A molecular mechanism underlies grass carp (Ctenopharyngodon idella) TARBP2 regulating PKR-mediated cell apoptosis.

Interferon-inducible double-stranded RNA-dependent protein kinase (PKR) is one of the key antiviral arms in the innate immune system. The activated PKR performs its antiviral function by inhibiting protein translation and inducing apoptosis. In our previous study, we identified grass carp TARBP2 as an inhibitor of PKR activity, thereby suppressing cell apoptosis. This study aimed to explore the effects of grass carp TARBP2 on PKR activity and cell apoptosis. Grass carp TARBP2 comprises two N-terminal dsRBDs and a C-terminal C4 domain. Subcellular localization analysis conducted in CIK cells revealed that TARBP2-FL (full-length TARBP2), TARBP2-Δ1 (lack of the first dsRBD), and TARBP2-Δ2 (lack of the second dsRBD) are predominantly located in the cytoplasm, while TARBP2-Δ3 (lack of the two dsRBDs) is distributed both in the nucleus and cytoplasm. Colocalization and immunoprecipitation assays confirmed the interaction of TARBP2-FL, TARBP2-Δ1, and TARBP2-Δ2 with PKR, while TARBP2-Δ3 showed no binding. Furthermore, our findings suggested that the inhibitory effect of TARBP2-Δ1 or TARBP2-Δ2 on the PKR-eIF2α pathway is depressed compared to TARBP2-FL. In cell apoptosis assays, it was observed that TARBP2-FL inhibits PKR-mediated cell apoptosis. TARBP2-Δ1 or TARBP2-Δ2 exhibits decreased inhibition to PKR-mediated cell apoptosis, whereas TARBP2-Δ3 nearly completely loses this inhibitory effect. These findings highlight the critical importance of two dsRBDs of TARBP2 in interaction with PKR, as well as in the inhibition of PKR activity, resulting in the suppression of cell apoptosis triggered by prolonged PKR activation.

A Combined Transcriptomic and Proteomic Analysis of Monkeypox Virus A23 Protein on HEK293T Cells.

Monkeypox virus (MPXV) is a cross-kingdom pathogen infecting both humans and wildlife, which poses a significant health risk to the public. Although MPXV attracts broad attention, there is a lack of adequate studies to elucidate pathogenic mechanisms associated with viral infections. In this study, a high-throughput RNA sequencing (RNA-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach was used to explore the transcriptional and metabolic responses of MPXV A23 protein to HEK293T cells. The protein-protein interactions and signaling pathways were conducted by GO and KEGG analyses. The localization of A23 protein in HEK293T cells was detected by immunofluorescence. A total of 648 differentially expressed genes (DEGs) were identified in cells by RNA-Seq, including 314 upregulated genes and 334 downregulated genes. Additionally, liquid chromatography-tandem mass spectrometry (LC-MS/MS) detected 115 cellular proteins that interact with the A23 proteins. Transcriptomic sequencing analysis revealed that transfection of MPXV A23 protein modulated genes primarily associated with cellular apoptosis and DNA damage repair. Proteomic analysis indicated that this protein primarily interacted with host ribosomal proteins and histones. Following the identification of the nuclear localization sequence RKKR within the A23 protein, a truncated mutant A23ΔRKKR was constructed to investigate the subcellular localization of A23 protein. The wild-type A23 protein exhibits a significantly higher nuclear-to-cytoplasmic ratio, exceeding 1.5, in contrast to the mutant A23ΔRKKR, which has a ratio of approximately 1. Immunofluorescence assays showed that the A23 protein was mainly localized in the nucleus. The integration of transcriptomics and proteomics analysis provides a comprehensive understanding of the interaction between MPXV A23 protein and the host. Our findings highlight the potential role of this enzyme in suppressing host antiviral immune responses and modulating host gene expression.

Comprehensive analysis of PPP4C's impact on prognosis, immune microenvironment, and immunotherapy response in lung adenocarcinoma using single-cell sequencing and multi-omics.

Background: Elevated PPP4C expression has been associated with poor prognostic implications for patients suffering from lung adenocarcinoma (LUAD). The extent to which PPP4C affects immune cell infiltration in LUAD, as well as the importance of associated genes in clinical scenarios, still requires thorough investigation. Methods: In our investigation, we leveraged both single-cell and comprehensive RNA sequencing data, sourced from LUAD patients, in our analysis. This study also integrated datasets of immune-related genes from InnateDB into the framework. Our expansive evaluation employed various analytical techniques; these included pinpointing differentially expressed genes, constructing WGCNA, implementing Cox proportional hazards models. We utilized these methods to investigate the gene expression profiles of PPP4C within the context of LUAD and to clarify its potential prognostic value for patients. Subsequent steps involved validating the observed enhancement of PPP4C expression in LUAD samples through a series of experimental approaches. The array comprised immunohistochemistry staining, Western blotting, quantitative PCR, and a collection of cell-based assays aimed at evaluating the influence of PPP4C on the proliferative and migratory activities of LUAD cells. Results: In lung cancer, elevated expression levels of PPP4C were observed, correlating with poorer patient prognoses. Validation of increased PPP4C levels in LUAD specimens was achieved using immunohistochemical techniques. Experimental investigations have substantiated the role of PPP4C in facilitating cellular proliferation and migration in LUAD contexts. Furthermore, an association was identified between the expression of PPP4C and the infiltration of immune cells in these tumors. A prognostic framework, incorporating PPP4C and immune-related genes, was developed and recognized as an autonomous predictor of survival in individuals afflicted with LUAD. This prognostic tool has demonstrated considerable efficacy in forecasting patient survival and their response to immunotherapeutic interventions. Conclusion: The involvement of PPP4C in LUAD is deeply intertwined with the tumor's immune microenvironment. PPP4C's over-expression is associated with negative clinical outcomes, promoting both tumor proliferation and spread. A prognostic framework based on PPP4C levels may effectively predict patient prognoses in LUAD, as well as the efficacy of immunotherapy strategy. This research sheds light on the mechanisms of immune interaction in LUAD and proposes a new strategy for treatment.

Transcriptome and proteomic analysis of mpox virus F3L-expressing cells.

Background: Monkeypox or mpox virus (mpox) is a double-stranded DNA virus that poses a significant threat to global public health security. The F3 protein, encoded by mpox, is an apoenzyme believed to possess a double-stranded RNA-binding domain (dsRBD). However, limited research has been conducted on its function. In this study, we present data on the transcriptomics and proteomics of F3L-transfected HEK293T cells, aiming to enhance our comprehension of F3L. Methods: The gene expression profiles of pCAGGS-HA-F3L transfected HEK293T cells were analyzed using RNA-seq. Proteomics was used to identify and study proteins that interact with F3L. Real-time PCR was used to detect mRNA levels of several differentially expressed genes (DEGs) in HEK293T cells (or Vero cells) after the expression of F3 protein. Results: A total of 14,822 genes were obtained in cells by RNA-Seq and 1,672 DEGs were identified, including 1,156 up-regulated genes and 516 down-regulated genes. A total of 27 cellular proteins interacting with F3 proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and 19 cellular proteins with large differences in abundance ratios were considered to be candidate cellular proteins. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that the DEGs were significantly enriched in immune-related pathways, including type I interferon signaling pathway, response to virus, RIG-I-like receptor signaling pathway, NOD-like receptor signaling pathway, etc. Moreover, some selected DEGs were further confirmed by real-time PCR and the results were consistent with the transcriptome data. Proteomics data show that cellular proteins interacting with F3 proteins are mainly related to RNA splicing and protein translation. Conclusions: Our analysis of transcriptomic and proteomic data showed that (1) F3L up-regulates the transcript levels of key genes in the innate immune signaling pathway, such as RIGI, MDA5, IRF5, IRF7, IRF9, ISG15, IFNA14, and elicits a broad spectrum of antiviral immune responses in the host. F3L also increases the expression of the FOS and JNK genes while decreasing the expression of TNFR2, these factors may ultimately induce apoptosis. (2) F3 protein interacts with host proteins involved in RNA splicing and protein translation, such as SNRNP70, POLR2H, HNRNPA1, DDX17, etc. The findings of this study shed light on the function of the F3 protein.

Grass carp (Ctenopharyngodon idella) deacetylase SIRT1 targets p53 to suppress apoptosis in a KAT8 dependent or independent manner.

Sirtuin1 (SIRT1) is known as a deacetylase to control various physiological processes. In mammals, SIRT1 inhibits apoptotic process, but the detailed mechanism is not very clear. Here, our study revealed that grass carp (Ctenopharyngodon idella) SIRT1 (CiSIRT1, MN125614.1) inhibits apoptosis through targeting p53 in a KAT8-dependent or a KAT8-independent manner. In CIK cells, CiSIRT1 over-expression results in significant decrease of some apoptotic gene expressions, including Bax/Bcl2, caspase3 and caspase9, whereas CiKAT8 or Cip53 facilitates the induction of apoptosis. Because CiSIRT1 separately interacted with CiKAT8 and Cip53, we speculated that CiSIRT1 blocked apoptosis may be by virtue of KAT8-p53 axis or directly by p53. In a KAT8-dependent manner, CiSIRT1 interacted with CiKAT8, then reduced the acetylation of CiKAT8 and subsequently promoted its degradation. Then, CiKAT8 acetylated p53 and induced p53-mediated apoptosis. MYST domain of CiKAT8 was critical in this pathway. In a KAT8-independent manner, CiSIRT1 also inhibited p53-induced apoptosis by directly deacetylating p53 and promoting the degradation of p53. Generally, these findings uncovered two pathways in which CiSIRT1 decreases the acetylation of p53 via a KAT8-dependent or a KAT8-independent manner.

Conjugation of chitopentaose with ß-lactoglobulin using Maillard reaction, and its effect on the allergic desensitization in vivo.

The conjugation of chitopentaose (CHP) on ß-lactoglobulin (ßLg) via Maillard reaction was used to desensitize ßLg. The stable ßLg-CHP conjugate (ßC-4) was formed at 4 h incubation, which contains 5 CHP attached molecules and a conjugated degree of 42 %. The conjugation promoted the thermal stability and emulsifying properties of ßLg, and inhibited the immunoglobulin E (IgE) combining capacity by decreasing the content of ß-sheet in ßLg. Moreover, ßLg-CHP conjugates were imparted with anti-oxidant properties and anti-inflammatory activities. Further, the combined action of inhibited IgE combining capacity and anti-inflammatory activities improved the allergy desensitization in ßLg sensitized mice. The results showed that overexpressed IgE and inflammatory factors, unbalanced Th1-/Th2- immune cytokines were significantly attenuated after ßLg was conjugated with CHP, avoiding the inflammatory lesions in spleen and colon. Additionally, the adverse changes in gut microbiota were alleviated in ßC-4 group with a decrease of Bacteroidetes and increase of Firmicutes at phylum level and the probiotic bacteria of Lactobacillaceae was significantly improved at the family level. Thus, the conjugation of CHP can desensitize allergic reaction caused by ßLg.

Genome-Wide Identification and Expression Analysis of the RADIALIS-like Gene Family in Camellia sinensis.

The RADIALIS-like (RL) proteins are v-myb avian myeloblastosis viral oncogene homolog (MYB)-related transcription factors (TFs), and are involved in many biological processes, including metabolism, development, and response to biotic and abiotic stresses. However, the studies on the RL genes of Camellia sinensis are not comprehensive enough. Therefore, we undertook this study and identified eight CsaRLs based on the typical conserved domain SANT Associated domain (SANT) of RL. These genes have low molecular weights and theoretical pI values ranging from 5.67 to 9.76. Gene structure analysis revealed that six CsaRL genes comprise two exons and one intron, while the other two contain a single exon encompassing motifs 1 and 2, and part of motif 3. The phylogenetic analysis divided one hundred and fifty-eight RL proteins into five primary classes, in which CsaRLs clustered in Group V and were homologous with CssRLs of the Shuchazao variety. In addition, we selected different tissue parts to analyze the expression profile of CsaRLs, and the results show that almost all genes displayed variable expression levels across tissues, with CsaRL1a relatively abundant in all tissues. qRT-PCR (real-time fluorescence quantitative PCR) was used to detect the relative expression levels of the CsaRL genes under various abiotic stimuli, and it was found that CsaRL1a expression levels were substantially higher than other genes, with abscisic acid (ABA) causing the highest expression. The self-activation assay with yeast two-hybrid system showed that CsaRL1a has no transcriptional activity. According to protein functional interaction networks, CsaRL1a was well connected with WIN1-like, lysine histidine transporter-1-like, ß-amylase 3 chloroplastic-like, carbonic anhydrase-2-like (CA2), and carbonic anhydrase dnaJC76 (DJC76). This study adds to our understanding of the RL family and lays the groundwork for further research into the function and regulatory mechanisms of the CsaRLs gene family in Camellia sinensis.

[Protein expression, purification and mouse antiserum preparation of monkeypox virus A23R].

Objective To express the monkeypox virus (MPXV) A23R protein in Escherichia coli and purify by Ni-NTA affinity column, and to prepare mouse antiserum against MPXV A23R. Methods The recombinant plasmid pET-28a-MPXV-A23R was constructed and transformed into Escherichia coli BL21 to induce the expression of A23R protein. After optimizing the conditions of expression, A23R protein was highly expressed. Recombinant A23R protein was purified by Ni-NTA affinity column and identified by Western blot analysis. The purified protein was used to immunize mice for preparing the A23R polyclonal antibody, and the antibody titer was detected by ELISA. Results The expression of A23R recombinant protein reached the peak under the induced conditions of 0.6 mmol/L isopropyl-ß-D-thiogalactoside (IPTG), 37 DegreesCelsius and 20 hours. The purity of the protein was about 96.07% and was identified by Western blot analysis. The mice were immunized with recombinant protein, and the titer of antibody reached 1:102 400 at the 6th week after immunization. Conclusion MPXV A23R is expressed highly and purified with a high purity and its antiserum from mouse is obtained with a high titre.

Performance evaluation of E-nose and E-tongue combined with machine learning for qualitative and quantitative assessment of bear bile powder.

Bear bile powder (BBP) is a valuable animal-derived product with a huge adulteration problem on market. It is a crucially important task to identify BBP and its counterfeit. Electronic sensory technologies are the inheritance and development of traditional empirical identification. Considering that each drug has its own specific odor and taste characteristics, electronic tongue (E-tongue), electronic nose (E-nose) and GC-MS were used to evaluate the aroma and taste of BBP and its common counterfeit. Two active components of BBP, namely tauroursodeoxycholic acid (TUDCA) and taurochenodeoxycholic acid (TCDCA) were measured and linked with the electronic sensory data. The results showed that bitterness was the main flavor of TUDCA in BBP, saltiness and umami were the main flavor of TCDCA. The volatiles detected by E-nose and GC-MS were mainly aldehydes, ketones, alcohols, hydrocarbons, carboxylic acids, heterocyclic, lipids, and amines, mainly earthy, musty, coffee, bitter almond, burnt, pungent odor descriptions. Four different machine learning algorithms (backpropagation neural network, support vector machine, K-nearest neighbor, and random forest) were used to identify BBP and its counterfeit, and the regression performance of these four algorithms was also evaluated. For qualitative identification, the algorithm of random forest has shown the best performance, with 100% accuracy, precision, recall and F1-score. Also, the random forest algorithm has the best R2 and the lowest RMSE in terms of quantitative prediction.

Biosynthesis and the Transcriptional Regulation of Terpenoids in Tea Plants (Camellia sinensis).

Terpenes, especially volatile terpenes, are important components of tea aroma due to their unique scents. They are also widely used in the cosmetic and medical industries. In addition, terpene emission can be induced by herbivory, wounding, light, low temperature, and other stress conditions, leading to plant defense responses and plant-plant interactions. The transcriptional levels of important core genes (including HMGR, DXS, and TPS) involved in terpenoid biosynthesis are up- or downregulated by the MYB, MYC, NAC, ERF, WRKY, and bHLH transcription factors. These regulators can bind to corresponding cis-elements in the promoter regions of the corresponding genes, and some of them interact with other transcription factors to form a complex. Recently, several key terpene synthesis genes and important transcription factors involved in terpene biosynthesis have been isolated and functionally identified from tea plants. In this work, we focus on the research progress on the transcriptional regulation of terpenes in tea plants (Camellia sinensis) and thoroughly detail the biosynthesis of terpene compounds, the terpene biosynthesis-related genes, the transcription factors involved in terpene biosynthesis, and their importance. Furthermore, we review the potential strategies used in studying the specific transcriptional regulation functions of candidate transcription factors that have been discriminated to date.

Identification of the Authenticity and Geographical Origin of Bear Bile Powder by Using High Performance Liquid Chromatography - Charged Aerosol Detector Fingerprints Combined with Chemometrics.

Bear bile powder (BBP) is a rare animal-derived traditional Chinese medicine, and it has been widely used to treat visual disorders and hepatobiliary diseases in East Asia. However, there is still a lack of reliable quality control methods for BBP. This study was designed to establish a comprehensive quality map of BBP based on bile acids. High-performance liquid chromatography coupled with charged aerosol detector (HPLC-CAD) was used for fingerprint establishment and quantitative analysis of BBP. The similarities of HPLC-CAD chromatograms for 50 batches of BBP were more than 0.95, while the similarities of reference chromatograms between 6 other animal bile and BBP were low than 0.7. Additionally, five bile acids in BBP, including tauroursodeoxycholic acid, taurocholic acid, taurochenodeoxycholic acid, ursodesoxycholic acid, and chenodeoxycholic acid, were simultaneously quantified. This method has been validated with good regression as well as satisfactory precision, sensitivity, stability, repeatability, and accuracy. Using this method, the contents of five bile acids in BBP samples from five producing areas were determined and compared. Furthermore, Fisher linear discriminant analysis was performed to discriminate the geographic origins of BBP. The result demonstrated that HPLC-CAD fingerprint combined with multi-components quantification is an effective and reliable method for quality control of BBP, it could be a meaningful reference for the quality evaluation of medicinal bile.

Study of the in vitro properties of oligopeptides from whey protein isolate with high Fisher's ratio and their ability to prevent allergic response to ß-lactoglobulin in vivo.

High Fisher ratio oligopeptides (HFOPs) with molecular weight range from 100 to 800 Da derived from whey protein isolate (WPI) were used to prevent the allergic response induced by ß-lactoglobulin (ßLg) in vivo due to their anti-inflammatory activities to lipopolysaccharide (LPS) treated RAW 264.7 cells and anti-allergicproperties to anti-DNP mouse IgE sensitized RBL-2H3 cells in vitro. The results showed theoverexpressed immunoglobulin E (IgE), unbalanced Th1-/Th2-type immune cytokines and inflammatory factors in ßLg-allergic mice were significantly attenuated by oral administration of HFOPs, resulting in the prevention of inflammatory lesions in spleen and colonic histopathology. Moreover, HFOPs increased ratio of Bacteroidetes/Firmicutes at phylum level in sensitive mice, and improved the abundance of Lactobacillaceae at family level to maintain oral tolerance against ßLg and prevented allergic response. The use of HFOPs may provide a potential alternative for preventing the milk allergy induced by WPI.

Chlorphoxim induces neurotoxicity in zebrafish embryo through activation of oxidative stress.

It is known that chlorphoxim is a broad-spectrum and high-effective pesticide. With the wide use in agricultural practice, chlorphoxim residue is also frequently detected in water, but its potential toxicity to aquatic life is still unclear. In this study, zebrafish is used as a model to detect the toxicity of chlorphoxim. Our results showed that exposure of high concentration of chlorphoxim at 96 h post-fertilization (hpf) resulted in a high mortality and pericardium edema rate, a low hatchability rate and heart rate. The nervous system damage, swimming behavior alteration and acetylcholinesterase (AChE) inhibition were measured in zebrafish embryos after a 6 days post-fertilization (dpf) of chlorphoxim exposure. The expression of neural-related genes is abnormal in zebrafish embryos. Chlorphoxim exposure significantly increases oxidative stress in zebrafish embryos by inhibiting antioxidant enzyme (SOD and CAT) and activating reactive oxygen species (ROS). As expected, chlorphoxim exposure induces apoptosis by enhancing the expression of apoptotic genes (Bax, Bcl2, and p53). Astaxanthin (ATX), an effective antioxidant, was found to be able to rescue the neurotoxicity of chlorphoxim through relieving oxidative stress and apoptosis. Altogether, the results showed that chlorphoxim exposure led to severe neurotoxicity to zebrafish embryos, which was contributed to a more comprehensive understanding of the safety use of the organophosphorus pesticide.

Rapid discrimination of the authenticity and geographical origin of bear bile powder using stable isotope ratio and elemental analysis.

Bear bile powder (BBP) is one of the most famous traditional Chinese medicines derived from animals. It has a long history of medicinal use and is widely used in the treatment of hepatobiliary and ophthalmic diseases. Due to its similar morphological characterizations and chemical composition compared with other bile powders, it is difficult to accurately identify its authenticity. In addition, there are very few methods that could analyze the geographical origins of BBP. In this study, elemental analysis isotope ratio mass spectrometry (EA-IRMS) and inductively coupled plasma mass spectrometry (ICP-MS) were used to determine stable isotope ratios and elemental contents, respectively. Combined these variables with chemometrics, the discrimination models were established successfully for identifying the authenticity and geographical origins of BBP. Meanwhile, the discrimination markers were identified by calculating the variable importance for the projection (VIP) value of each variable. A total of 13 discrimination markers (δ13C, δ15N, C, Li, Mg, K, Ca, Cr, Ni, Zn, As, Se, and Sr) were used to further establish the fingerprint of BBP. According to similarity analysis, the authenticity and geographical origins of BBP could be identified without chemometrics. In conclusion, the present study established a reliable method for authenticity identification and origin traceability of BBP, which will provide references for the quality control of bile medicines.

Long-read sequencing revealed alterations of microbial relationship between tongue coating and gastric mucosa in patients with gastric intestinal metaplasia / 数字中医药（英文）

Objective@#To explore the microbial correlation between oral tongue coating (TC) and gastric mucosa (GM) in patients with gastric intestinal metaplasia (GIM).@*Methods@#The present study recruited 1360 volunteers for upper gastrointestinal cancer screening. The microbiota in TC and GM were profiled by long-read sequencing of full-length 16S rRNA gene. The microbial diversity, community structure, and linear discriminant analysis effect size (LEfSe) were analyzed by the software Visual Genomics. SparCC correlation analysis was used to construct the commensal network and the graphical display was conducted by R software.@*Results@#The population included 44 patients with precancerous GIM, and 28 matched controls with negative rapid urease test (RUT) and non-symptomatic chronic superficial gastritis (CSG). No significant difference in diversity was observed between GIM patients and controls in TC or GM microbiota (P > 0.05). Patients had a higher percentage of 41 – 60 co-occurring operational taxonomic units (OTUs) between TC and GM than controls (34.1% vs. 25.0%) (P < 0.05). The LEfSe showed that TC Prevotella melaninogenica and three gastric Helicobacter species (i.e., Helicobacter pylori, Helicobacter pylori XZ274, and Helicobacter pylori 83) were enriched in patients with GIM. Furthermore, GIM patients with positive RUT had a lower percentage of co-occurring OTUs over 20 (P < 0.05), and lower abundances of gastric Veillonella, Pseudonocardia, and Mesorhizobium than those with negative RUT (P < 0.05). The commensal network between TC and GM was more complex in GIM patients than in controls. GIM patients with positive RUT demonstrated more bacterial correlations between TC and GM than those with negative RUT. Finally, the serum ratio of PG-I/II was negatively correlated with three gastric Helicobacter species (Helicobacter pylori, Helicobacter pylori XZ274, and Helicobacter pylori 83) in patients with negative RUT (P < 0.05), and negatively correlated with two TC species (Fusobacterium nucleatum subsp. nucleatum and Campylobacter showae) in patients with positive RUT (P < 0.05).@*Conclusion@#The development of GIM potentiated the commensal network between oral TC and GM, providing microbial evidence of the correlation between TC and the stomach.

Weifuchun alters tongue flora and decreases serum trefoil factor I levels in gastric intestinal metaplasia: A CONSORT-compliant article.

OBJECTIVE: To explore the molecular mechanisms of Weifuchun in the treatment of gastric intestinal metaplasia (GIM), we designed a preclinical pilot study to examine potential markers of disease progression based on alterations in the tongue flora. METHODS: Total 27 patients with GIM were treated with Weifuchun for 4 weeks and 26 volunteers as controls. Tongue coating bacteria were profiled using 16S rDNA high-throughput sequencing. Serum pepsinogen I and II levels were detected using the latex immunoturbidimetric assay. The levels of serum trefoil factor I was detected by ELISA. Microplate-based quantification was used to detect serum total bile acid (TBA). RESULTS: After treatment, the relative abundance of 4 dominant tongue coating genera (Granulicatella, Gemella, Lachnoanaerobaculum, and Neisseria) increased significantly wheras Alloprevotella, [Eubacterium] nodatum group, Prevotell, and Ruminococcaceae UCG-014 decreased (P < .05). The results showed that Alloprevotella and 3 rare tongue coating genera (Lautropia, Treponema 2, and Aliihoeflea) might be potential markers or target flora for the treatment of GIM. Kyoto encyclopedia of genes and genomes (KEGG) function prediction analysis showed that Weifuchun may regulate bile secretion and folate biosynthesis in patients with GIM. The level of serum trefoil factor I decreased significantly in response to Weifuchun treatment, which was consistent with the decrease in folate biosynthesis predicted by KEGG. CONCLUSION: Weifuchun may restore the balance of tongue flora by decreasing the levels of serum trefoil factor I, thereby providing a new way to measuring the underlying effectiveness and potential mechanisms of action of this traditional Chinese medicinal compound in the treatment of GIM.

Diflubenzuron Induces Cardiotoxicity in Zebrafish Embryos.

Diflubenzuron is an insecticide that serves as a chitin inhibitor to restrict the growth of many harmful larvae, including mosquito larvae, cotton bollworm and flies. The residue of diflubenzuron is often detected in aquaculture, but its potential toxicity to aquatic organisms is still obscure. In this study, zebrafish embryos (from 6 h to 96 h post-fertilization, hpf) were exposed to different concentrations of diflubenzuron (0, 0.5, 1.5, 2.5, 3.5 and 4.5 mg/L), and the morphologic changes, mortality rate, hatchability rate and average heart rate were calculated. Diflubenzuron exposure increased the distance between the venous sinus and bulbar artery (SV-BA), inhibited proliferation of myocardial cells and damaged vascular development. In addition, diflubenzuron exposure also induced contents of reactive oxygen species (ROS) and malondialdehyde (MDA) and inhibited the activity of antioxidants, including SOD (superoxide dismutase) and CAT (catalase). Moreover, acridine orange (AO) staining showed that diflubenzuron exposure increased the apoptotic cells in the heart. Q-PCR also indicated that diflubenzuron exposure promoted the expression of apoptosis-related genes (bax, bcl2, p53, caspase3 and caspase9). However, the expression of some heart-related genes were inhibited. The oxidative stress-induced apoptosis damaged the cardiac development of zebrafish embryos. Therefore, diflubenzuron exposure induced severe cardiotoxicity in zebrafish embryos. The results contribute to a more comprehensive understanding of the safety use of diflubenzuron.

Intratumoral synthesis of transformable metal-phenolic nanoaggregates with enhanced tumor penetration and retention for photothermal immunotherapy.

Rationale: Effective photothermal therapy (PTT) remains a great challenge due to the difficulties of delivering photothermal agents with both deep penetration and prolonged retention at tumor lesion spatiotemporally. Methods: Here, we report an intratumoral self-assembled nanostructured aggregate named FerH, composed of a natural polyphenol and a commercial iron supplement. FerH assemblies possess size-increasing dynamic kinetics as a pseudo-stepwise polymerization from discrete nanocomplexes to microscale aggregates. Results: The nanocomplex can penetrate deeply into solid tumors, followed by prolonged retention (> 6 days) due to the in vivo growth into nanoaggregates in the tumor microenvironment. FerH performs a targeting ablation of tumors with a high photothermal conversion efficiency (60.2%). Importantly, an enhanced immunotherapeutic effect on the distant tumor can be triggered when co-administrated with checkpoint-blockade PD-L1 antibody. Conclusions: Such a therapeutic approach by intratumoral synthesis of metal-phenolic nanoaggregates can be instructive to address the challenges associated with malignant tumors.

Role of arginine supplementation on muscular metabolism and flesh quality of Pacific white shrimp (Litopenaeus vannamei) reared in freshwater.

It is no doubt that the improvement of flesh quality of Pacific white shrimp (Litopenaeus vannamei) reared in freshwater contributes to its development potential in aquaculture. In this study, we aimed to explore the effect of arginine supplementation on the flesh quality of L. vannamei reared in freshwater and its mechanism. L. vannamei were randomly fed with three diets for 56 days, of which arginine level was 10.15 g kg-1 (arginine-deficient diet), 21.82 g kg-1 (arginine-optimal diet), and 32.46 g kg-1 (arginine-excessive diet), respectively. Each diet was randomly assigned to triplicate tanks, and each tank was stocked with 35 shrimps (initial weight: 1.70 ± 0.02 g). Results showed the arginine-optimal diet increased the weight gain, flesh percentage, crude protein and flavor amino acid contents in muscle, and improved the flesh hardness by conversing fast myofibers to slow myofibers, increasing myofiber density and myofibrillar length, and promoting ornithine and collagen synthesis. The arginine-optimal diet influenced the purine metabolic pathway by reducing hypoxanthine, xanthine, and inosine contents. Ornithine, citrulline, and glutamate were identified as the key metabolites affecting flesh quality traits after arginine treatments. Only increasing the levels of dietary arginine did not result in an increase in endogenous creatine synthesis in muscle and hepatopancreas. Overall, the arginine-optimal diet improved the flesh quality traits of L. vannamei reared in freshwater due to the enhanced muscular hardness, protein deposition, and flavor, which may be contributing to the transformation of muscle fiber type and increase in protein synthesis by the metabolites of arginine (ornithine, citrulline, and glutamate).

Grass carp (Ctenopharyngodon idella) DYRK2 modulates cell apoptosis through phosphorylating p53.

In mammals, DYRK2 increases p53 phosphorylation level by interacting with it and then promotes cell apoptosis. However, the function of fish DYRK2 has not yet been elucidated. In this paper, we cloned and identified the coding sequence (CDS) of a grass carp DYRK2 (CiDYRK2) which is 1773 bp in length and encodes 590 amino acids. SMART predictive analysis showed that CiDYRK2 possesses a serine/threonine kinase domain. Subsequently, we used the dsRNA analog polyinosinic-polycytidylic acid (poly (I:C) and Grass carp reovirus (GCRV) to stimulate grass carp and CIK cells for different times and found that CiDYRK2 mRNA was significantly up-regulated both in fish tissues and cells. To explore the function of CiDYRK2, we carried out overexpression and knockdown experiments of CiDYRK2 in CIK cells. Real-time quantitative PCR (Q-PCR), TdT-mediated dUTP nick end labeling (TUNEL) assay and flow cytometry were used to detect the ratio of BAX/BCL-2 mRNA, the number of TUNEL positive cells, the proportion of Annexin V-positive cells respectively. The results showed that CiDYRK2 significantly up-regulated BAX/Bcl-2 mRNA ratio and increased the number of TUNEL-positive cells, as well as the proportion of Annexin V-positive cells. On the contrary, knock-down of CiDYRK2 significantly down-regulated BAX/Bcl-2 mRNA ratio in the cells. Therefore, CiDYRK2 promoted cell apoptosis. To study the molecular mechanism by which CiDYRK2 promoting cell apoptosis, subcellular localization and immunoprecipitation experiments were used to study the relationship between grass carp DYRK2 and the pro-apoptotic protein p53. The results showed that CiDYRK2 and Cip53 were located and co-localized in the nucleus. Co-immunoprecipitation experiment also showed that CiDYRK2 and Cip53 can bind with each other. We further found that DYRK2 can increase the phosphorylation level of p53. In a word, our results showed that grass carp DYRK2 induces cell apoptosis by increasing the phosphorylation level of p53.