With the increasing accumulation of plastic pollutants in various environments,research on microorganisms(including bacteria,fungi,and algae)with plastic degradation capabilities has gained significant attention.Howev...With the increasing accumulation of plastic pollutants in various environments,research on microorganisms(including bacteria,fungi,and algae)with plastic degradation capabilities has gained significant attention.However,only a limited number of microbial plastic-degrading enzymes have been identified to date.This highlights that the degradation mechanisms employed by many plastic-degrading microorganisms,particularly filamentous fungi,remain insufficiently explored.In this study,we utilized a versatile fungal plasmid(pCT74)to express green fluorescent protein(GFP)in a marine-derived fungus Alternaria alternata strain FB1 with plastic degradation capabilities.Upon evaluating the degradation effect of polyester-type polyurethane(PU)film,we observed that different transformants exhibited three kinds of activities(the same,reduced,or enhanced degradation capability)compared to the FB1 wild-type strain.Further analysis of the plasmid fragment insertion sites in different transformants revealed that pCT74 integrates randomly into the genome of the host fungus.Notably,a direct correlation was found between the plasmid insertion site and the degradation capability of the corresponding transformant.Our findings not only redefine the potential applications of plasmid pCT74 in filamentous fungi but also show a novel research approach to identifying key enzymes involved in plastic degradation by fungi.展开更多
Insects represent emerging sources of bioactive peptides and functional materials.Mantidis Oötheca(Sang-Piao-Xiao in Chinese,SPX)serves as an insect-derived medicine for treating kidney disease.This study demonst...Insects represent emerging sources of bioactive peptides and functional materials.Mantidis Oötheca(Sang-Piao-Xiao in Chinese,SPX)serves as an insect-derived medicine for treating kidney disease.This study demonstrated that supernatant(SPX)improved kidney function in adriamycin(ADR)-induced nephropathy mice model.Transcriptomic analysis revealed that SPX inhibited complement activation by targeting the MASP1-C3/C3a receptor(C3aR)pathway.Peptidomic analysis identified 304 peptides from SPX,with 49 peptides selected for evaluation using prediction tools and molecular docking with complement core protein C3.Three peptides(PMGFPFDR,FNDPK,AAQFFNR)exhibiting docking scores below-8.0 were synthesized to verify complement inhibition and anti-fibrotic activities.The synthetic peptide AAQFFNR demonstrated complement inhibitory activity,with an inhibitory complement hemolytic 50%(ICH_(50))value of 24.54μmol·L^(-1),and exhibited superior protective effects in ADR-induced HK-2 cells.Surface plasmon resonance(SPR)assay revealed direct interaction between AAQFFNR and complement C3 with K_(d)value of 16.8μmol·L^(-1).The reno-protective effect of AAQFFNR was subsequently verified in ADR-induced mice.This research provides initial evidence that complement C3-inhibiting peptides from insects demonstrate potential in preventing nephropathy through in silico and in vivo validation approaches.展开更多
Diabetic retinopathy(DR)is a leading cause of vision loss among working-age populations,with early screening significantly reducing the risk of blindness.However,resource-limited regions often face challenges in DR sc...Diabetic retinopathy(DR)is a leading cause of vision loss among working-age populations,with early screening significantly reducing the risk of blindness.However,resource-limited regions often face challenges in DR screening due to a shortage of ophthalmologists.This study reports the implementation and outcomes of the Chinese local standard DB52/T 1726-2023,Regulations for the application of diabetic retinopathy screening artificial intelligence,in Cambodian healthcare institutions.A pilot DR screening program with independent operational capability is established by providing a non-mydriatic fundus camera and deploying a localized diabetic retinopathy artificial intelligence(DR-AI)screening platform at the Cambodia-Kingdom Friendship Hospital in Phnom Penh,along with comprehensive training.From January to August 2025,a total of 565 patients with type 2 diabetes were screened,yielding a DR detection rate of 26.0%(147 cases).Research findings demonstrate that applying mature Chinese DR-AI screening standards and technological solutions through international collaboration in regions with a scarcity of ophthalmic professionals is both feasible and effective.This project serves as a reference for promoting DR-AI in resource-constrained countries and regions,highlighting its significant potential to leverage AI in addressing the global burden of chronic diseases and advancing the modernization of health systems.展开更多
[Objective] The study aimed to obtain attenuated strain of Haemophilus parasuis.[Method] Tn5 transposon technology was used to construct a library of mutants.Positive mutants were screened by kanamycin resistance.Fals...[Objective] The study aimed to obtain attenuated strain of Haemophilus parasuis.[Method] Tn5 transposon technology was used to construct a library of mutants.Positive mutants were screened by kanamycin resistance.False positive was identified by PCR and then removed.Mice were infected to detect the virulence of mutants.The bionomics of attenuated strains were detected,too.[Result] The attenuated mutants showed similar reproductive activity to that of wild strain.The virulence of mutants was still stable after 30 passages.[Conclusion] This study provided foundation for exploring the virulence factors and pathogenic mechanism of HPS.展开更多
The maize mutant gene Vestigial glume 1(Vg1) has been fine-mapped to a narrow region by map-based cloning and the candidate gene for Vg1 spanned 19.5 kb. Here we report Vg1 genomic fosmid library construction and scre...The maize mutant gene Vestigial glume 1(Vg1) has been fine-mapped to a narrow region by map-based cloning and the candidate gene for Vg1 spanned 19.5 kb. Here we report Vg1 genomic fosmid library construction and screening. The fosmid library of Vg1 consisted of574,000 clones with an average insert size of 36.4 kb, representing 7.9-fold coverage of the maize genome. Fosmid stability assays indicated that clones were stable during propagation in the fosmid system. Using Vg1 candidate gene-specific primers, a positive clone was successfully identified. This discovery will pave the way for identifying the function of Vg1 in maize development.展开更多
The simplicity of the CRISPR/Cas9 system of genome engineering has opened up the possibility of performing genome-wide targeted mutagenesis in cell lines,enabling screening for cellular phenotypes resulting from genet...The simplicity of the CRISPR/Cas9 system of genome engineering has opened up the possibility of performing genome-wide targeted mutagenesis in cell lines,enabling screening for cellular phenotypes resulting from genetic aberrations.Drosophila cells have proven to be highly effective in identifying genes involved in cellular processes through similar screens using partial knockdown by RNAi.This is in part due to the lower degree of redundancy between genes in this organism,whilst still maintaining highly conserved gene networks and orthologs of many human disease-causing genes.The ability of CRISPR to generate genetic loss of function mutations not only increases the magnitude of any effect over currently employed RNAi techniques,but allows analysis over longer periods of time which can be critical for certain phenotypes.In this study,we have designed and built a genome-wide CRISPR library covering 13,501 genes,among which 8989 genes are targeted by three or more independent single guide RNAs（sg RNAs）.Moreover,we describe strategies to monitor the population of guide RNAs by high throughput sequencing（HTS）.We hope that this library will provide an invaluable resource for the community to screen loss of function mutations for cellular phenotypes,and as a source of guide RNA designs for future studies.展开更多
Gayal is a rare semi-wild bovine species found in the Indo-China. They can graze grasses, including bamboo leaves, as well as reeds and other plant species, and grow to higher mature live weights than Yunnan Yellow ca...Gayal is a rare semi-wild bovine species found in the Indo-China. They can graze grasses, including bamboo leaves, as well as reeds and other plant species, and grow to higher mature live weights than Yunnan Yellow cattle maintained in similar harsh environments. The aim of this study was to identify specific cellulase in the gayal rumen. A metagenomic fosmid library was constructed using genomic DNA isolated from the ruminal contents of four adult gayals. This library contained 38400 clones with an average insert size of 35.5 kb. The Umcel-1 gene was isolated from this library. Investigation of the cellulase activity of 24 random clones led to the identification of the Umcel-1 gene, which exhibited the most potent cellulase activity. Sequencing the Umcel-1 gene revealed that it contained an open reading frame of 942 base pairs that encoded a product of 313 amino acids. The putative gene Umcel-1 product belonged to the glycosyl hydrolase family 5 and showed the highest homology to the cellulase （GenBank accession no. YP_004310852.1 ） from Clostridium lentocellum DSM 5427, with 44% identity and 62% similarity. The Umcel-1 gene was heterologously expressed in Escherichia coil BL21, and recombinant Umcel-1 was purified. The activity of purified recombinant Umcel-1 was assessed, and the results revealed that it hydrolyzed carboxymethyl cellulose with optimal activity at pH 5.5 and 45~C. To our knowledge, this study provides the first evidence for a cellulase produced by bacteria in gayal rumen.展开更多
The present study reports an improved PCR-based technique that allows quick and effective screening of cDNA libraries. First, the cDNA library was arrayed as follow: about 3 X 10’ cDNA clones were multiplied as indiv...The present study reports an improved PCR-based technique that allows quick and effective screening of cDNA libraries. First, the cDNA library was arrayed as follow: about 3 X 10’ cDNA clones were multiplied as individual plaques on solid medium in 24-well culture dishes at 1 200 plaque forming units per well. The phage suspension of each well was transferred to an individual microcentrifuge tube in 72-tube box. Then, box pool, row pools and column pools were set up that respectively represent a 72-tube box, rows and columns within the box. To screen a specific target cDNA,primers specific for novel ESTs ob- tained in our laboratory were employed to conduct PCR in a hierarchy mode. PCR began with the box pools, resulting in the identification of some Positive box pools. Then PCR went down to the row and col- umn pools of the positive box. The intersection of the positive row (s) and column (s) revealed the candi- date positive tubes. The specificity of PCR products were meanwhile checked by restriction enzyme diges- tion. Finally, hybridization was carried out to get single specific cDNA clomes from the positive tubes. This PCR-based technique features high specificity, high efficiency and less-cost in large-scale cDNA library screening. Our initial implementation of the technique resulted in the isolation of three longer different cD- NA clones from a human fetal brain cDNA library. Thus this improved technique can serve as an alterna-tive to the time-consuming and laborious conventional hybridization-based method for screening cDNA li-brary.展开更多
To study the bioactive metabolites produced by sponge-derived uncultured symbionts, a metagenomic DNA library of the symbionts of sponge Gelliodes gracilis was constructed. The average size of DNA inserts in the libra...To study the bioactive metabolites produced by sponge-derived uncultured symbionts, a metagenomic DNA library of the symbionts of sponge Gelliodes gracilis was constructed. The average size of DNA inserts in the library was 20 kb. This library was screened for antibiotic activity using paper disc assaying. Two clones displayed the antibacterial activity against Micrococcus tetragenus. The metabolites of these two clones were analyzed through HPLC. The result showed that their metabolites were quite different from those of the host E. coli DNA and the host containing vector pHZ132. This study may present a new approach to exploring bioactive metabolites of sponge symbionts.展开更多
The breeding of herbicide-resistant wheat varieties has helped control weeds in wheat fields economically and effectively.Imidazolinone (IMI) herbicides are popular as they have low toxicity in mammals,are effective a...The breeding of herbicide-resistant wheat varieties has helped control weeds in wheat fields economically and effectively.Imidazolinone (IMI) herbicides are popular as they have low toxicity in mammals,are effective at small doses,and exhibit broad-spectrum herbicidal action in the field.Therefore,the isolation and genetic and molecular characterization of IMI-resistant wheat mutants will enhance weed management in wheat fields.In the present study,352 IMI-resistant plants were isolated by genetic screening from a mutant pool prepared by EMS-based random mutagenesis.Cloning of the mutated genes from the IMI-resistant plants indicated that ten taals alleles had been isolated,and mutation in one of three Ta ALS homolog genes conferred IMI resistance,and such a mutation is a dominant trait.Further analysis showed that taals-d exhibited the greatest IMI resistance,whereas taals-b exhibited the weakest resistance to IMI among three homologous taals mutants.In terms of IMI resistance,the taals triple mutant was stronger than the taals double mutants,and the taals double mutants were stronger than the single mutants,indicating a dose-dependent effect of the Ta ALS mutation on IMI resistance in wheat.Biochemical analysis indicated that the mutation in Ta ALS increased the tolerance of Ta ALS to inhibition by IMI.Our work details the genetic and molecular characterization of als wheat mutants,provides a foundation for understanding IMI resistance and breeding wheat varieties with herbicide resistance,and indicates that genetic screening using a mutagenized pool is an effective and important means of breeding crops with additional desired agricultural traits.展开更多
The regulation of protein stability is a fundamental issue for biophysical processes,but there has not previously been a convenient and unbiased method of identifying regulators of protein stability.However,as reporte...The regulation of protein stability is a fundamental issue for biophysical processes,but there has not previously been a convenient and unbiased method of identifying regulators of protein stability.However,as reported in the article entitled "A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25 A," recently published in Cell Discovery,our team developed a protein stability regulators screening assay(Pro-SRSA) by combining the whole-genome clustered regularly interspaced short palindromic repeats Cas9(CRISPR-Cas9) library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability.Based on our findings,we are confident that this efficient and unbiased screening method at the genome scale will be used by researchers worldwide to identify regulators of protein stability.展开更多
When devoured by macrophages,Mycobacterium tuberculosis remains persistent in macrophages and gains energy through the glyoxylate bypass to maintain its long-term existence in host cells.Therefore it is possible to st...When devoured by macrophages,Mycobacterium tuberculosis remains persistent in macrophages and gains energy through the glyoxylate bypass to maintain its long-term existence in host cells.Therefore it is possible to stop persistent infections by interdicting the glyoxylate bypass in which the isocitrate lyase（ICL） is the key rate-limiting enzyme and a persistence factor.ICL is the target of anti-TB（TB：tubercular） drugs,which could screen ICL out and effectively inhibit the activity of ICL in Mycobacterium tuberculosis,and because of this,anti-TB drugs can be used to kill persistent Mycobacterium tuberculosis.In this study,the ICL gene of the Mycobacterium tuberculosis H37Rv was cloned successfully and recombinant protein with bioactivity was obtained through the enzyme characteristic appraisal.The specific activity of the recombined ICL is 24μmol·mg-1·min-1.The recombined ICL protein was used as the target,and phages which can specifically combine to ICL were screened in the phage 7 peptide library.According to the results of the ELISA and DNA sequence detection,eventually three 7-peptide chains were synthesized.Then the peptide chains were reacted with ICL,respectively,to detect their inhibitory effects on ICL.The results show that all the three 7-peptide chains possessed varying inhibitory effects on the activity of ICL.This study provided lead compounds for the research and development of new peptide anti-TB drugs.展开更多
Objective:Osteosarcoma is the most common primary malignant bone tumor.However,the survival of patients with osteosarcoma has remained unchanged during the past 30 years,owing to a lack of efficient therapeutic target...Objective:Osteosarcoma is the most common primary malignant bone tumor.However,the survival of patients with osteosarcoma has remained unchanged during the past 30 years,owing to a lack of efficient therapeutic targets.Methods:We constructed a kinome-targeting CRISPR-Cas9 library containing 507 kinases and 100 nontargeting controls and screened the potential kinase targets in osteosarcoma.The CRISPR screening sequencing data were analyzed with the Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout(MAGeCK)Python package.The functional data were applied in the 143B cell line through lenti-CRISPR-mediated gene knockout.The clinical significance of kinases in the survival of patients with osteosarcoma was analyzed in the R2:Genomics Analysis and Visualization Platform.Results:We identified 53 potential kinase targets in osteosarcoma.Among these targets,we analyzed 3 kinases,TRRAP,PKMYT1,and TP53RK,to validate their oncogenic functions in osteosarcoma.PKMYT1 and TP53RK showed higher expression in osteosarcoma than in normal bone tissue,whereas TRRAP showed no significant difference.High expression of all 3 kinases was associated with relatively poor prognosis in patients with osteosarcoma.Conclusions:Our results not only offer potential therapeutic kinase targets in osteosarcoma but also provide a paradigm for functional genetic screening by using a CRISPR-Cas9 library,including target design,library construction,screening workflow,data analysis,and functional validation.This method may also be useful in potentially accelerating drug discovery for other cancer types.展开更多
The purpose of this study was to screen peptides that can specifically bind to human hepatocellular carcinoma （hHCC） cells using phage display of random peptide library in order to develope a peptide-based carrier f...The purpose of this study was to screen peptides that can specifically bind to human hepatocellular carcinoma （hHCC） cells using phage display of random peptide library in order to develope a peptide-based carrier for the diagnosis or therapy of hHCC. A peptide 12-mer phage display library was employed and 4 rounds of subtractive panning were performed using the hHCC cell line HepG2 as the target. After panning, the phages that specifically bound to and internalized in hHCC cells were selected. The selected phages demonstrated highly specific affinity to HepG2 cells analyzed by ELISA and immunofluorescence analysis. 57.3% of the selected phage clones displayed repeated sequence FLLEPHLMDTSM, and 4 amino acid residues, FLEP were extremely conservative. Based on the sequencing results, a 16-mer peptide （WH-16） was synthesized. The competitive EL1SA showed that the binding of the phage clones displayed sequence FLLEPHLMDTSM to HepG2 cells was efficiently inhibited by WH-16. Our findings indicate that cellular binding of phage is mediated via its displayed peptide and the synthesized 16-mer peptide may have the potential to be a delivery carrier in target diagnosis or therapy for hHCC.展开更多
Objective: To clone the human counterpart of rat ZA73, EST cloned from rat tracheal epithelial (RTE) neoplastic transformed cell model induced by (a-particles radiation by using mRNA differential display. Methods: Acc...Objective: To clone the human counterpart of rat ZA73, EST cloned from rat tracheal epithelial (RTE) neoplastic transformed cell model induced by (a-particles radiation by using mRNA differential display. Methods: According to the sequence of rat ZA73, a probe was biotin-labeled to screen human cDNA library, and then the gene sequence was extended by RACE (rapid amplification of cDNA ends). Result: Human gene HRNT-1 (GenBank Accession Number: AF223393) is 4.256 kb in length, with an ORF located in the region between 254 and 3013 bp. 5′ UTS (untranslated sequences) is 253 bp, 3′ UTS is 1243 bp. Conclusion: The combination of cDNA library screening with biotin-labeled probes and RACE is an effective method to clone full-length cDNA, especially for sequences longer than 2 kb.展开更多
Microcystis species identification is essential for ecological studies and water bloom control.Immunoassays are more specific and convenient and several approaches have been used to develop for diagnosing harmful red ...Microcystis species identification is essential for ecological studies and water bloom control.Immunoassays are more specific and convenient and several approaches have been used to develop for diagnosing harmful red tide algae.Howeve r,inve stigations onMicrocystis identification using immunological approaches are still in the initial stage.In this study.Microcystis aeruginosa PCC7806 lysates were utilized as coated antigens to enrich and screen specific Microcystis nanobodies from a human domain antibody display library.After three rounds of enrichment,10 positive monoclonal particles were isolated from the library and the most two positive nanobodies(DAb2 and Dab3)were effectively produced in Escherichia coli BL21.Finally,the DAb2 showed specific immune binding to different Microcystis by the immuno-dot blot assay.This antibody could be used to establish an immunological method to identify Microcystis.展开更多
An antibody phage library was screened with two target molecules, IFNα-2a and FGFR-GST, and the KD value of each round of panning was measured. It was found that the apparent KD values decreased along with each addit...An antibody phage library was screened with two target molecules, IFNα-2a and FGFR-GST, and the KD value of each round of panning was measured. It was found that the apparent KD values decreased along with each additional panning round, which indicates the increase of the binding affinity between the phage and the target molecules. This result shows that the KD value is a reliable intrinsic parameter and a new method for screening efficiency detection is thus provided.展开更多
OBJECTIVE To utilize a structure-based lead optimization approach to generate novel natural product-like typeⅡ inhibitors of JAK2 using the DOLPHIN protocol.METHODS Initially,the DOLHPIN computational protocol was em...OBJECTIVE To utilize a structure-based lead optimization approach to generate novel natural product-like typeⅡ inhibitors of JAK2 using the DOLPHIN protocol.METHODS Initially,the DOLHPIN computational protocol was employed to convert an active(DFG-in)conformation of JAK2 into a typeⅡ-compatible conformation,which was used as a model for the structure-based virtual screening of 150 000 natural product-like compounds in silico.The novel biflavonoid analogues were designed and synthesized based on the structure of lead compound and then tested for JAK2 and STAT3 inhibitory activity,cytotoxicity and HCV antiviral activity.RESULTS The top eleven highest-scoring compounds were generated from the initial high-throughput virtual screening.Amentoflavone 1a,a biflavonoid from the Chinese plant Gingko biloba,emerged as a promising candidate for further biological evaluation.In dose-response experiments,amentoflavone 1ainhibited JAK2 activity in a concentration dependent fashion with an IC50 value of 5μmol·L-1.As a proof-of-concept,we designed nine analogues 1b-1j with the addition of one or more aliphatic side chains to the biflavonoid scaffold of 1a.The octyl(C8)analogue 1bdisplayed superior potency against JAK2 activity and HCV activity compared to the parent compound 1a,validating the structure-based lead optimization approach used in this study.Moreover,kinetic analysis indicated that analogue 1bexhibited a non-competitive mode of inhibition,suggesting that this compound may be a putative typeⅡ inhibitor of JAK2.CONCLUSION Amentoflavone 1ahas been identified as a JAK2 inhibitor by structure-based virtual screening of a natural product library.In silico optimization using the DOLPHIN model yielded analogues with enhanced potency against JAK2 activity and HCV activity in cellulo.Molecular modeling and kinetic experiments suggested that the analogues may function as typeⅡ inhibitors of JAK2.展开更多
The plant mitochondrial genome displays complex features, particularly in terms of cytoplasmic male sterility （CMS）. Therefore, research on the cotton mitochondrial genome may provide important information for analy...The plant mitochondrial genome displays complex features, particularly in terms of cytoplasmic male sterility （CMS）. Therefore, research on the cotton mitochondrial genome may provide important information for analyzing genome evolution and exploring the molecular mechanism of CMS. In this paper, we present a preliminary study on the mitochondrial genome of sea island cotton （Gossypium barbadense） based on positive clones from the bacterial artificial chromosome （BAC） library. Thirty-five primers designed with the conserved sequences of functional genes and exons of mitochondria were used to screen positive clones in the genome library of the sea island cotton variety called Pima 90-53. Ten BAC clones were obtained and verified for further study. A contig was obtained based on six overlapping clones and subsequently laid out primarily on the mitochondrial genome. One BAC clone, clone 6 harbored with the inserter of approximate 115 kb mtDNA sequence, in which more than 10 primers fragments could be amplified, was sequenced and assembled using the Solexa strategy. Fifteen mitochondrial functional genes were revealed in clone 6 by gene annotation. The characteristics of the syntenic gene/exon of the sequences and RNA editing were preliminarily predicted.展开更多
基金Supported by the Science and Technology Innovation Project of Laoshan Laboratory(Nos.2022QNLM030004-3,LSKJ202203103)the NSFC Innovative Group Grant(No.42221005)+5 种基金the Key Collaborative Research Program of the Alliance of International Science Organizations(No.ANSO-CR-KP-2022-08)the Shandong Provincial Natural Science Foundation(No.ZR2021ZD28)the Major Research Plan of the National Natural Science Foundation(No.92351301)the Strategic Priority Research Program of the Chinese Academy of Sciences(No.XDA22050301)the Taishan Scholars Program(No.tstp20230637)the Qingdao Natural Science Foundation(No.23-2-1-182-zyyd-jch)。
文摘With the increasing accumulation of plastic pollutants in various environments,research on microorganisms(including bacteria,fungi,and algae)with plastic degradation capabilities has gained significant attention.However,only a limited number of microbial plastic-degrading enzymes have been identified to date.This highlights that the degradation mechanisms employed by many plastic-degrading microorganisms,particularly filamentous fungi,remain insufficiently explored.In this study,we utilized a versatile fungal plasmid(pCT74)to express green fluorescent protein(GFP)in a marine-derived fungus Alternaria alternata strain FB1 with plastic degradation capabilities.Upon evaluating the degradation effect of polyester-type polyurethane(PU)film,we observed that different transformants exhibited three kinds of activities(the same,reduced,or enhanced degradation capability)compared to the FB1 wild-type strain.Further analysis of the plasmid fragment insertion sites in different transformants revealed that pCT74 integrates randomly into the genome of the host fungus.Notably,a direct correlation was found between the plasmid insertion site and the degradation capability of the corresponding transformant.Our findings not only redefine the potential applications of plasmid pCT74 in filamentous fungi but also show a novel research approach to identifying key enzymes involved in plastic degradation by fungi.
基金supported by the National Natural Science Foundation of China(No.82104353)China Postdoctoral Science Foundation funded project(No.2022M711680).
文摘Insects represent emerging sources of bioactive peptides and functional materials.Mantidis Oötheca(Sang-Piao-Xiao in Chinese,SPX)serves as an insect-derived medicine for treating kidney disease.This study demonstrated that supernatant(SPX)improved kidney function in adriamycin(ADR)-induced nephropathy mice model.Transcriptomic analysis revealed that SPX inhibited complement activation by targeting the MASP1-C3/C3a receptor(C3aR)pathway.Peptidomic analysis identified 304 peptides from SPX,with 49 peptides selected for evaluation using prediction tools and molecular docking with complement core protein C3.Three peptides(PMGFPFDR,FNDPK,AAQFFNR)exhibiting docking scores below-8.0 were synthesized to verify complement inhibition and anti-fibrotic activities.The synthetic peptide AAQFFNR demonstrated complement inhibitory activity,with an inhibitory complement hemolytic 50%(ICH_(50))value of 24.54μmol·L^(-1),and exhibited superior protective effects in ADR-induced HK-2 cells.Surface plasmon resonance(SPR)assay revealed direct interaction between AAQFFNR and complement C3 with K_(d)value of 16.8μmol·L^(-1).The reno-protective effect of AAQFFNR was subsequently verified in ADR-induced mice.This research provides initial evidence that complement C3-inhibiting peptides from insects demonstrate potential in preventing nephropathy through in silico and in vivo validation approaches.
基金funded by the Chronic Disease Management Research Project of National Health Commission Capacity Building and Continuing Education Center 2025(GWJJMB202510024146)the Post-Subsidy Project for Standard Development of Guizhou Provincial Market Supervision and Administration Bureau 2025(DB52/T1726-2023)the Guizhou Provincial Health Commission Science and Technology Fund Project(gzwkj2024-076,gzwkj2026-146).
文摘Diabetic retinopathy(DR)is a leading cause of vision loss among working-age populations,with early screening significantly reducing the risk of blindness.However,resource-limited regions often face challenges in DR screening due to a shortage of ophthalmologists.This study reports the implementation and outcomes of the Chinese local standard DB52/T 1726-2023,Regulations for the application of diabetic retinopathy screening artificial intelligence,in Cambodian healthcare institutions.A pilot DR screening program with independent operational capability is established by providing a non-mydriatic fundus camera and deploying a localized diabetic retinopathy artificial intelligence(DR-AI)screening platform at the Cambodia-Kingdom Friendship Hospital in Phnom Penh,along with comprehensive training.From January to August 2025,a total of 565 patients with type 2 diabetes were screened,yielding a DR detection rate of 26.0%(147 cases).Research findings demonstrate that applying mature Chinese DR-AI screening standards and technological solutions through international collaboration in regions with a scarcity of ophthalmic professionals is both feasible and effective.This project serves as a reference for promoting DR-AI in resource-constrained countries and regions,highlighting its significant potential to leverage AI in addressing the global burden of chronic diseases and advancing the modernization of health systems.
基金Supported by National High Technology Research and Development Program of China(2006AA10A206)National Natural Science Foundation of China(31001072)the Fund of Beijing Academy of Agriculture and Forestry Sciences for Young Scholars(QNJJ201012)~~
文摘[Objective] The study aimed to obtain attenuated strain of Haemophilus parasuis.[Method] Tn5 transposon technology was used to construct a library of mutants.Positive mutants were screened by kanamycin resistance.False positive was identified by PCR and then removed.Mice were infected to detect the virulence of mutants.The bionomics of attenuated strains were detected,too.[Result] The attenuated mutants showed similar reproductive activity to that of wild strain.The virulence of mutants was still stable after 30 passages.[Conclusion] This study provided foundation for exploring the virulence factors and pathogenic mechanism of HPS.
基金supported by a Chongqing Postdoctoral Science Foundation funded project (Xm201344)China Postdoctoral Science Foundation funded project (2014M552303)Fundamental Research Funds for the Central Universities (XDJK2013C023, 2362015xk05)
文摘The maize mutant gene Vestigial glume 1(Vg1) has been fine-mapped to a narrow region by map-based cloning and the candidate gene for Vg1 spanned 19.5 kb. Here we report Vg1 genomic fosmid library construction and screening. The fosmid library of Vg1 consisted of574,000 clones with an average insert size of 36.4 kb, representing 7.9-fold coverage of the maize genome. Fosmid stability assays indicated that clones were stable during propagation in the fosmid system. Using Vg1 candidate gene-specific primers, a positive clone was successfully identified. This discovery will pave the way for identifying the function of Vg1 in maize development.
基金the European Research Council(DARCGENs,project number 249869)the Medical Research Council for financial support
文摘The simplicity of the CRISPR/Cas9 system of genome engineering has opened up the possibility of performing genome-wide targeted mutagenesis in cell lines,enabling screening for cellular phenotypes resulting from genetic aberrations.Drosophila cells have proven to be highly effective in identifying genes involved in cellular processes through similar screens using partial knockdown by RNAi.This is in part due to the lower degree of redundancy between genes in this organism,whilst still maintaining highly conserved gene networks and orthologs of many human disease-causing genes.The ability of CRISPR to generate genetic loss of function mutations not only increases the magnitude of any effect over currently employed RNAi techniques,but allows analysis over longer periods of time which can be critical for certain phenotypes.In this study,we have designed and built a genome-wide CRISPR library covering 13,501 genes,among which 8989 genes are targeted by three or more independent single guide RNAs（sg RNAs）.Moreover,we describe strategies to monitor the population of guide RNAs by high throughput sequencing（HTS）.We hope that this library will provide an invaluable resource for the community to screen loss of function mutations for cellular phenotypes,and as a source of guide RNA designs for future studies.
基金supported by the National Natural Science Foundation of China (31160467, 31360562, 31160449 and 31260543)
文摘Gayal is a rare semi-wild bovine species found in the Indo-China. They can graze grasses, including bamboo leaves, as well as reeds and other plant species, and grow to higher mature live weights than Yunnan Yellow cattle maintained in similar harsh environments. The aim of this study was to identify specific cellulase in the gayal rumen. A metagenomic fosmid library was constructed using genomic DNA isolated from the ruminal contents of four adult gayals. This library contained 38400 clones with an average insert size of 35.5 kb. The Umcel-1 gene was isolated from this library. Investigation of the cellulase activity of 24 random clones led to the identification of the Umcel-1 gene, which exhibited the most potent cellulase activity. Sequencing the Umcel-1 gene revealed that it contained an open reading frame of 942 base pairs that encoded a product of 313 amino acids. The putative gene Umcel-1 product belonged to the glycosyl hydrolase family 5 and showed the highest homology to the cellulase （GenBank accession no. YP_004310852.1 ） from Clostridium lentocellum DSM 5427, with 44% identity and 62% similarity. The Umcel-1 gene was heterologously expressed in Escherichia coil BL21, and recombinant Umcel-1 was purified. The activity of purified recombinant Umcel-1 was assessed, and the results revealed that it hydrolyzed carboxymethyl cellulose with optimal activity at pH 5.5 and 45~C. To our knowledge, this study provides the first evidence for a cellulase produced by bacteria in gayal rumen.
基金National Natural Science Fund!(39392900 ) 863 High-tech Project Fund of China!(102-10-03-02 )
文摘The present study reports an improved PCR-based technique that allows quick and effective screening of cDNA libraries. First, the cDNA library was arrayed as follow: about 3 X 10’ cDNA clones were multiplied as individual plaques on solid medium in 24-well culture dishes at 1 200 plaque forming units per well. The phage suspension of each well was transferred to an individual microcentrifuge tube in 72-tube box. Then, box pool, row pools and column pools were set up that respectively represent a 72-tube box, rows and columns within the box. To screen a specific target cDNA,primers specific for novel ESTs ob- tained in our laboratory were employed to conduct PCR in a hierarchy mode. PCR began with the box pools, resulting in the identification of some Positive box pools. Then PCR went down to the row and col- umn pools of the positive box. The intersection of the positive row (s) and column (s) revealed the candi- date positive tubes. The specificity of PCR products were meanwhile checked by restriction enzyme diges- tion. Finally, hybridization was carried out to get single specific cDNA clomes from the positive tubes. This PCR-based technique features high specificity, high efficiency and less-cost in large-scale cDNA library screening. Our initial implementation of the technique resulted in the isolation of three longer different cD- NA clones from a human fetal brain cDNA library. Thus this improved technique can serve as an alterna-tive to the time-consuming and laborious conventional hybridization-based method for screening cDNA li-brary.
基金This work was supported by High Tech R&D Program of China(Grant Nos.2002AA628130 and 2003AA624020)the National Natural Science Foundation of China(30171102)+2 种基金the Fund for Cheung Kong Scholar from the Cheung Kong Scholar Program of Ministry of Education of Chinathe Fund from the Natural Science Foundation of Shandong Province(No.Z2001C01)the High Tech R&D Program of Shandong Province(No.0121100107).The authors would like to thank Professor Li Jinhe of Institute of 0ceanology,Chinese Academy of Sciences,for the identification of the sponge.
文摘To study the bioactive metabolites produced by sponge-derived uncultured symbionts, a metagenomic DNA library of the symbionts of sponge Gelliodes gracilis was constructed. The average size of DNA inserts in the library was 20 kb. This library was screened for antibiotic activity using paper disc assaying. Two clones displayed the antibacterial activity against Micrococcus tetragenus. The metabolites of these two clones were analyzed through HPLC. The result showed that their metabolites were quite different from those of the host E. coli DNA and the host containing vector pHZ132. This study may present a new approach to exploring bioactive metabolites of sponge symbionts.
基金financially supported by the National Key Research and Development Program of China (2017YFD0101001)Beijing Municipal Government Science Foundation (IDHT20170513)Peking University Institute of Advanced Agricultural Sciences。
文摘The breeding of herbicide-resistant wheat varieties has helped control weeds in wheat fields economically and effectively.Imidazolinone (IMI) herbicides are popular as they have low toxicity in mammals,are effective at small doses,and exhibit broad-spectrum herbicidal action in the field.Therefore,the isolation and genetic and molecular characterization of IMI-resistant wheat mutants will enhance weed management in wheat fields.In the present study,352 IMI-resistant plants were isolated by genetic screening from a mutant pool prepared by EMS-based random mutagenesis.Cloning of the mutated genes from the IMI-resistant plants indicated that ten taals alleles had been isolated,and mutation in one of three Ta ALS homolog genes conferred IMI resistance,and such a mutation is a dominant trait.Further analysis showed that taals-d exhibited the greatest IMI resistance,whereas taals-b exhibited the weakest resistance to IMI among three homologous taals mutants.In terms of IMI resistance,the taals triple mutant was stronger than the taals double mutants,and the taals double mutants were stronger than the single mutants,indicating a dose-dependent effect of the Ta ALS mutation on IMI resistance in wheat.Biochemical analysis indicated that the mutation in Ta ALS increased the tolerance of Ta ALS to inhibition by IMI.Our work details the genetic and molecular characterization of als wheat mutants,provides a foundation for understanding IMI resistance and breeding wheat varieties with herbicide resistance,and indicates that genetic screening using a mutagenized pool is an effective and important means of breeding crops with additional desired agricultural traits.
基金supported by grants from the Key Project of Guangzhou(No.1561000151)the Yangtze River Scholarship(No.85000-52121100)+1 种基金the National Nature Science Foundation in China(No.81530081,31571395)the 973 project(No.2012CB967000)
文摘The regulation of protein stability is a fundamental issue for biophysical processes,but there has not previously been a convenient and unbiased method of identifying regulators of protein stability.However,as reported in the article entitled "A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25 A," recently published in Cell Discovery,our team developed a protein stability regulators screening assay(Pro-SRSA) by combining the whole-genome clustered regularly interspaced short palindromic repeats Cas9(CRISPR-Cas9) library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability.Based on our findings,we are confident that this efficient and unbiased screening method at the genome scale will be used by researchers worldwide to identify regulators of protein stability.
基金Supported by the Technology Development Funds of Education Department of Jilin Province,China(No.2008110)
文摘When devoured by macrophages,Mycobacterium tuberculosis remains persistent in macrophages and gains energy through the glyoxylate bypass to maintain its long-term existence in host cells.Therefore it is possible to stop persistent infections by interdicting the glyoxylate bypass in which the isocitrate lyase（ICL） is the key rate-limiting enzyme and a persistence factor.ICL is the target of anti-TB（TB：tubercular） drugs,which could screen ICL out and effectively inhibit the activity of ICL in Mycobacterium tuberculosis,and because of this,anti-TB drugs can be used to kill persistent Mycobacterium tuberculosis.In this study,the ICL gene of the Mycobacterium tuberculosis H37Rv was cloned successfully and recombinant protein with bioactivity was obtained through the enzyme characteristic appraisal.The specific activity of the recombined ICL is 24μmol·mg-1·min-1.The recombined ICL protein was used as the target,and phages which can specifically combine to ICL were screened in the phage 7 peptide library.According to the results of the ELISA and DNA sequence detection,eventually three 7-peptide chains were synthesized.Then the peptide chains were reacted with ICL,respectively,to detect their inhibitory effects on ICL.The results show that all the three 7-peptide chains possessed varying inhibitory effects on the activity of ICL.This study provided lead compounds for the research and development of new peptide anti-TB drugs.
基金This work was funded by the National Key Research and Development Program of China(Grant No.2016YFA0500304to T.K.)the Science and Technology Program of Guangzhou,(Grant Nos.202002020092 and 201607020038 to T.K.)+2 种基金the National Nature Science Foundation in China(NSFC)(Grant Nos.81772922 to Y.W.,81702890 to X.W.,81530081,31571395 to T.K.)the Guangdong Natural Science Foundation Team Project(Grant No.2014A030312015 to T.K.)the Natural Science Foundation of Guangdong Province(Grant No.2016A030310218 to W.Y.).
文摘Objective:Osteosarcoma is the most common primary malignant bone tumor.However,the survival of patients with osteosarcoma has remained unchanged during the past 30 years,owing to a lack of efficient therapeutic targets.Methods:We constructed a kinome-targeting CRISPR-Cas9 library containing 507 kinases and 100 nontargeting controls and screened the potential kinase targets in osteosarcoma.The CRISPR screening sequencing data were analyzed with the Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout(MAGeCK)Python package.The functional data were applied in the 143B cell line through lenti-CRISPR-mediated gene knockout.The clinical significance of kinases in the survival of patients with osteosarcoma was analyzed in the R2:Genomics Analysis and Visualization Platform.Results:We identified 53 potential kinase targets in osteosarcoma.Among these targets,we analyzed 3 kinases,TRRAP,PKMYT1,and TP53RK,to validate their oncogenic functions in osteosarcoma.PKMYT1 and TP53RK showed higher expression in osteosarcoma than in normal bone tissue,whereas TRRAP showed no significant difference.High expression of all 3 kinases was associated with relatively poor prognosis in patients with osteosarcoma.Conclusions:Our results not only offer potential therapeutic kinase targets in osteosarcoma but also provide a paradigm for functional genetic screening by using a CRISPR-Cas9 library,including target design,library construction,screening workflow,data analysis,and functional validation.This method may also be useful in potentially accelerating drug discovery for other cancer types.
基金a grant from National Natural Science Foundation of China(N0.30171062)
文摘The purpose of this study was to screen peptides that can specifically bind to human hepatocellular carcinoma （hHCC） cells using phage display of random peptide library in order to develope a peptide-based carrier for the diagnosis or therapy of hHCC. A peptide 12-mer phage display library was employed and 4 rounds of subtractive panning were performed using the hHCC cell line HepG2 as the target. After panning, the phages that specifically bound to and internalized in hHCC cells were selected. The selected phages demonstrated highly specific affinity to HepG2 cells analyzed by ELISA and immunofluorescence analysis. 57.3% of the selected phage clones displayed repeated sequence FLLEPHLMDTSM, and 4 amino acid residues, FLEP were extremely conservative. Based on the sequencing results, a 16-mer peptide （WH-16） was synthesized. The competitive EL1SA showed that the binding of the phage clones displayed sequence FLLEPHLMDTSM to HepG2 cells was efficiently inhibited by WH-16. Our findings indicate that cellular binding of phage is mediated via its displayed peptide and the synthesized 16-mer peptide may have the potential to be a delivery carrier in target diagnosis or therapy for hHCC.
基金China States Key Basic Research Program Grant (No. G1998051208) and National Natural Science Foundation of China (No. 39990600).
文摘Objective: To clone the human counterpart of rat ZA73, EST cloned from rat tracheal epithelial (RTE) neoplastic transformed cell model induced by (a-particles radiation by using mRNA differential display. Methods: According to the sequence of rat ZA73, a probe was biotin-labeled to screen human cDNA library, and then the gene sequence was extended by RACE (rapid amplification of cDNA ends). Result: Human gene HRNT-1 (GenBank Accession Number: AF223393) is 4.256 kb in length, with an ORF located in the region between 254 and 3013 bp. 5′ UTS (untranslated sequences) is 253 bp, 3′ UTS is 1243 bp. Conclusion: The combination of cDNA library screening with biotin-labeled probes and RACE is an effective method to clone full-length cDNA, especially for sequences longer than 2 kb.
基金Supported by the National Natural Science Foundation of China(Nos.31971561,31370217,31701724)the Research Program of Jiangsu Province of China。
文摘Microcystis species identification is essential for ecological studies and water bloom control.Immunoassays are more specific and convenient and several approaches have been used to develop for diagnosing harmful red tide algae.Howeve r,inve stigations onMicrocystis identification using immunological approaches are still in the initial stage.In this study.Microcystis aeruginosa PCC7806 lysates were utilized as coated antigens to enrich and screen specific Microcystis nanobodies from a human domain antibody display library.After three rounds of enrichment,10 positive monoclonal particles were isolated from the library and the most two positive nanobodies(DAb2 and Dab3)were effectively produced in Escherichia coli BL21.Finally,the DAb2 showed specific immune binding to different Microcystis by the immuno-dot blot assay.This antibody could be used to establish an immunological method to identify Microcystis.
文摘An antibody phage library was screened with two target molecules, IFNα-2a and FGFR-GST, and the KD value of each round of panning was measured. It was found that the apparent KD values decreased along with each additional panning round, which indicates the increase of the binding affinity between the phage and the target molecules. This result shows that the KD value is a reliable intrinsic parameter and a new method for screening efficiency detection is thus provided.
基金The project upported by Hong Kong Baptist University(FRG2/12-13/021and FRG2/13-14/008)Centre for Cancer and Inflammation Research,School of Chinese Medicine(CCIR-SCM,HKBU)+4 种基金the Health and Medical Research Fund(HMRF/11101212and HMRF/13121482)the Research Grants Council(HKBU/201811,HKBU/204612and HKBU/201913)the French National Research Agency/Research Grants Council Joint Research Scheme(A-HKBU201/12)the State Key Laboratory of Synthetic Chemistry,the Science and Technology Development Fund,Macao SAR(103/2012/A3)the University of Macao〔MYRG091(Y3-L2)-ICMS12-LCH,MYRG121(Y3-L2)-ICMS12-LCH,MRG007/LCH/2014/ICMS and MRG023/LCH/2013/ICMS〕
文摘OBJECTIVE To utilize a structure-based lead optimization approach to generate novel natural product-like typeⅡ inhibitors of JAK2 using the DOLPHIN protocol.METHODS Initially,the DOLHPIN computational protocol was employed to convert an active(DFG-in)conformation of JAK2 into a typeⅡ-compatible conformation,which was used as a model for the structure-based virtual screening of 150 000 natural product-like compounds in silico.The novel biflavonoid analogues were designed and synthesized based on the structure of lead compound and then tested for JAK2 and STAT3 inhibitory activity,cytotoxicity and HCV antiviral activity.RESULTS The top eleven highest-scoring compounds were generated from the initial high-throughput virtual screening.Amentoflavone 1a,a biflavonoid from the Chinese plant Gingko biloba,emerged as a promising candidate for further biological evaluation.In dose-response experiments,amentoflavone 1ainhibited JAK2 activity in a concentration dependent fashion with an IC50 value of 5μmol·L-1.As a proof-of-concept,we designed nine analogues 1b-1j with the addition of one or more aliphatic side chains to the biflavonoid scaffold of 1a.The octyl(C8)analogue 1bdisplayed superior potency against JAK2 activity and HCV activity compared to the parent compound 1a,validating the structure-based lead optimization approach used in this study.Moreover,kinetic analysis indicated that analogue 1bexhibited a non-competitive mode of inhibition,suggesting that this compound may be a putative typeⅡ inhibitor of JAK2.CONCLUSION Amentoflavone 1ahas been identified as a JAK2 inhibitor by structure-based virtual screening of a natural product library.In silico optimization using the DOLPHIN model yielded analogues with enhanced potency against JAK2 activity and HCV activity in cellulo.Molecular modeling and kinetic experiments suggested that the analogues may function as typeⅡ inhibitors of JAK2.
基金supported by the Key Project Founded by Ministry of Education, China (107012)the National Natural Science Foundation of China (31171591)
文摘The plant mitochondrial genome displays complex features, particularly in terms of cytoplasmic male sterility （CMS）. Therefore, research on the cotton mitochondrial genome may provide important information for analyzing genome evolution and exploring the molecular mechanism of CMS. In this paper, we present a preliminary study on the mitochondrial genome of sea island cotton （Gossypium barbadense） based on positive clones from the bacterial artificial chromosome （BAC） library. Thirty-five primers designed with the conserved sequences of functional genes and exons of mitochondria were used to screen positive clones in the genome library of the sea island cotton variety called Pima 90-53. Ten BAC clones were obtained and verified for further study. A contig was obtained based on six overlapping clones and subsequently laid out primarily on the mitochondrial genome. One BAC clone, clone 6 harbored with the inserter of approximate 115 kb mtDNA sequence, in which more than 10 primers fragments could be amplified, was sequenced and assembled using the Solexa strategy. Fifteen mitochondrial functional genes were revealed in clone 6 by gene annotation. The characteristics of the syntenic gene/exon of the sequences and RNA editing were preliminarily predicted.
