of CgLITAF changed slightly after LPS stimulation. When the siRNA of CgLITAF was injected to inhibit its expression, the apoptotic level of haemocytes decreased observably after LPS stimulation. Consistently, the transcripts of CgTNF3 and CgTNF4 (LOC105343080, LOC105341146), the apoptotic-related molecules including CgBax, CgCytochrome c, CgCaspase9 and CgCaspase3, were significantly suppressed in the CgLITAF-RNAi oysters. While the mRNA expression level of CgBcl was enhanced significantly in the CgLITAF-RNAi oysters. These results indicated that CgLITAF promoted haemocyte apoptosis by regulating the expression of apoptotic-related factors, suggesting its important role in the immune response of oysters.

Programmable Biosynthesis of Plant-Derived 4'-Deoxyflavone Glycosides by an Unconventional Yeast Consortium.

Previous data established 4'-deoxyflavone glycosides (4'-DFGs) as important pharmaceutical components in the roots of rare medical plants like Scutellaria baicalensis Georgi. Extracting these compounds from plants involves land occupation and is environmentally unfriendly. Therefore, a modular ("plug-and-play") yeast-consortium platform is developed to synthesize diverse 4'-DFGs de novo. By codon-optimizing glycosyltransferase genes from different organisms for Pichia pastoris, six site-specific glycosylation chassis are generated to be capable of biosynthesizing 18 different 4'-DFGs. Cellular factories showed increased 4'-DFG production (up to 18.6-fold) due to strengthened synthesis of UDP-sugar precursors and blocked hydrolysis of endogenous glycosides. Co-culturing upstream flavone-synthesis-module cells with downstream glycoside-transformation-module cells alleviated the toxicity of 4'-deoxyflavones and enabled high-level de novo synthesis of 4'-DFGs. Baicalin is produced at the highest level (1290.0 mg L-1 ) in a bioreactor by controlling the consortium through carbon-source shifting. These results provide a valuable reference for biosynthesizing plant-derived 4'-DFGs and other glycosides with potential therapeutic applications.

Network Pharmacology and Experimental Validation Explore the Pharmacological Mechanisms of Herb Pair for Treating Rheumatoid Arthritis.

OBJECTIVE: This study aimed to elucidate the multitarget mechanism of the Mori Ramulus - Taxilli Herba (MT) herb pair in treating rheumatoid arthritis (RA). METHODS: The targets of the herb pair and RA were predicted from databases and screened through cross-analysis. The core targets were obtained using protein-protein interaction (PPI) network analysis. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed. Finally, animal experiments were conducted to validate the anti-RA effect and mechanism of this herb pair. RESULTS: This approach successfully identified 9 active compounds of MT that interacted with 6 core targets (AKT1, TNF, IL6, TP53, VEGFA, and IL1ß). Pathway and functional enrichment analyses revealed that MT had significant effects on the TNF and IL-17 signaling pathways. The consistency of interactions between active components and targets in these pathways was confirmed through molecular docking. Moreover, the potential therapeutic effect of MT was verified in vivo, demonstrating its ability to effectively relieve inflammation by regulating these targeted genes and pathways. CONCLUSION: The present work suggests that the therapeutic effect of MT herb pair on RA may be attributed to its ability to regulate the TNF signaling pathway and IL-17 signaling pathway.

Chrysin 7-O-ß-D-glucuronide, a dual inhibitor of SARS-CoV-2 3CLpro and PLpro, for the prevention and treatment of COVID-19.

The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) resulted in the coronavirus disease 2019 (COVID-19) pandemic. Given the advent of subvariants, there is an urgent need to develop novel drugs. The aim of this study was to find SARS-CoV-2 inhibitors from Scutellaria baicalensis Georgi targeting the proteases 3CLpro and PLpro. After screening 25 flavonoids, chrysin 7-O-ß-D-glucuronide was found to be a potent inhibitor of SARS-CoV-2 on Vero E6 cells, with half-maximal effective concentration of 8.72 µM. Surface plasmon resonance assay, site-directed mutagenesis and enzymatic activity measurements indicated that chrysin-7-O-ß-D-glucuronide inhibits SARS-CoV-2 by binding to H41 of 3CLpro, and K157 and E167 of PLpro. Hydrogen-deuterium exchange mass spectrometry analysis showed that chrysin-7-O-ß-D-glucuronide changes the conformation of PLpro. Finally, chrysin 7-O-ß-D-glucuronide was shown to have anti-inflammatory activity, mainly due to reduction of the levels of the pro-inflammatory cytokines interleukin (IL)-1ß and IL-6.

The alleviating effect of Scutellaria amoena extract on the regulation of gut microbiota and its metabolites in NASH rats by inhibiting the NLRP3/ASC/caspase-1 axis.

Background: Scutellaria amoena (SA) is the root of S. amoena C.H. Wright of Labiatae, also known as Scutellaria southwestern. This is mainly distributed in Sichuan, Yunnan, and Guizhou in China. In southwest China, SA is used as an alternative method to genuine medicine for the treatment of allergy, diarrhea, inflammation, hepatitis, and bronchitis. Thus far, studies on the effects of SA on non-alcoholic steatohepatitis (NASH) are lacking. This paper investigated the effect of SA on the regulation of gut microbiota and its metabolites in NASH rats by inhibiting the NOD-like receptor 3 (NLRP3)/apoptosis-associated speck-like protein (ASC)/caspase-1 axis. Methods: A NASH rat model was induced by a high-fat diet (HFD) for 12 weeks, and rats were orally given different doses of SA extracts (150 and 300 mg/kg/d) for 6 weeks. Changes in histological parameters, body weight, organ indexes, cytokines, and biochemical parameters related to NLRP3 in NASH rats were checked. 16S rRNA gene sequencing and UPLC-MS/MS technology were used to analyze the changes in the gut microbiota composition and its metabolites in NASH rats. Results: SA significantly inhibited the HFD-induced increase in body weight, lipid levels, and inflammatory infiltration. SA notably inhibited the HFD-induced increase in the upper and lower factors of NLRP3, such as transforming growth factor (TGF)-ß, tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-18, pro-IL-18, IL-1ß, pro-IL-1ß, NLRP3, ASC, and caspase-1. Additionally, mRNA expressions of caspase-1, NLRP3, and ASC were significantly downregulated after SA treatment. The results of the intestinal flora showed that SA could increase the diversity of flora and change its structure and composition in NASH rats by reducing Firmicutes/Bacteroidetes (F/B) ratio, Blautia (genus), Lachospiraceae (family), and Christensenellaceae R-7 group (genus), and increasing Muribaculaceae (family) and Bacteroides (genus). The metabolomics revealed that 24 metabolites were possibly the key metabolites for SA to regulate the metabolic balance of NASH rats, including chenodeoxycholic acid, xanthine, and 9-OxoODE. Nine metabolic pathways were identified, including primary bile acid biosynthesis, bile secretion, purine metabolism, and secondary bile acid biosynthesis. Conclusion: SA can regulate the intestinal microbial balance and metabolic disorder by inhibiting the NLRP3/ASC/caspase-1 axis to relieve NASH.

Novel NIR-II fluorescent probes for biliary atresia imaging.

Biliary atresia is a rare infant disease that predisposes patients to liver transplantation and death if not treated in time. However, early diagnosis is challenging because the clinical manifestations and laboratory tests of biliary atresia overlap with other cholestatic diseases. Therefore, it is very important to develop a simple, safe and reliable method for the early diagnosis of biliary atresia. Herein, a novel NIR-II fluorescence probe, HZL2, with high quantum yield, excellent biocompatibility, low cytotoxicity and rapid excretion through the liver and gallbladder was developed based on the oil/water partition coefficient and permeability. A simple fecal sample after injection of HZL2 can be used to efficiently identify the success of the mouse model of biliary atresia for the first time, allowing for an early diagnosis of the disease. This study not only developed a simple and safe method for the early diagnosis of biliary atresia with great potential in clinical translation but also provides a research tool for the development of pathogenesis and therapeutic medicines for biliary atresia.

Insights into the missing apiosylation step in flavonoid apiosides biosynthesis of Leguminosae plants.

Apiose is a natural pentose containing an unusual branched-chain structure. Apiosides are bioactive natural products widely present in the plant kingdom. However, little is known on the key apiosylation reaction in the biosynthetic pathways of apiosides. In this work, we discover an apiosyltransferase GuApiGT from Glycyrrhiza uralensis. GuApiGT could efficiently catalyze 2″-O-apiosylation of flavonoid glycosides, and exhibits strict selectivity towards UDP-apiose. We further solve the crystal structure of GuApiGT, determine a key sugar-binding motif (RLGSDH) through structural analysis and theoretical calculations, and obtain mutants with altered sugar selectivity through protein engineering. Moreover, we discover 121 candidate apiosyltransferase genes from Leguminosae plants, and identify the functions of 4 enzymes. Finally, we introduce GuApiGT and its upstream genes into Nicotiana benthamiana, and complete de novo biosynthesis of a series of flavonoid apiosides. This work reports an efficient phenolic apiosyltransferase, and reveals mechanisms for its sugar donor selectivity.

Research Progress of Metformin in the Treatment of Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is one of the most common malignancies and has a high mortality, posing a great threat to both human physical and mental health. With the advancement of scientific research, a variety of cancer therapies have been used for OSCC treatment. However, the prognosis of OSCC shows no significant improvement. Metformin has been recognized as the first-line drug for the treatment of diabetes, and recent studies have shown that metformin has a remarkable suppressive effect on tumor progression. Metformin can not only affect the energy metabolism of tumor cells but also play an antitumor role by modulating the tumor microenvironment and cancer stem cells. In this review, the molecular mechanism of metformin and its anticancer mechanism in OSCC are summarized. In addition, this article summarizes the side effects of metformin and the future prospects of its application in the treatment of OSCC.

Characterization and structure-based protein engineering of a regiospecific saponin acetyltransferase from Astragalus membranaceus.

Acetylation contributes to the bioactivity of numerous medicinally important natural products. However, little is known about the acetylation on sugar moieties. Here we report a saponin acetyltransferase from Astragalus membranaceus. AmAT7-3 is discovered through a stepwise gene mining approach and characterized as the xylose C3'/C4'-O-acetyltransferse of astragaloside IV (1). To elucidate its catalytic mechanism, complex crystal structures of AmAT7-3/1 and AmAT7-3A310G/1 are obtained, which reveal a large active pocket decided by a specific sequence AADAG. Combining with QM/MM computation, the regiospecificity of AmAT7-3 is determined by sugar positioning modulated by surrounding amino acids including #A310 and #L290. Furthermore, a small mutant library is built using semi-rational design, where variants A310G and A310W are found to catalyze specific C3'-O and C4'-O acetylation, respectively. AmAT7-3 and its variants are also employed to acetylate other bioactive saponins. This work expands the understanding of saponin acetyltransferases, and provide efficient catalytic tools for saponin acetylation.

Role and clinical significance of immunogenic cell death biomarkers in chemoresistance and immunoregulation of head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignancies in the whole world, with little improvement in the 5-year survival rate due to the occurrence of chemoresistance. With the increasing interests in tumor immune microenvironment, immunogenic cell death (ICD)-induced chemotherapy has shown promising results in enhancing sensitivity to immune checkpoint inhibitors (ICI) and improving the efficiency of tumor immunotherapy. This review summarizes the role of key ICD biomarkers and their underlying molecular mechanisms in HNSCC chemoresistance. The results showed that ICD initiation could significantly improve the survival and prognosis of patients. ICD and its biomarker could also serve as molecular markers for tumor diagnosis and prognosis. Moreover, key components of DAMPs including CALR, HGMB1, and ATP are involved in the regulation of HNSCC chemo-sensitivity, confirming that the key biomarkers of ICD can also be developed into new targets for regulating HNSCC chemoresistance. This review clearly illustrates the theoretical basis for the hypothesis that ICD biomarkers are therapeutic targets involved in HNSCC progression, chemoresistance, and even immune microenvironment regulation. The compilation and investigation may provide new insights into the molecular therapy of HNSCC.

A circadian clock protein cryptochrome inhibits the expression of inflammatory cytokines in Chinese mitten crab (Eriocheir sinensis).

Cryptochrome (Cry), as important flavoprotein, plays a key role in regulating the innate immune response, such as the release of inflammatory cytokines. In the present study, a cryptochrome homologue (EsCry) was identified from Chinese mitten crab Eriocheir sinensis, which contained a typical DNA photolyase domain, a FAD binding domain. The transcripts of EsCry were highly expressed at 11:00, and lowest at 3:00 within one day, while those of Interleukin enhancer binding factor (EsILF), Lipopolysaccharide-induced TNF-alpha factor (EsLITAF), Tumor necrosis factor (EsTNF) and Interleukin-16 (EsIL-16) showed a rhythm expression pattern contrary to EsCry. After EsCry was knocked down by dsEsCry injection, mRNA transcripts of Timeless (EsTim), Cycle (EsCyc), Circadian locomotor output cycles kaput (EsClock), Period (EsPer), and EsLITAF, EsTNF, EsILF, EsIL-16, as well as phosphorylation level of Dorsal significantly up-regulated. The transcripts of EsLITAF, EsTNF, EsILF, and EsIL-16 in EsCry-RNAi crabs significantly down-regulated after injection of NF-κB inhibitor. The interactions of EsCyc and EsCry, EsCyc and Dorsal were observed in vitro. These results indicated that EsCry negatively regulated the expression of the cytokine TNF and IL-16 via inhibiting their transcription factor LITAF and ILF through NF-κB signaling pathway, which provide evidences to better understand the circadian regulation mechanism of cytokine production in crabs.

A highly selective C-rhamnosyltransferase from Viola tricolor and insights into its mechanisms.

C-Glycosides are important natural products with various bioactivities. In plant biosynthetic pathways, the C-glycosylation step is usually catalyzed by C-glycosyltransferases (CGTs), and most of them prefer to accept uridine 5'-diphosphate glucose (UDP-Glc) as sugar donor. No CGTs favoring UDP-rhamnose (UDP-Rha) as sugar donor has been reported, thus far. Herein, we report the first selective C-rhamnosyltransferase VtCGTc from the medicinal plant Viola tricolor. VtCGTc could efficiently catalyze C-rhamnosylation of 2-hydroxynaringenin 3-C-glucoside, and exhibited high selectivity towards UDP-Rha. Mechanisms for the sugar donor selectivity of VtCGTc were investigated by molecular dynamics (MD) simulations and molecular mechanics with generalized Born and surface area solvation (MM/GBSA) binding free energy calculations. Val144 played a vital role in recognizing UDP-Rha, and the V144T mutant could efficiently utilize UDP-Glc. This work provides a new and efficient approach to prepare flavonoid C-rhamnosides such as violanthin and iso-violanthin.