β-Conglycinin is one of the major allergens existed in soybean.N-Glycans attached to theβ-conglycinin influenced the immunoreactivity and antigen presenting efficiency ofβ-conglycinin.In this study,we described a n...β-Conglycinin is one of the major allergens existed in soybean.N-Glycans attached to theβ-conglycinin influenced the immunoreactivity and antigen presenting efficiency ofβ-conglycinin.In this study,we described a new method used to release and collect the N-glycans fromβ-conglycinin,and the N-glycans existed in linear epitopes ofβ-conglycinin were identified.Glycopeptides hydrolyzed fromβ-conglycinin were purified by cotton hydrophilic chromatography.Trifluoromethylsulfonic acid was then used to release glycans from glycopeptides,and new glycopeptides containing one single N-acety1-D-glucosamine(G1 cNAc)moiety were then utilized for mass spectrometry.Five glycosylation sites(Asn-199,Asn-455,Asn-215,Asn-489 and Asn-326)and 22 kinds of glycopeptides were identified.It is noteworthy that the peptide VVN^(#)ATSNL(where^(#)represents for the glycosylation site)was analyzed to be both glycopeptide and linear epitope.Our results provided a new method for the N-glycoform analysis of food allergens,and laid a foundation for understanding the relationship between glyco sylation and food allergy.展开更多
Cell-free expression systems have emerged as a versatile and powerful platform for metabolic engineering,biosynthesis and synthetic biology studies.Nevertheless,successful examples of the synthesis of complex natural ...Cell-free expression systems have emerged as a versatile and powerful platform for metabolic engineering,biosynthesis and synthetic biology studies.Nevertheless,successful examples of the synthesis of complex natural products using this system are still limited.Bicyclomycin,a structurally unique and complex diketopiperazine alkaloid,is a clinically promising antibiotic that selectively inhibits the transcription termination factor Rho.Here,we established a modular cell-free expression system with cascade catalysis for the biosynthesis of bicyclomycin from a chemically synthesized cyclodipeptide.The six cell-free expressed biosynthetic enzymes,including five iron-andα-ketoglutarate-dependent dioxygenases and one cytochrome P450 monooxygenase,were active in converting their substrates to the corresponding products.The co-expressed enzymes in the cell-free module were able to complete the related partial pathway.In vitro biosynthesis of bicyclomycin was also achieved by reconstituting the entire biosynthetic pathways(i.e.,six enzymes)using the modular cell-free expression system.This study demonstrates that the modular cell-free expression system can be used as a robust and promising platformforthe biosynthesis of complex antibiotics.展开更多
Ovarian cancer(OC)is one of the most lethal malignancies of the female reproduc-tive system.OC patients are usually diagnosed at advanced stages due to the lack of early diag-nosis.The standard treatment for OC includ...Ovarian cancer(OC)is one of the most lethal malignancies of the female reproduc-tive system.OC patients are usually diagnosed at advanced stages due to the lack of early diag-nosis.The standard treatment for OC includes a combination of debulking surgery and platinum-taxane chemotherapy,while several targeted therapies have recently been approved for maintenance treatment.The vast majority of OC patients relapse with chemoresistant tu-mors after an initial response.Thus,there is an unmet clinical need to develop new therapeu-tic agents to overcome the chemoresistance of OC.The anti-parasite agent niclosamide(NA)has been repurposed as an anti-cancer agent and exerts potent anti-cancer activities in human cancers including OC.Here,we investigated whether NA could be repurposed as a therapeutic agent to overcome cisplatin-resistant(CR)in human OC cells.To this end,we first established two CR lines SKOV3CR and OVCAR8CR that exhibit the essential biological characteristics of cisplatin resistance in human cancer.We showed that NA inhibited cell proliferation,sup-pressed cell migration,and induced cell apoptosis in both CR lines at a low micromole range.Mechanistically,NA inhibited multiple cancer-related pathways including AP1,ELK/SRF,HIF1,and TCF/LEF,in SKOV3CR and OVCAR8CR cells.NA was further shown to effectively inhibit xenograft tumor growth of SKOV3CR cells.Collectively,our findings strongly suggest that NA may be repurposed as an efficacious agent to combat cisplatin resistance in chemoresistant hu-man OC,and further clinical trials are highly warranted.展开更多
The authors regret that an image assembly(copy/paste)error in Figure 3D,in which the image for the organoid of"Primary MiCs"group was erroneously duplicated with an image of primary MICs that was previously ...The authors regret that an image assembly(copy/paste)error in Figure 3D,in which the image for the organoid of"Primary MiCs"group was erroneously duplicated with an image of primary MICs that was previously published.The corrected figure is shown below.As shown in the corrected Figure 3D,this error does not adversely impact the conclusion of the original work.The authors would like to apologise forany inconvenience caused.展开更多
The treatment of cancer mainly involves surgical excision supplemented by radiotherapy and chemotherapy.Chemotherapy drugs act by interfering with tumor growth and inducing the death of cancer cells.Anti-tumor drugs w...The treatment of cancer mainly involves surgical excision supplemented by radiotherapy and chemotherapy.Chemotherapy drugs act by interfering with tumor growth and inducing the death of cancer cells.Anti-tumor drugs were developed to induce apoptosis,but some patient’s show apoptosis escape and chemotherapy resistance.Therefore,other forms of cell death that can overcome the resistance of tumor cells are important in the context of cancer treatment.Ferroptosis is a newly discovered iron-dependent,non-apoptotic type of cell death that is highly negatively correlated with cancer development.Ferroptosis is mainly caused by the abnormal increase in iron-dependent lipid reactive oxygen species and the imbalance of redox homeostasis.This review summarizes the progression and regulatory mechanism of ferroptosis in cancer and discusses its possible clinical applications in cancer diagnosis and treatment.展开更多
Notch is a cellecell signaling pathway that is involved in a host of activities including development,oncogenesis,skeletal homeostasis,and much more.More specifically,recent research has demonstrated the importance of...Notch is a cellecell signaling pathway that is involved in a host of activities including development,oncogenesis,skeletal homeostasis,and much more.More specifically,recent research has demonstrated the importance of Notch signaling in osteogenic differentiation,bone healing,and in the development of the skeleton.The craniofacial skeleton is complex and understanding its development has remained an important focus in biology.In this review we briefly summarize what recent research has revealed about Notch signaling and the current understanding of how the skeleton,skull,and face develop.We then discuss the crucial role that Notch plays in both craniofacial development and the skeletal system,and what importance it may play in the future.展开更多
As multipotent progenitor cells,mesenchymal stem cells(MSCs)can renew themselves and give rise to multiple lineages including osteoblastic,chondrogenic and adipogenic lineages.It’s previously shown that BMP9 is the m...As multipotent progenitor cells,mesenchymal stem cells(MSCs)can renew themselves and give rise to multiple lineages including osteoblastic,chondrogenic and adipogenic lineages.It’s previously shown that BMP9 is the most potent BMP and induces osteogenic and adipogenic differentiation of MSCs.However,the molecular mechanism through which BMP9 regulates MSC differentiation remains poorly understood.Emerging evidence indicates that noncoding RNAs,especially microRNAs,may play important roles in regulating MSC differentiation and bone formation.As highly conserved RNA binding proteins,Argonaute(AGO)proteins are essential components of the multi-protein RNA-induced silencing complexes(RISCs),which are critical for small RNA biogenesis.Here,we investigate possible roles of AGO proteins in BMP9-induced lineage-specific differentiation of MSCs.We first found that BMP9 upregulated the expression of Ago1,Ago2 and Ago3 in MSCs.By engineering multiplex siRNA vectors that express multiple siRNAs targeting individual Ago genes or all four Ago genes,we found that silencing individual Ago expression led to a decrease in BMP9-induced early osteogenic marker alkaline phosphatase(ALP)activity in MSCs.Furthermore,we demonstrated that simultaneously silencing all four Ago genes significantly diminished BMP9-induced osteogenic and adipogenic differentiation of MSCs and matrix mineralization,and ectopic bone formation.Collectively,our findings strongly indicate that AGO proteins and associated small RNA biogenesis pathway play an essential role in mediating BMP9-induced osteogenic differentiation of MSCs.展开更多
Intestinal cancers are developed from intestinal epithelial stem cells(ISCs)in intestinal crypts through a multi-step process involved in genetic mutations of oncogenes and tumor suppressor genes.ISCs play a key role ...Intestinal cancers are developed from intestinal epithelial stem cells(ISCs)in intestinal crypts through a multi-step process involved in genetic mutations of oncogenes and tumor suppressor genes.ISCs play a key role in maintaining the homeostasis of gut epithelium.In 2009,Sato et al established a three-dimensional culture system,which mimicked the niche microenvironment by employing the niche factors,and successfully grew crypt ISCs into organoids or Mini-guts in vitro.Since then,the intestinal organoid technology has been used to delineate cellular signaling in ISC biology.However,the cultured organoids consist of heterogeneous cell populations,and it was technically challenging to introduce genomic changes into three-dimensional organoids.Thus,there was a technical necessity to develop a twodimensional ISC culture system for effective genomic manipulations.In this study,we established a conditionally immortalized mouse intestinal crypt(ciMIC)cell line by using a piggyBac transposon-based SV40 T antigen expression system.We showed that the ciMICs maintained long-term proliferative activity under two-dimensional niche factor-containing culture condition,retained the biological characteristics of intestinal epithelial stem cells,and could form intestinal organoids in three-dimensional culture.While in vivo cell implantation tests indicated that the ciMICs were non-tumorigenic,the ciMICs overexpressing oncogenic b-catenin and/or KRAS exhibited high proliferative activity and developed intestinal adenoma-like pathological features in vivo.Collectively,these findings strongly suggested that the engineered ciMICs should be used as a valuable tool cell line to dissect the genetic and/or epigenetic underpinnings of intestinal tumorigenesis.展开更多
Plasmid DNA(pDNA)isolation from bacterial cells is one of the most common and critical steps in molecular cloning and biomedical research.Almost all pDNA purification in-volves disruption of bacteria,removal of membra...Plasmid DNA(pDNA)isolation from bacterial cells is one of the most common and critical steps in molecular cloning and biomedical research.Almost all pDNA purification in-volves disruption of bacteria,removal of membrane lipids,proteins and genomic DNA,purifi-cation of pDNA from bulk lysate,and concentration of pDNA for downstream applications.While many liquid-phase and solid-phase pDNA purification methods are used,the final pDNA preparations are usually contaminated with varied degrees of host RNA,which cannot be completely digested by RNase A.To develop a simple,cost-effective,and yet effective method for RNA depletion,we investigated whether commercially available size selection magnetic beads(SSMBs),such as Mag-Bind®TotalPure NGS Kit(or Mag-Bind),can completely deplete bacterial RNA in pDNA preparations.In this proof-of-principle study,we demonstrated that,compared with RNase A digestion and two commercial plasmid affinity purification kits,the SSMB method was highly efficient in depleting contaminating RNA from pDNA minipreps.Gene transfection and bacterial colony formation assays revealed that pDNA purified from SSMB method had superior quality and integrity to pDNA samples cleaned up by RNase A digestion and/or commercial plasmid purification kits.We further demonstrated that the SSMB method completely depleted contaminating RNA in large-scale pDNA samples.Furthermore,the Mag-bind-based SSMB method costs only 5-10%of most commercial plasmid purification kits on a per sample basis.Thus,the reported SSMB method can be a valuable and inexpensive tool for the removal of bacterial RNA for routine pDNA preparations.展开更多
基金funded by National Natural Science Foundation of China(31870798,31972024)Shaanxi Province Innovation Capability Support Plan-Science and Technology Innovation Team(2020TD-044)+2 种基金Key Laboratory of Glycobiology and Glycoengineering of Xi’an(2019219514SYS010CG032)Natural Science Project of Shaanxi Provincial Department of Education(21JK0929)Science and Technology Resources Sharing Platform Project of Science and Technology Department in Shaanxi Province(2022PT-46)。
文摘β-Conglycinin is one of the major allergens existed in soybean.N-Glycans attached to theβ-conglycinin influenced the immunoreactivity and antigen presenting efficiency ofβ-conglycinin.In this study,we described a new method used to release and collect the N-glycans fromβ-conglycinin,and the N-glycans existed in linear epitopes ofβ-conglycinin were identified.Glycopeptides hydrolyzed fromβ-conglycinin were purified by cotton hydrophilic chromatography.Trifluoromethylsulfonic acid was then used to release glycans from glycopeptides,and new glycopeptides containing one single N-acety1-D-glucosamine(G1 cNAc)moiety were then utilized for mass spectrometry.Five glycosylation sites(Asn-199,Asn-455,Asn-215,Asn-489 and Asn-326)and 22 kinds of glycopeptides were identified.It is noteworthy that the peptide VVN^(#)ATSNL(where^(#)represents for the glycosylation site)was analyzed to be both glycopeptide and linear epitope.Our results provided a new method for the N-glycoform analysis of food allergens,and laid a foundation for understanding the relationship between glyco sylation and food allergy.
基金supported in part by grants from the National Key Research and Development Program of China(2022YFC2303100)the National Natural Science Foundation of China(22207117).
文摘Cell-free expression systems have emerged as a versatile and powerful platform for metabolic engineering,biosynthesis and synthetic biology studies.Nevertheless,successful examples of the synthesis of complex natural products using this system are still limited.Bicyclomycin,a structurally unique and complex diketopiperazine alkaloid,is a clinically promising antibiotic that selectively inhibits the transcription termination factor Rho.Here,we established a modular cell-free expression system with cascade catalysis for the biosynthesis of bicyclomycin from a chemically synthesized cyclodipeptide.The six cell-free expressed biosynthetic enzymes,including five iron-andα-ketoglutarate-dependent dioxygenases and one cytochrome P450 monooxygenase,were active in converting their substrates to the corresponding products.The co-expressed enzymes in the cell-free module were able to complete the related partial pathway.In vitro biosynthesis of bicyclomycin was also achieved by reconstituting the entire biosynthetic pathways(i.e.,six enzymes)using the modular cell-free expression system.This study demonstrates that the modular cell-free expression system can be used as a robust and promising platformforthe biosynthesis of complex antibiotics.
基金supported in part by research grants from the National Institutes of Health(No.CA226303 to TCH,No.DE030480 to RRR)supported by the Medical Scientist Training Program of the National Institutes of Health(USA)(No.T32 GM007281)+2 种基金supported in part by The University of Chicago Cancer Center Support Grant(No.P30CA014599)the National Center for Advancing Translational Sciences of the National Institutes of Health(USA)(No.UL1 TR000430)supported by the Mabel Green Myers Research Endowment Fund and The University of Chicago Orthopaedics Alumni Fund.
文摘Ovarian cancer(OC)is one of the most lethal malignancies of the female reproduc-tive system.OC patients are usually diagnosed at advanced stages due to the lack of early diag-nosis.The standard treatment for OC includes a combination of debulking surgery and platinum-taxane chemotherapy,while several targeted therapies have recently been approved for maintenance treatment.The vast majority of OC patients relapse with chemoresistant tu-mors after an initial response.Thus,there is an unmet clinical need to develop new therapeu-tic agents to overcome the chemoresistance of OC.The anti-parasite agent niclosamide(NA)has been repurposed as an anti-cancer agent and exerts potent anti-cancer activities in human cancers including OC.Here,we investigated whether NA could be repurposed as a therapeutic agent to overcome cisplatin-resistant(CR)in human OC cells.To this end,we first established two CR lines SKOV3CR and OVCAR8CR that exhibit the essential biological characteristics of cisplatin resistance in human cancer.We showed that NA inhibited cell proliferation,sup-pressed cell migration,and induced cell apoptosis in both CR lines at a low micromole range.Mechanistically,NA inhibited multiple cancer-related pathways including AP1,ELK/SRF,HIF1,and TCF/LEF,in SKOV3CR and OVCAR8CR cells.NA was further shown to effectively inhibit xenograft tumor growth of SKOV3CR cells.Collectively,our findings strongly suggest that NA may be repurposed as an efficacious agent to combat cisplatin resistance in chemoresistant hu-man OC,and further clinical trials are highly warranted.
文摘The authors regret that an image assembly(copy/paste)error in Figure 3D,in which the image for the organoid of"Primary MiCs"group was erroneously duplicated with an image of primary MICs that was previously published.The corrected figure is shown below.As shown in the corrected Figure 3D,this error does not adversely impact the conclusion of the original work.The authors would like to apologise forany inconvenience caused.
基金This study was supported by The National Natural Science Foundation of China(No.81904231,82072978,82072979)the China Postdoctoral Science Foundation(No.2020M672369)+1 种基金the Natural Science Foundation of Hubei Province(No.2020CFB861)the Postdoctoral Innovation Practice Post in Hubei Province(No.34).
文摘The treatment of cancer mainly involves surgical excision supplemented by radiotherapy and chemotherapy.Chemotherapy drugs act by interfering with tumor growth and inducing the death of cancer cells.Anti-tumor drugs were developed to induce apoptosis,but some patient’s show apoptosis escape and chemotherapy resistance.Therefore,other forms of cell death that can overcome the resistance of tumor cells are important in the context of cancer treatment.Ferroptosis is a newly discovered iron-dependent,non-apoptotic type of cell death that is highly negatively correlated with cancer development.Ferroptosis is mainly caused by the abnormal increase in iron-dependent lipid reactive oxygen species and the imbalance of redox homeostasis.This review summarizes the progression and regulatory mechanism of ferroptosis in cancer and discusses its possible clinical applications in cancer diagnosis and treatment.
基金the National Institutes of Health(CA226303to TCH)the U.S.Department of Defense(OR130096 to JMW)+5 种基金the Scoliosis Research Society(TCH and MJL)the Pritzker-Northshore Fellowship at The University of Chicagothe Medical Scientist Training Program of the National Institutes of Health(T32 GM007281)The University of Chicago Cancer Center Support Grant(P30CA014599)the National Center for Advancing Translational Sciences of the National Institutes of Health through Grant Number UL1 TR000430the Mabel Green Myers Research Endowment Fund and The University of Chicago Orthopaedics Alumni Fund。
文摘Notch is a cellecell signaling pathway that is involved in a host of activities including development,oncogenesis,skeletal homeostasis,and much more.More specifically,recent research has demonstrated the importance of Notch signaling in osteogenic differentiation,bone healing,and in the development of the skeleton.The craniofacial skeleton is complex and understanding its development has remained an important focus in biology.In this review we briefly summarize what recent research has revealed about Notch signaling and the current understanding of how the skeleton,skull,and face develop.We then discuss the crucial role that Notch plays in both craniofacial development and the skeletal system,and what importance it may play in the future.
基金The reported work was supported in part by research grants from the National Institutes of Health(CA226303 to TCH,and AR072731 to JY)the Chicago Biomedical Consortium with support from the Searle Funds at The Chicago Community Trust(RRR),and the Scoliosis Research Society(TCH and MJL)+2 种基金WW was supported by the Medical Scientist Training Program of the National Institutes of Health(T32 GM007281)This project was also supported in part by The University of Chicago Cancer Center Support Grant(P30CA014599)the National Center for Advancing Translational Sciences(NCATS)of the National Institutes of Health(NIH)through Grant Number 5UL1TR002389-02 that funds the Institute for Translational Medicine(ITM).TCH was supported by the Mabel Green Myers Research Endowment Fund and The University of Chicago Orthopaedics Alumni Fund.
文摘As multipotent progenitor cells,mesenchymal stem cells(MSCs)can renew themselves and give rise to multiple lineages including osteoblastic,chondrogenic and adipogenic lineages.It’s previously shown that BMP9 is the most potent BMP and induces osteogenic and adipogenic differentiation of MSCs.However,the molecular mechanism through which BMP9 regulates MSC differentiation remains poorly understood.Emerging evidence indicates that noncoding RNAs,especially microRNAs,may play important roles in regulating MSC differentiation and bone formation.As highly conserved RNA binding proteins,Argonaute(AGO)proteins are essential components of the multi-protein RNA-induced silencing complexes(RISCs),which are critical for small RNA biogenesis.Here,we investigate possible roles of AGO proteins in BMP9-induced lineage-specific differentiation of MSCs.We first found that BMP9 upregulated the expression of Ago1,Ago2 and Ago3 in MSCs.By engineering multiplex siRNA vectors that express multiple siRNAs targeting individual Ago genes or all four Ago genes,we found that silencing individual Ago expression led to a decrease in BMP9-induced early osteogenic marker alkaline phosphatase(ALP)activity in MSCs.Furthermore,we demonstrated that simultaneously silencing all four Ago genes significantly diminished BMP9-induced osteogenic and adipogenic differentiation of MSCs and matrix mineralization,and ectopic bone formation.Collectively,our findings strongly indicate that AGO proteins and associated small RNA biogenesis pathway play an essential role in mediating BMP9-induced osteogenic differentiation of MSCs.
文摘Intestinal cancers are developed from intestinal epithelial stem cells(ISCs)in intestinal crypts through a multi-step process involved in genetic mutations of oncogenes and tumor suppressor genes.ISCs play a key role in maintaining the homeostasis of gut epithelium.In 2009,Sato et al established a three-dimensional culture system,which mimicked the niche microenvironment by employing the niche factors,and successfully grew crypt ISCs into organoids or Mini-guts in vitro.Since then,the intestinal organoid technology has been used to delineate cellular signaling in ISC biology.However,the cultured organoids consist of heterogeneous cell populations,and it was technically challenging to introduce genomic changes into three-dimensional organoids.Thus,there was a technical necessity to develop a twodimensional ISC culture system for effective genomic manipulations.In this study,we established a conditionally immortalized mouse intestinal crypt(ciMIC)cell line by using a piggyBac transposon-based SV40 T antigen expression system.We showed that the ciMICs maintained long-term proliferative activity under two-dimensional niche factor-containing culture condition,retained the biological characteristics of intestinal epithelial stem cells,and could form intestinal organoids in three-dimensional culture.While in vivo cell implantation tests indicated that the ciMICs were non-tumorigenic,the ciMICs overexpressing oncogenic b-catenin and/or KRAS exhibited high proliferative activity and developed intestinal adenoma-like pathological features in vivo.Collectively,these findings strongly suggested that the engineered ciMICs should be used as a valuable tool cell line to dissect the genetic and/or epigenetic underpinnings of intestinal tumorigenesis.
基金supported in part by research grants from the China Postdoctoral Science Foundation(2019M663446 to ZZ)the Postdoctoral Program of the Natural Science Foundation of Chongqing,China(cstc2019jcyj-bsh0006 to ZZ)+6 种基金WW was supported by the Medical Scientist Training Program of the National Institutes of Health(T32 GM007281)This project was also supported in part by The University of Chicago Cancer Center Support Grant(P30CA014599)the National Center for Advancing Translational Sciences of the National Institutes of Health through Grant Number UL1 TR000430TCH was supported by the Mabel Green Myers Research Endowment Fund and The University of Chicago Orthopaedics Alumni Fund.Funding sources were not involved in the study designin the collection,analysis and interpretation of datain the writing of the reportand in the decision to submit the paper for publication.
文摘Plasmid DNA(pDNA)isolation from bacterial cells is one of the most common and critical steps in molecular cloning and biomedical research.Almost all pDNA purification in-volves disruption of bacteria,removal of membrane lipids,proteins and genomic DNA,purifi-cation of pDNA from bulk lysate,and concentration of pDNA for downstream applications.While many liquid-phase and solid-phase pDNA purification methods are used,the final pDNA preparations are usually contaminated with varied degrees of host RNA,which cannot be completely digested by RNase A.To develop a simple,cost-effective,and yet effective method for RNA depletion,we investigated whether commercially available size selection magnetic beads(SSMBs),such as Mag-Bind®TotalPure NGS Kit(or Mag-Bind),can completely deplete bacterial RNA in pDNA preparations.In this proof-of-principle study,we demonstrated that,compared with RNase A digestion and two commercial plasmid affinity purification kits,the SSMB method was highly efficient in depleting contaminating RNA from pDNA minipreps.Gene transfection and bacterial colony formation assays revealed that pDNA purified from SSMB method had superior quality and integrity to pDNA samples cleaned up by RNase A digestion and/or commercial plasmid purification kits.We further demonstrated that the SSMB method completely depleted contaminating RNA in large-scale pDNA samples.Furthermore,the Mag-bind-based SSMB method costs only 5-10%of most commercial plasmid purification kits on a per sample basis.Thus,the reported SSMB method can be a valuable and inexpensive tool for the removal of bacterial RNA for routine pDNA preparations.