Molecular identification,expression pattern,and in-vitro bioactivity analysis of insulin-like growth factor 2 in olive flounder Paralichthys olivaceus

Insulin-like growth factors(IGFs)are key regulators of development and growth.Here,we characterized the igf2 gene from olive flounder(Paralichthys olivaceus)and determined its temporal and spatial expression.We set up an in-vitro protein expression system in eukaryotic human embryonic kidney(HEK293 T)cells and explored its effects on cell proliferation.The flounder igf2 cDNA contained a 648-bp open reading frame(ORF)encoding a protein of 215 amino acids(aa),which spanned the complete signal peptide(47 aa),mature peptide(70 aa),and E domain(98 aa).In adult flounder,igf2 mRNA was detected in all selected tissues.In early development,igf2 mRNA was detected throughout development from unfertilized eggs to hatching-stage embryos.In-situ hybridization analysis indicated that igf2 mRNA was specially expressed in the brain region,floor plate,hypochord,otic vesicle,and pectoral fin during embryogenesis.Western blotting analysis indicated that the soluble recombinant flounder IGF2 protein was successfully produced through eukaryotic expression in HEK293T cells.In addition,the recombinant IGF2 protein significantly promoted the proliferation of human cervical carcinoma(HeLa)and HEK293T cells.These results provide new information about the structural and functional conservation,expression patterns,and biological activity of the igf2 in teleosts.

Understanding water conservation vs. prof ligation traits in vegetable legumes through a physio-transcriptomic-functional approach

Abstract Vegetable soybean and cowpea are related warm-season legumes showing contrasting leaf water use behaviors under similar root drought stresses,whose mechanisms are not well understood.Here we conducted an integrative phenomic-transcriptomic study on the two crops grown in a feedback irrigation system that enabled precise control of soil water contents.Continuous transpiration rate monitoring demonstrated that cowpea used water more conservatively under earlier soil drought stages,but tended to maintain higher transpiration under prolonged drought.Interestingly,we observed a soybean-specific transpiration rate increase accompanied by phase shift under moderate soil drought.Time-series transcriptomic analysis suggested a dehydration avoidance mechanism of cowpea at early soil drought stage,in which the VuHAI3 and VuTIP2;3 genes were suggested to be involved.Multifactorial gene clustering analysis revealed different responsiveness of genes to drought,time of day and their interactions between the two crops,which involved species-dependent regulation of the circadian clock genes.Gene network analysis identified two co-expression modules each associated with transpiration rate in cowpea and soybean,including a pair of negatively correlated modules between species.Module hub genes,including the ABA-degrading gene GmCYP707A4 and the trehalose-phosphatase/synthase gene VuTPS9 were identified.Intermodular network analysis revealed putative co-players of the hub genes.Transgenic analyses verified the role of VuTPS9 in regulating transpiration rate under osmotic stresses.These findings propose that species-specific transcriptomic reprograming in leaves of the two crops suffering similar soil drought was not only a result of the different drought resistance level,but a cause of it.

Multiple Isoforms of Olive Flounder (Paralichthys olivaceus) Pax3a and Pax3b Display Differential Regulations on Myogenic Differentiation

Paired box 3(Pax3)is a critical upstream regulator of the onset of myogenesis.We have previously identified two spliced isoforms of pax3a(pax3a-1 and pax3a-2)and three spliced isoforms of pax3b(pax3b-1,pax3b-2,and pax3b-3)in olive flounder,but their roles in myogenesis are unknown.In this study,we investigated their cellular localization,transcriptional activity on myod gene regulation,and roles in myogenesis.Different Pax3a and Pax3b isoforms revealed various subcellular localizations,which were related to their corresponding protein structures.Pax3a-1,Pax3a-2,and Pax3b-1 promoted the transcriptional activity of myod to dif-ferent degrees,whereas Pax3b-2 and Pax3b-3 had a slight inhibitory or no effect.The pairwise interaction analysis demonstrated the synergistic effect of Pax3b-1 and Pax3b-3 on myod transcriptional activity.The overexpression of different pax3a and pax3b isoforms differentially altered the spatial expression patterns of myod and differentially regulated the expression levels of their target genes(myod,myf5,and c-met)in zebrafish embryonic myogenesis.In addition,the different flounder myod promoter-driven pax3a/3b isoform expression vectors were successfully introduced into the skeletal muscles of juvenile flounder by electroporation.How-ever,none of them could change the mRNA expression levels of mstn,myf5,myod,myogenin,pax7a,and pax7b in the electroporated muscles.These results suggest that different Pax3a and Pax3b isoforms may precisely and collaboratively regulate embryonic myogenesis,but their roles in juvenile myogenesis are uncertain.

An ultra-sensitive and easy-to-use assay for sensing human UGT1A1 activities in biological systems

The human UDP-glucuronosyltransferase 1A1(UGT1A1),one of the most essential conjugative enzymes,is responsible for the metabolism and detoxification of bilirubin and other endogenous substances,as well as many different xenobiotic compounds.Deciphering UGT1A1 relevance to human diseases and characterizing the effects of small molecules on the activities of UGT1A1 requires reliable tools for probing the function of this key enzyme in complex biological matrices.Herein,an easy-to-use assay for highly-selective and sensitive monitoring of UGT1A1 activities in various biological matrices,using liquid chromatography with fluorescence detection(LC-FD),has been developed and validated.The newly developed LC-FD based assay has been confirmed in terms of sensitivity,specificity,precision,quantitative linear range and stability.One of its main advantages is lowering the limits of detection and quantification by about 100-fold in comparison to the previous assay that used the same probe substrate,enabling reliable quantification of lower amounts of active enzyme than any other method.The precision test demonstrated that both intra-and inter-day variations for this assay were less than 5.5%.Furthermore,the newly developed assay has also been successfully used to screen and characterize the regulatory effects of small molecules on the expression level of UGT1A1 in living cells.Ove rall,an easy-to-use LC-FD based assay has been developed for ultra-sensitive UGT1A1 activities measurements in various biological systems,providing an inexpensive and practical approach for exploring the role of UGT1A1 in human diseases,interactions with xenobiotics,and characterization modulatory effects of small molecules on this conjugative enzyme.

Analytical methodologies for sensing catechol-O-methyltransferase activity and their applications

Mammalian catechol-O-methyltransferases(COMT)are an important class of conjugative enzymes,which play a key role in the metabolism and inactivation of catechol neurotransmitters,catechol estrogens and a wide range of endobiotics and xenobiotics that bear the catechol group.Currently,COMT inhibitors are used in combination with levodopa for the treatment of Parkinson’s disease in clinical practice.The crucial role of COMT in human health has raised great interest in the development of more practical assays for highly selective and sensitive detection of COMT activity in real samples,as well as for rapid screening and characterization of COMT inhibitors as drug candidates.This review summarizes recent advances in analytical methodologies for sensing COMT activity and their applications.Several lists of biochemical assays for measuring COMT activity,including the probe substrates,along with their analytical conditions and kinetic parameters,are presented.Finally,the challenges and future perspectives in the field,such as visualization of COMT activity in vivo and in situ,are highlighted.Collectively,this review article overviews the practical assays for measuring COMT activities in complex biological samples,which will strongly facilitate the investigations on the relevance of COMT to human diseases and promote the discovery of COMT inhibitors via high-throughput screening.

Simulated Sunlight Irradiation Based Photocatalytic Degradation of Acrylonitrile by Sn-F Co-doped TiO 2/SiO 2 Nano Powder Catalyst

This study proved the significance of simulated sunlight irradiation response capability of Sn-F co-doped TiO_(2)/SiO_(2)(Sn-F-TiO_(2)/SiO_(2))photocatalysts,which were prepared by a simple sol-gel method and were evaluated by acrylonitrile degradation for photocatalytic activity.The synthesized catalysts were characterized by X-ray Diffraction(XRD),Scanning Electron Microscopy(SEM),Energy Dispersive Spectrometer(EDS),X-ray Photoelectron Spectroscopy(XPS),Brunauer-Emmett-Teller(BET),Ultraviolet-Visible Absorption spectroscopy(UV-Vis),and Photoluminescence Spectroscopy(PL).UV-Visible spectroscopy demonstrated that Sn doping caused remarkable red shift in TiO_(2),which significantly increased the absorption efficiency of the catalysts.The XPS results showed that Sn was successfully doped into the TiO_(2) lattice.The photocatalytic degradation of acrylonitrile indicated that the Sn-F-TiO_(2)/SiO_(2) photocatalysts exhibited excellent photocatalytic activity when being annealed at 550℃for 2 h.The degradation rate of acrylonitrile reached 67.7%after irradiation under simulated sunlight for 6 min,and the hole was the most important active species.

Establishment and application of national reference panels for SARS-CoV-2 antigen detection kit

To develop a national reference panel for severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)antigen detection kit and establish a quality standard.The cultures of SARS-CoV-2 and other pathogens were collected to establish a national reference panel for SARS-CoV-2 antigen detection.The stability and homogeneity of the reference panel were evaluated.Based on World Health Organization(WHO)guidance and nucleic acid quantitative results,a quality standard reference panel was established.Currently,three generations of SARS-CoV-2 antigen national reference materials with batch numbers 370095–202001,370095–202202,and 370095–202203 have been successfully established.These national reference panels comprised 8 positive samples,20 negative samples,1 repetitive sample,and 1 lower detection limit sample.The stability and homogeneity of the reference panel meet the requirements.The quality standards are as follows:the positive and negative coincidence rates are 8/8 and 20/20,respectively.The 10 test results of the medium and low-concentration repetitive reference materials should be positive,and the color rendering should be uniform(or the coefficient of variance should not be higher than 20.0%).The lower detection limit should be at least 5×105 U/mL(equivalent to copies/mL),and higher concentrations above the lower detection limit must be positive.A national reference panel for the SARS-CoV-2 antigen detection kit has been established.As the standard of SARS-CoV-2 antigen reagents,the reference panel has played a crucial role in the pre-marketing quality evaluation and post-marketing quality supervision in China.

Multicenter evaluation of a simple and sensitive nucleic acid self-testing for SARS-CoV-2

A rapid and accurate COVID-19 diagnosis is a prerequisite for blocking the source of infection as soon as possible and taking the appropriate medical action.Herein,we developed GeneClick,a device for nucleic acid self-testing of SARS-CoV-2,consisting of three modules:a sampling kit,a microfluidic chip-based disposable cartridge,and an amplification reader.In addition,we evaluated the clinical performance of GeneClick using 2162 nasal swabs collected at three medical institutions,using three commercial RT-qPCR kits and an antigen self-test as references.Compared to RT-qPCR,the sensitivity and specificity of the GeneClick assay were 97.93%and 99.72%,respectively,with a kappa value of 0.979(P<0.01).Of the 2162 samples,2076 were also tested for SARS-CoV-2 antigens.Among the 314 positive samples identified by GeneClick assay,63 samples were undetected by antigen tests.Overall,the GeneClick nucleic acid self-test demonstrated higher accuracy than the antigen-based detection.Based on the additional features,including simple operation,affordable price,portable device,and reliability of smartphone APP-driven sampling and result reporting,GeneClick offers a powerful tool for field-based SARS-CoV-2 detection in primary healthcare institutions or at-home use.

Targeting CD47-SIRPα axis for Hodgkin and non-Hodgkin lymphoma immunotherapy

The interaction between cluster of differentiation 47(CD47)and signal regulatory proteinα(SiRPa)protects healthy cells from macrophage attack,which is crucial for maintain-ing immune homeostasis.Overexpression of CD47 occurs widely across various tumor cell types and transmits the"don't eat me"signal to macrophages to avoid phagocytosis through binding to SIRPa.Blockade of the CD47-SIRPa axis is therefore a promising approach for cancer treat-ment.Lymphoma is the most common hematological malignancy and is an area of unmet clin-ical need.This review mainly described the current strategies targeting the CD47-SIRPa axis,including antibodies,SiRPaFc fusion proteins,small molecule inhibitors,and peptides both in preclinical studies and clinical trials with Hodgkin lymphoma and non-Hodgkin lymphoma.

Mast cell degranulation-triggered by SARS-CoV-2 induces tracheal-bronchial epithelial inflammation and injury

SARS-CoV-2 infection-induced hyper-inflammation is a key pathogenic factor of COVID-19.Our research,along with others',has demonstrated that mast cells(MCs)play a vital role in the initiation of hyper-inflammation caused by SARS-CoV-2.In previous study,we observed that SARS-CoV-2 infection induced the accumulation of MCs in the peri-bronchus and bronchioalveolar-duct junction in humanized mice.Additionally,we found that MC degranulation triggered by the spike protein resulted in inflammation in alveolar epithelial cells and capillary endothelial cells,leading to subsequent lung injury.The trachea and bronchus are the routes for SARS-CoV-2 transmission after virus inhalation,and inflammation in these regions could promote viral spread.MCs are widely distributed throughout the respiratory tract.Thus,in this study,we investigated the role of MCs and their degranulation in the development of inflammation in tracheal-bronchial epithelium.Histological analyses showed the accumulation and degranulation of MCs in the peri-trachea of humanized mice infected with SARS-CoV-2.MC degranulation caused lesions in trachea,and the formation of papillary hyperplasia was observed.Through transcriptome analysis in bronchial epithelial cells,we found that MC degranulation significantly altered multiple cellular signaling,particularly,leading to upregulated immune responses and inflammation.The administration of ebastine or loratadine effectively suppressed the induction of inflammatory factors in bronchial epithelial cells and alleviated tracheal injury in mice.Taken together,our findings confirm the essential role of MC degranulation in SARS-CoV-2-induced hyper-inflammation and the subsequent tissue lesions.Furthermore,our results support the use of ebastine or loratadine to inhibit SARS-CoV-2-triggered degranulation,thereby preventing tissue damage caused by hyper-inflammation.

Sensing carboxylesterase 1 in living systems by a practical and isoformspecific fluorescent probe

Carboxylesterase 1(CES1), one of the most abundant serine hydrolases in mammals, has drawn much attentions in recent years, owing to this enzyme involves in many physiological processes via hydrolysis of both endogenous esters and xenobiotic esters. Herein, to real-time monitor the activities of CES1 in various biological systems, a practical and iso form-specific fluorescent probe was developed on the basis of the substrate preference of CES1, as well as the structural and optical properties of BODIPY dyes. After screening of a panel of BODIPY ester derivatives, probe 1 displayed the best combination of specificity,sensitivity, enzymatic kinetics and applicability for monitoring CES1 activities in real samples. This probe was successfully used to detect CESl activities in several biological systems including tissue preparations,living cells, tissue slices and zebrafish. Furthermore, the biomedical applications of probe 1 for screening of CES1 inhibitors were also demonstrated using tissue preparations or living cells as enzyme sources. In summary, a practical and broadly applicable tool for real-time monitoring CES1 in biological systems was developed and well-characterized, which held great promise for further investigations on CES1-associated drug discovery, clinical practice and fundamental research.

Selective Insertion in Copolymerization of Ethylene and Styrene Catalyzed by Half-Titanocene System Bearing Ketimide Ligand： A Theoretical Study

The copolymerization of ethylene and styrene can be efficiently carried out by using Cp＊TiCl2（N=CtBu2）/ MAO （Cp＊=η5-C5Me5） system, yielding the poly（ethylene-co-styrene）s with isolated styrene units. In order to in- vestigate the reasons for formation of the structure, the mechanism of copolymerization, especially the selective in- sertion of ethylene and styrene, is studied in detail by density functional theory （DFT） method. At the initiation stage, insertion of ethylene is kinetically more favorable than insertion of styrene, and insertion of styrene kinet- ically and thermodynamically prefers 2,1-insertion. That is different from the conventional half-titanocene system, in which the 1,2-insertion is favorable. At chain propagation stage, the computational results suggest that the con- tinuous insertion of styrene is hard to occur at room temperature due to the high free energy barriers （28.90 and 35.04 kcal/mol for 1,2-insertion, and 29.15 and 34.00 kcal/mol for 2,1-insertion） and thermodynamically unfavora- ble factors in two different conditions. That is mainly attributed to the steric hindrance between the coming styrene and chain-end styrene or ketimide ligand. The computational results are in good agreement with the experimental data.

A Survey on Automatic Delineation of Radiotherapy Target Volume based on Machine Learning

Radiotherapy is one of the main treatment methods for cancer,and the delineation of the radiotherapy target area is the basis and premise of precise treatment.Artificial intelligence technology represented by machine learning has done a lot of research in this area,improving the accuracy and efficiency of target delineation.This article will review the applications and research of machine learning in medical image matching,normal organ delineation and treatment target delineation according to the procudures of doctors to delineate the target volume,and give an outlook on the development prospects.

Herbal drug discovery for the treatment of nonalcoholic fatty liver disease

Few medications are available for meeting the increasing disease burden of nonalcoholic fatty liver disease(NAFLD)and its progressive stage,nonalcoholic steatohepatitis(NASH).Traditional herbal medicines(THM)have been used for centuries to treat indigenous people with various symptoms but without clarified modern-defined disease types and mechanisms.In modern times,NAFLD was defined as a common chronic disease leading to more studies to understand NAFLD/NASH pathology and progression.THM have garnered increased attention for providing therapeutic candidates for treating NAFLD.In this review,a new model called"multiple organs-multiple hits"is proposed to explain mechanisms of NASH progression.Against this proposed model,the effects and mechanisms of the frequentlystudied THM-yielded single anti-NAFLD drug candidates and multiple herb medicines are reviewed,among which silymarin and berberine are already under U.S.FDA-sanctioned phase 4 clinical studies.Furthermore,experimental designs for anti-NAFLD drug discovery from THM in treating NAFLD are discussed.The opportunities and challenges of reverse pharmacology and reverse pharmacokinetic concepts-guided strategies for THM modernization and its global recognition to treat NAFLD are highlighted.Increasing mechanistic evidence is being generated to support the beneficial role of THM in treating NAFLD and anti-NAFLD drug discovery.

Network pharmacology analysis to explore mechanism of Three Flower Tea against nonalcoholic fatty liver disease with experimental support using high-fat diet-induced rats

Objective:Nonalcoholic fatty liver disease(NAFLD)has become a common chronic liver disease that is harmful to human health.Moreover,there is currently no FDA-approved first-line drug for the treatment of nonalcoholic steatohepatitis(NASH)or NAFLD.Traditional Chinese medicine(TCM)is widely used to ameliorate liver diseases,such as the traditional ancient recipe called Three Flower Tea(TFT),which consists of double rose(Rosa rugosa),white chrysanthemum(Chrysanthemum morifolium),and Daidaihua(Citrus aurantium).However,the mechanisms of the action of TFT are not clear.Therefore,this study aimed to elucidate the mechanisms of TFT against NAFLD in high-fat diet(HFD)-induced rats.Methods:This study utilized bioinformatics and network pharmacology to establish the active and potential ingredient-target networks of TFT.Furthermore,a protein–protein interaction(PPI)network was constructed,and enrichment analysis was performed to determine the key targets of TFT against NAFLD.Furthermore,an animal experiment was conducted to evaluate the therapeutic effect and confirm the key targets of TFT against NAFLD.Results:A total of 576 NAFLD-related genes were searched in Gene Cards,and under the screening criteria of oral bioavailability(OB)≥30%and drug-likeness(DL)≥0.18,a total of 19 active ingredients and 210 targets were identified in TFT.Network pharmacology analysis suggested that 55 matching targets in PPIs were closely associated with roles for NAFLD treatment.Through the evaluation of network topology parameters,four key central genes,PPARγ,SREBP,AKT,and RELA,were identified.Furthermore,animal experiments indicated that TFT could reduce plasma lipid profiles,hepatic lipid profiles and hepatic fat accumulation,improve liver function,suppress inflammatory factors,and reduce oxidative stress.Through immunoblotting and immunofluorescence analysis,PPARγ,SREBP,AKT,and RELA were confirmed as targets of TFT in HFD-induced rats.Conclusion:In summary,our results indicate that TFT can prevent and treat NAFLD via multiple targets,including lipid accumulation,antioxidation,insulin sensitivity,and inflammation.

Field Validation of a Rapid Recombinase Aided Amplification Assay for SARS-CoV-2 RNA at Customs-Zhejiang Province,China,January 2021

ABSTRACT Introduction:The best approach to preventing the importation of coronavirus disease 2019(COVID-19)is enhancing the detection capacity at customs.The rapid detection is of utmost importance and therefore highly demanded.Methods:We conducted a field validation study of a duplex real-time reverse transcription recombinaseaided amplification(RT-RAA)assay in Zhoushan and Hangzhou customs,in Zhejiang Province,China.