The brain tumor perivascular niche(PVN),the region in the vicinity of microvessels is a prime location for brain tumor stem-like cells(BTSCs)[1].Tumor microvasculature creates a complex microenvironment consisting of ...The brain tumor perivascular niche(PVN),the region in the vicinity of microvessels is a prime location for brain tumor stem-like cells(BTSCs)[1].Tumor microvasculature creates a complex microenvironment consisting of various cell types,the extracellular matrix,and soluble factors that mediate cell-cell interaction.The brain tumor PVN controls maintenance,expansion,and differentiation of BTSCs via direct cell contact or paracrine signaling cues.BTSCs often receive bidirectional crosstalk from endothelial cells and other cell types in the niche[2].In addition,the perivascular zone may serve as a path for tumor cells to migrate over long distances(3,4)Unlike other solid tumors,glioblastoma multiforme(GBM)cells rarely metastasize to other organs,but they can invade the entire brain by migrating along specific brain tissue structures,such as blood vessels or white matter tracts,leading to high rates of relapse.Despite the success in modeling diffuse brain tumors in both genetically-modified and patient-derived xenograft(PDX)animals,there is an unmet need for an in vitro system that can bridge conventional cell culture and animal models by mimicking not only the anatomy but also the function of the PVN to study the dynamics of BTSCs.In this presentation,I will describe the use of a microvasculature-on-a-chip system as a PVN model to evaluate the dynamics of BTSCs ex vivo from 10 glioblastoma patients [5].We observed that BTSCs preferentially localize in the perivascular zone.Live cell tracking showed that the cells residing in the vicinity of microvessels had the lowest motility,while a fraction of cells on the microvessels unexpectedly possessed the highest motility and migrated over the longest distance.These results indicate that the perivascular zone is a niche for BTSCs,while the microvascular tracks are also a path for long-distance tumor cell migration and invasion.Additionally,the degree of co-localization between tumor cells and microvessels varied significantly across patients.To validate the results from our microvasculature-on-a-chip system,we used single-cell transcriptome sequencing(10 patients and 21,750 single cells in total)to identify the subtype of each tumor cell.The co-localization coefficient was found to correlate positively with proneural(stem-like)or mesenchymal(invasive)but not classical(proliferative)tumor cells.Furthermore,we found that a gene signature profile including PDGFRA correlated strongly with the'homing'of brain tumor cells to the PVN.Our findings demonstrated that ex vivo dynamics of human brain tumor cells in a microvasculature-on-a-chip model can recapitulate in vivo tumor cell dynamics,heterogeneity,and subtypes,representing a new route to the study of human tumor cell biology and uncover patient-specific tumor cell functions.Acknowledgments:We thank Drs.Laura Niklason,Eric Holland,Franziska Michor,and Frank Szulzewsky for scientific discussion.We thank Misha Guy,Vladimir Polejaev,Zhenting Jiang,and Alice Yun for suggestions and help on the simulation computing and SEM/confocal imaging process.This research was supported by the Packard Fellowship for Science and Engineering(R.F.),National Science Foundation CAREER Award CBET-1351443(R.F.),U54 CA193461(R.F.),U54CA209992(Sub-Project ID:7297 to R.F.),R01 NS095817(J.Z.),Yale Cancer Center Co-Pilot Grant(to R.F.).The molds for microfluidic devices were fabricated in the Yale School of Engineering and Applied Science cleanroom.Sequencing was performed at the Yale Center for Genome Analysis(YCGA)facility.Data was analyzed at Yale High Performance Computing(HPC)center.Super resolution confocal imaging was performed at Yale Center for Cellular and Molecular Imaging(CCMI).展开更多
An improved method for showing Gc band by sulfosalicylic acidafter IEF is reported. This method provides us an effective mean to identify theGc subtypes and its variants within 4 h without using anti-serum for immunof...An improved method for showing Gc band by sulfosalicylic acidafter IEF is reported. This method provides us an effective mean to identify theGc subtypes and its variants within 4 h without using anti-serum for immunofix-ation. And new variants Gc<sup>1c3</sup> and Gc<sup>2c7</sup> are discovered in Chinese people, andtheir frequencies are 0. 0008 and 0. 0004 respectively.展开更多
Glial glutamate transporter-1(GLT-1)is the predominant subtype of glutamate transporters and is responsible for the clearance of extracellular glutamate and for limiting the concentration of extracellular glutamate.Ou...Glial glutamate transporter-1(GLT-1)is the predominant subtype of glutamate transporters and is responsible for the clearance of extracellular glutamate and for limiting the concentration of extracellular glutamate.Our previous studies have shown that the up-regulation of GLT-1 expression plays an important role展开更多
The HA1 gene of H3N2 subtype swine influenza virus(SIV)was cloned into the expression plasmid pET-30a,the recombinant plasmid was named pET-HAl.This was transformed into E.coli BL21(DE3),and expressed by induction wit...The HA1 gene of H3N2 subtype swine influenza virus(SIV)was cloned into the expression plasmid pET-30a,the recombinant plasmid was named pET-HAl.This was transformed into E.coli BL21(DE3),and expressed by induction with IPTG.The expressed HA protein was identified by SDS-PAGE and Western blotting which showed the protein to be 42kDa and was immunoreactive.The purified HA protein was used to establish the indirect ELIS A for detection of the antibodies,specifically against the H3 subtype of SIV.The assay has excellent specificity,sensitivity and reproducibility.When 96 serum samples,randomly collected from the field,were evaluated in parallel by this new ELISA using recombinant HA1 and a routine HI test,the coincidental rate between the two tests was 86.5%.These results show that the recombinant HAl-based ELISA is specific,sensitive and easy to perform for the serological diagnosis of SIV infection.展开更多
Ducks inoculated intravenously or via the ocular-nasal-oral-cloacal routes with a highly pathogenic avian influenza virus,A/duck/Guangdong/220/2004(H5N1),developed systemic hyperemia,congestion,hemorrhage,thrombosis a...Ducks inoculated intravenously or via the ocular-nasal-oral-cloacal routes with a highly pathogenic avian influenza virus,A/duck/Guangdong/220/2004(H5N1),developed systemic hyperemia,congestion,hemorrhage,thrombosis and edema in various organs,as well as necrosis or apoptosis in the parenchyma of the heart,liver,spleen,lungs,kidneys,pancreas,brain,thymus and bursa of Fabricius.The main manifestations were angiitis,necrotic pancreatitis,atrophic necrotic thymitis and bursitis Fabricii,splenitis,tracheitis,hemorrhagic bronchointerstitial pneumonia,viral myocarditis,nonsuppurative encephalitis,focal viral hepatitis,ulcerative enteritis,renal tubule interstitial nephritis,and intraglomerular mesangial cell hyperplastic glomerular nephritis.The results demonstrated that the mechanism of pathogenesis involved cellular necrosis and apoptosis,and that death of the ducks was caused by severe pathologic trauma occurring in multiple visceral organs.展开更多
OBJECTIVE Dopamine receptors(DRs) are involved in the development and treatment of many neuropsychiatric disorders.Currently available dopaminergic drugs modulate both DRD2 and DRD3,leading to side effects and uncerta...OBJECTIVE Dopamine receptors(DRs) are involved in the development and treatment of many neuropsychiatric disorders.Currently available dopaminergic drugs modulate both DRD2 and DRD3,leading to side effects and uncertainty as to the roles each DR subtype plays physiologically.Our lab employed high throughput screening paradigms to discover highly selective modulators for the DRD3.METHODS The NIH Molecular Libraries Program 400,000 + small molecule library was screened using the Discove Rx Path Hunter?β-arrestin assay for compounds that activate the DRD3 without effects on the DRD2.Confirmation and counter-screens assessed selectivity and mechanisms of action.We identified 62 potential agonists,and chose the most promising to perform a structure-activity relationship(SAR) study to increase potency while maintaining selectivity.The lead compound identified through this process,ML417,was also characterized using bioluminescence resonance energy transfer(BRET)-based β-arrestin recruitment and G-protein activation assays as well as p-ERK assays.Potential neuroprotective properties of this compound were assessed using a SHSY5 Y neuronal cell model.RESULTS ML417 displays potent,DRD3-selective agonist activity in multiple functional assays.Binding and functional GPCR screens(>165 receptors) show ML417 has limited cross-reactivity with other GPCRs.ML417 also displays superior(compared to the reference compound pramipexole),dose-dependent protection against a decrease in neurite length induced by 10 μmol·L^(-1) of the neurotoxin,6-hydroxydopamine,in the SHSY5 Y cell model.CONCLUSION We have discovered and characterized ML417,a potent and highly selective DRD3 agonist.This compound will be useful as a research tool,and may prove useful as a therapeutic drug lead.展开更多
To construct a recombinant adenovirus shuttle plasmid pDC315-H5HA-EGFP,the HA gene of A/Swine/Fujian/1/2001(H5N1) was amplified by RT-PCR and then inserted into adenovirus shuttle plasmid pDC315.A replication-defectiv...To construct a recombinant adenovirus shuttle plasmid pDC315-H5HA-EGFP,the HA gene of A/Swine/Fujian/1/2001(H5N1) was amplified by RT-PCR and then inserted into adenovirus shuttle plasmid pDC315.A replication-defective recombinant adenovirus expressing the HA gene(rAd-H5HA-EGFP) was generated by co-transfecting the recombinant shuttle plasmid pDC315-H5HA-EGFP and the genomic plasmid pBHGlox△E1,E3Cre in HEK293 cells.The recombinant adenovirus was confirmed by PCR,RT-PCR and Western blot assay.These results demonstrated that HA protein was properly expressed by the rAd-H5HA-EGFP in HEK293 cells and had natural biological activities.The TCID<sub>50</sub> of the rAd-H5HA- EGFP was assessed to be 2.26×10<sup>10</sup>/mL after propagation and purification.Immunization of BALB/ c mice indicated that rAd-H5HA-EGFP induced HI antibodies and protected mice from replication of the challenge virus in their lungs.展开更多
文摘The brain tumor perivascular niche(PVN),the region in the vicinity of microvessels is a prime location for brain tumor stem-like cells(BTSCs)[1].Tumor microvasculature creates a complex microenvironment consisting of various cell types,the extracellular matrix,and soluble factors that mediate cell-cell interaction.The brain tumor PVN controls maintenance,expansion,and differentiation of BTSCs via direct cell contact or paracrine signaling cues.BTSCs often receive bidirectional crosstalk from endothelial cells and other cell types in the niche[2].In addition,the perivascular zone may serve as a path for tumor cells to migrate over long distances(3,4)Unlike other solid tumors,glioblastoma multiforme(GBM)cells rarely metastasize to other organs,but they can invade the entire brain by migrating along specific brain tissue structures,such as blood vessels or white matter tracts,leading to high rates of relapse.Despite the success in modeling diffuse brain tumors in both genetically-modified and patient-derived xenograft(PDX)animals,there is an unmet need for an in vitro system that can bridge conventional cell culture and animal models by mimicking not only the anatomy but also the function of the PVN to study the dynamics of BTSCs.In this presentation,I will describe the use of a microvasculature-on-a-chip system as a PVN model to evaluate the dynamics of BTSCs ex vivo from 10 glioblastoma patients [5].We observed that BTSCs preferentially localize in the perivascular zone.Live cell tracking showed that the cells residing in the vicinity of microvessels had the lowest motility,while a fraction of cells on the microvessels unexpectedly possessed the highest motility and migrated over the longest distance.These results indicate that the perivascular zone is a niche for BTSCs,while the microvascular tracks are also a path for long-distance tumor cell migration and invasion.Additionally,the degree of co-localization between tumor cells and microvessels varied significantly across patients.To validate the results from our microvasculature-on-a-chip system,we used single-cell transcriptome sequencing(10 patients and 21,750 single cells in total)to identify the subtype of each tumor cell.The co-localization coefficient was found to correlate positively with proneural(stem-like)or mesenchymal(invasive)but not classical(proliferative)tumor cells.Furthermore,we found that a gene signature profile including PDGFRA correlated strongly with the'homing'of brain tumor cells to the PVN.Our findings demonstrated that ex vivo dynamics of human brain tumor cells in a microvasculature-on-a-chip model can recapitulate in vivo tumor cell dynamics,heterogeneity,and subtypes,representing a new route to the study of human tumor cell biology and uncover patient-specific tumor cell functions.Acknowledgments:We thank Drs.Laura Niklason,Eric Holland,Franziska Michor,and Frank Szulzewsky for scientific discussion.We thank Misha Guy,Vladimir Polejaev,Zhenting Jiang,and Alice Yun for suggestions and help on the simulation computing and SEM/confocal imaging process.This research was supported by the Packard Fellowship for Science and Engineering(R.F.),National Science Foundation CAREER Award CBET-1351443(R.F.),U54 CA193461(R.F.),U54CA209992(Sub-Project ID:7297 to R.F.),R01 NS095817(J.Z.),Yale Cancer Center Co-Pilot Grant(to R.F.).The molds for microfluidic devices were fabricated in the Yale School of Engineering and Applied Science cleanroom.Sequencing was performed at the Yale Center for Genome Analysis(YCGA)facility.Data was analyzed at Yale High Performance Computing(HPC)center.Super resolution confocal imaging was performed at Yale Center for Cellular and Molecular Imaging(CCMI).
文摘An improved method for showing Gc band by sulfosalicylic acidafter IEF is reported. This method provides us an effective mean to identify theGc subtypes and its variants within 4 h without using anti-serum for immunofix-ation. And new variants Gc<sup>1c3</sup> and Gc<sup>2c7</sup> are discovered in Chinese people, andtheir frequencies are 0. 0008 and 0. 0004 respectively.
文摘Glial glutamate transporter-1(GLT-1)is the predominant subtype of glutamate transporters and is responsible for the clearance of extracellular glutamate and for limiting the concentration of extracellular glutamate.Our previous studies have shown that the up-regulation of GLT-1 expression plays an important role
基金supported by the Chinese National S&T Plan(2004BA519A55)
文摘The HA1 gene of H3N2 subtype swine influenza virus(SIV)was cloned into the expression plasmid pET-30a,the recombinant plasmid was named pET-HAl.This was transformed into E.coli BL21(DE3),and expressed by induction with IPTG.The expressed HA protein was identified by SDS-PAGE and Western blotting which showed the protein to be 42kDa and was immunoreactive.The purified HA protein was used to establish the indirect ELIS A for detection of the antibodies,specifically against the H3 subtype of SIV.The assay has excellent specificity,sensitivity and reproducibility.When 96 serum samples,randomly collected from the field,were evaluated in parallel by this new ELISA using recombinant HA1 and a routine HI test,the coincidental rate between the two tests was 86.5%.These results show that the recombinant HAl-based ELISA is specific,sensitive and easy to perform for the serological diagnosis of SIV infection.
基金supported by the Guangdong Province Science&Technology Hard Nut Project(2004A2090102)Guangdong Province Education Bureau Science Foundation Project(Z02003)
文摘Ducks inoculated intravenously or via the ocular-nasal-oral-cloacal routes with a highly pathogenic avian influenza virus,A/duck/Guangdong/220/2004(H5N1),developed systemic hyperemia,congestion,hemorrhage,thrombosis and edema in various organs,as well as necrosis or apoptosis in the parenchyma of the heart,liver,spleen,lungs,kidneys,pancreas,brain,thymus and bursa of Fabricius.The main manifestations were angiitis,necrotic pancreatitis,atrophic necrotic thymitis and bursitis Fabricii,splenitis,tracheitis,hemorrhagic bronchointerstitial pneumonia,viral myocarditis,nonsuppurative encephalitis,focal viral hepatitis,ulcerative enteritis,renal tubule interstitial nephritis,and intraglomerular mesangial cell hyperplastic glomerular nephritis.The results demonstrated that the mechanism of pathogenesis involved cellular necrosis and apoptosis,and that death of the ducks was caused by severe pathologic trauma occurring in multiple visceral organs.
基金supported by National Institute of Neurological Disorders and Stroke Intramural Research Program
文摘OBJECTIVE Dopamine receptors(DRs) are involved in the development and treatment of many neuropsychiatric disorders.Currently available dopaminergic drugs modulate both DRD2 and DRD3,leading to side effects and uncertainty as to the roles each DR subtype plays physiologically.Our lab employed high throughput screening paradigms to discover highly selective modulators for the DRD3.METHODS The NIH Molecular Libraries Program 400,000 + small molecule library was screened using the Discove Rx Path Hunter?β-arrestin assay for compounds that activate the DRD3 without effects on the DRD2.Confirmation and counter-screens assessed selectivity and mechanisms of action.We identified 62 potential agonists,and chose the most promising to perform a structure-activity relationship(SAR) study to increase potency while maintaining selectivity.The lead compound identified through this process,ML417,was also characterized using bioluminescence resonance energy transfer(BRET)-based β-arrestin recruitment and G-protein activation assays as well as p-ERK assays.Potential neuroprotective properties of this compound were assessed using a SHSY5 Y neuronal cell model.RESULTS ML417 displays potent,DRD3-selective agonist activity in multiple functional assays.Binding and functional GPCR screens(>165 receptors) show ML417 has limited cross-reactivity with other GPCRs.ML417 also displays superior(compared to the reference compound pramipexole),dose-dependent protection against a decrease in neurite length induced by 10 μmol·L^(-1) of the neurotoxin,6-hydroxydopamine,in the SHSY5 Y cell model.CONCLUSION We have discovered and characterized ML417,a potent and highly selective DRD3 agonist.This compound will be useful as a research tool,and may prove useful as a therapeutic drug lead.
基金supported by the Chinese National S&T Plan(2004BA519A55)Scientific Research Program of State Key Laboratory of Veterinary Biotechnology(NKLVBP200818)
文摘To construct a recombinant adenovirus shuttle plasmid pDC315-H5HA-EGFP,the HA gene of A/Swine/Fujian/1/2001(H5N1) was amplified by RT-PCR and then inserted into adenovirus shuttle plasmid pDC315.A replication-defective recombinant adenovirus expressing the HA gene(rAd-H5HA-EGFP) was generated by co-transfecting the recombinant shuttle plasmid pDC315-H5HA-EGFP and the genomic plasmid pBHGlox△E1,E3Cre in HEK293 cells.The recombinant adenovirus was confirmed by PCR,RT-PCR and Western blot assay.These results demonstrated that HA protein was properly expressed by the rAd-H5HA-EGFP in HEK293 cells and had natural biological activities.The TCID<sub>50</sub> of the rAd-H5HA- EGFP was assessed to be 2.26×10<sup>10</sup>/mL after propagation and purification.Immunization of BALB/ c mice indicated that rAd-H5HA-EGFP induced HI antibodies and protected mice from replication of the challenge virus in their lungs.
