W

W. for the introduction of extra glycopeptides as potential inhibitors of cathepsin L-dependent infections. Notably, considering that teicoplanin continues to be found in the center with low toxicity consistently, our function offers a guaranteeing potential customer for the procedure and prophylaxis of Ebola, MERS, and SARS pathogen infection. genus have already been determined, including (2,C6). Ebola pathogen infection qualified prospects to serious viral hemorrhagic fever in human beings and nonhuman primates. In March 2014, outbreaks of Ebola infections started in Guinea and triggered over 28,000 situations of infections and over 11,000 fatalities, which posed a serious threat to open public health world-wide. The Ebola pathogen genome includes seven genes that encode the NP, VP35, VP40, glycoprotein (GP), VP30, VP24, and RNA-dependent RNA polymerase (L) pathogen proteins. To infect web host cells, the Gps navigation of Ebola infections bind to connection substances such as for example 1 integrins initial, DC-SIGNs, L-SIGNs, lectins, TIM-1s, Tyro3 family members proteins, heparan sulfates, or folate receptor- (7,C13). Ebola infections are after that internalized by macropinocytosis and eventually transported through the first and past due endosomes as well as the endo/lysosomes (14,C16), where in fact the Ebola virus Gps navigation are cleaved by cathepsin L and eventually cathepsin B to expose the receptor-binding domains (17). After binding the precise receptor NPC1, Ebola infections discharge their genomes in to the cytoplasm from the web host cells (16, 18). Anti-EBOV medications and vaccines are in intensive advancement. Two guaranteeing vaccines, cAd3-EBOV and rVSVG-EBOV-GP, have been proven to render nonhuman primates resistant to Ebola pathogen infections and so are presently in clinical studies (19, 20). Furthermore, the anti-EBOV monoclonal antibody Zmapp, siRNAs, and various other compounds that may inhibit Ebola pathogen infections have been developed (21,C24). Furthermore, several clinically approved drugs were also reported to inhibit Ebola virus infections (25, 26). However, because the IC50 values of those drugs were relatively high, more anti-EBOV drugs with potent inhibitory activity are urgently needed. To facilitate their identification, the method of high throughput screening of clinically approved drugs, which could be applied immediately in the clinic, is a reasonable approach. In this study, we identified teicoplanin and several other glycopeptide antibiotics as Ebola virus entry inhibitors with high efficiency and low cytotoxicity, providing a promising means to effect the prophylaxis and treatment of Ebola virus infection. Experimental Procedures Cell Culture HEK293T, A549, HeLa, Huh7.5.1, and Madin-Darby canine kidney cell lines were maintained in Dulbecco’s modified Eagle’s medium (Gibco) with 10% fetal calf serum (Gibco), 100 units/ml penicillin, and 100 g/ml streptomycin (Gibco) at 37 C and 5% CO2. THP-1 cell lines were maintained in RPMI1640 medium (Gibco) with 10% fetal calf serum, 100 units/ml penicillin, and 100 g/ml streptomycin at 37 C and 5% CO2. Primary human umbilical vein endothelial cells were maintained in human endothelial-SFM (Gibco) with 30 ng/ml endothelial cell growth supplement (Merck Millipore), 20 ng/ml recombinant human FGF basic (146 amino acids) protein (R&D Systems), 20% fetal calf serum, 100 units/ml penicillin, and 100 g/ml streptomycin at 37 C and 5% CO2. Plasmids GP sequence of Zaire EBOV-2014 was chemically synthesized and inserted into pcDNA3.1 plasmid. The pHIV-luciferase and pCMV-VSV-G plasmids were obtained from Addgene, and the pCMV-R8.2 plasmid was kindly provided by Dr. Trono (27). The p4cis plasmid that encodes a luciferase reporter, VP40, GP and VP24, the pCAGGS-NP, pCAGGS-VP35, pCAGGS-VP30, pCAGGS-L, pCAGGS-T7, and pCAGGS-Tim1 plasmids were produced as described previously (28). Viruses Pseudotyped viruses were produced by the co-transfection of pHIV-luciferase, pCMV-R8.2, and different envelope plasmids into HEK293T cells that were 90% confluent in a 10-cm plate with Lipofectamine 2000 by Naspm trihydrochloride following the manufacturer’s instructions (Invitrogen). The amounts of plasmids were listed as follows: HIV-luc/Zaire EBOV-GP(2014) pseudotyped viruses: 4.5 g of pHIV-luciferase, 4.5 g of pCMV-R8.2, and 7.65 g of pcDNA3.1-Zaire EBOV-GP(2014); HIV-luc/VSV-G pseudotyped viruses: 4.5 g of pHIV-luciferase, 4.5 g of pCMV-R8.2, and 2.7 g of pCMV-VSV-G; HIV-luc/SARS-CoV-S pseudotyped viruses: 6.5 g of pHIV-luciferase, 8 g of pCMV-R8.2, and 20 g of pcDNA3.1-SARS-CoV-S; and HIV-luc/MERS-CoV-S pseudotyped viruses: 4.5 g of pHIV-luciferase, 4.5 g of pCMV-R8.2, and 10 g of pcDNA3.1-MERS-CoV-S. After 48 h, the supernatants which contain the pseudotyped viruses were filtered and collected through a 0.45-m pore-size filter (Pall) and stored at ?80 C until make use of. Ebola transcription- and replication-competent virus-like contaminants (trVLPs) had been made by the co-transfection of 250 ng of p4cis plasmids that encode a luciferase reporter gene, VP40, GP, and VP24, 250.(worth was dependant on a Student’s test. and serious acute respiratory symptoms (SARS) envelope pseudotyped infections aswell. Teicoplanin derivatives such as for example dalbavancin, oritavancin, and telavancin can inhibit the entrance of Ebola also, MERS, and SARS infections. Mechanistic research demonstrated that teicoplanin blocks Ebola trojan entrance by inhibiting the experience of cathepsin L particularly, opening a book avenue for the introduction of extra glycopeptides as potential inhibitors of cathepsin L-dependent Naspm trihydrochloride infections. Notably, considering that teicoplanin provides been found in the medical clinic with low toxicity consistently, our work offers a appealing potential customer for the prophylaxis and treatment of Ebola, MERS, and SARS trojan infection. genus have already been discovered, including (2,C6). Ebola trojan infection network marketing leads to serious viral hemorrhagic fever in human beings and nonhuman primates. In March 2014, outbreaks of Ebola infections started in Guinea and triggered over 28,000 situations of an infection and over 11,000 fatalities, which posed a serious threat to open public health world-wide. The Ebola trojan genome includes seven genes that encode the NP, VP35, VP40, glycoprotein (GP), VP30, VP24, and RNA-dependent RNA polymerase (L) trojan proteins. To infect web host cells, the Gps navigation of Ebola infections initial bind to connection molecules such as for example 1 integrins, DC-SIGNs, L-SIGNs, lectins, TIM-1s, Tyro3 family members proteins, heparan sulfates, or folate receptor- (7,C13). Ebola infections are after that internalized by macropinocytosis and eventually transported through the first and past due endosomes as well as the endo/lysosomes (14,C16), where in fact the Ebola virus Gps navigation are cleaved by cathepsin L and eventually cathepsin B to expose the receptor-binding domains (17). After binding the precise receptor NPC1, Ebola infections discharge their genomes in to the cytoplasm from the web host cells (16, 18). Anti-EBOV vaccines and medications are under comprehensive development. Two appealing vaccines, rVSVG-EBOV-GP and cAd3-EBOV, have already been proven to render nonhuman primates resistant to Ebola trojan infections and so are presently in clinical studies (19, 20). Furthermore, the anti-EBOV monoclonal antibody Zmapp, siRNAs, and various other compounds that may inhibit Ebola trojan infections have already been created (21,C24). Furthermore, many clinically approved medications had been also reported to inhibit Ebola trojan attacks (25, 26). Nevertheless, as the IC50 beliefs of those medications had been relatively high, even more anti-EBOV medications with powerful inhibitory activity are urgently required. To facilitate their id, the technique of high throughput testing of clinically accepted drugs, that could be applied instantly in the medical clinic, is an acceptable approach. Within this research, we discovered teicoplanin and many various other glycopeptide antibiotics as Ebola trojan entrance inhibitors with high performance and low cytotoxicity, offering a appealing means to impact the prophylaxis and treatment of Ebola trojan infection. Experimental Techniques Cell Lifestyle HEK293T, A549, HeLa, Huh7.5.1, and Madin-Darby dog kidney cell lines had been preserved in Dulbecco’s modified Eagle’s moderate (Gibco) with 10% fetal leg serum (Gibco), 100 systems/ml penicillin, and 100 g/ml streptomycin (Gibco) in 37 C and 5% CO2. THP-1 cell lines had been preserved in RPMI1640 moderate (Gibco) with 10% fetal leg serum, 100 systems/ml penicillin, and 100 g/ml streptomycin at 37 C and 5% CO2. Principal individual umbilical vein endothelial cells had been maintained in individual endothelial-SFM (Gibco) with 30 ng/ml endothelial cell development dietary supplement (Merck Millipore), 20 ng/ml recombinant individual FGF simple (146 proteins) proteins (R&D Systems), 20% fetal leg serum, 100 models/ml penicillin, and 100 g/ml streptomycin at 37 C and 5% CO2. Plasmids GP sequence of Zaire EBOV-2014 was chemically synthesized and inserted into pcDNA3.1 plasmid. The pHIV-luciferase and pCMV-VSV-G plasmids were obtained from Addgene, and the pCMV-R8.2 plasmid was kindly provided by Dr. Trono (27). The p4cis plasmid that encodes a luciferase reporter, VP40, GP and VP24, the pCAGGS-NP, pCAGGS-VP35, pCAGGS-VP30, pCAGGS-L, pCAGGS-T7, and pCAGGS-Tim1 plasmids were produced as explained previously (28). Viruses Pseudotyped viruses were produced by the co-transfection of pHIV-luciferase, pCMV-R8.2, and different envelope plasmids into HEK293T.show the mean values S.D. routinely been used in the medical center with low toxicity, our work provides a encouraging prospect for the prophylaxis and treatment of Ebola, MERS, and SARS computer virus infection. genus have been recognized, including (2,C6). Ebola computer virus infection prospects to severe viral hemorrhagic fever in humans and non-human primates. In March 2014, outbreaks of Ebola viruses began in Guinea and caused over 28,000 cases of contamination and over 11,000 deaths, which posed a severe threat to public health worldwide. The Ebola computer virus genome contains seven genes that encode the NP, VP35, VP40, glycoprotein (GP), VP30, VP24, and RNA-dependent RNA polymerase (L) computer virus proteins. To infect host cells, the GPs of Ebola viruses first bind to attachment molecules such as 1 integrins, DC-SIGNs, L-SIGNs, lectins, TIM-1s, Tyro3 family proteins, heparan sulfates, or folate receptor- (7,C13). Ebola viruses are then internalized by macropinocytosis and subsequently transported through the early and late endosomes and the endo/lysosomes (14,C16), where the Ebola virus GPs are cleaved by cathepsin L and subsequently cathepsin B to expose the receptor-binding domains (17). After binding the specific receptor NPC1, Ebola viruses release their genomes into the cytoplasm of the host cells (16, 18). Anti-EBOV vaccines and drugs are under considerable development. Two encouraging vaccines, rVSVG-EBOV-GP and cAd3-EBOV, have been shown to render non-human primates resistant to Ebola computer virus infections and are currently in clinical trials (19, 20). In addition, the anti-EBOV monoclonal antibody Zmapp, siRNAs, and other compounds that can inhibit Ebola computer virus infections have been developed (21,C24). Furthermore, several clinically approved drugs were also reported to inhibit Ebola computer virus infections (25, 26). However, because the IC50 values of those drugs were relatively high, more anti-EBOV drugs with potent inhibitory activity are urgently needed. To facilitate their identification, the method of high throughput screening of clinically approved drugs, which could be applied immediately in the medical center, is a reasonable approach. In this study, we recognized teicoplanin and several other glycopeptide antibiotics as Ebola computer virus access inhibitors with high efficiency and low cytotoxicity, providing a encouraging means to effect the prophylaxis and treatment of Ebola computer virus infection. Experimental Procedures Cell Culture HEK293T, A549, HeLa, Huh7.5.1, and Madin-Darby canine kidney cell lines were maintained in Dulbecco’s modified Eagle’s medium (Gibco) with 10% fetal calf serum (Gibco), 100 models/ml penicillin, and 100 g/ml streptomycin (Gibco) at 37 C and 5% CO2. THP-1 cell lines were managed in RPMI1640 medium (Gibco) with 10% fetal calf serum, 100 models/ml penicillin, and 100 g/ml streptomycin at 37 C and 5% CO2. Main human umbilical vein endothelial cells were maintained in human endothelial-SFM Rabbit Polyclonal to ENDOGL1 (Gibco) with 30 ng/ml endothelial cell growth product (Merck Millipore), 20 ng/ml recombinant human FGF basic (146 amino acids) protein (R&D Systems), 20% fetal calf serum, 100 models/ml penicillin, and 100 g/ml streptomycin at 37 C and 5% CO2. Plasmids GP sequence of Zaire EBOV-2014 was chemically synthesized and inserted into pcDNA3.1 plasmid. The pHIV-luciferase and pCMV-VSV-G plasmids were obtained from Addgene, and the pCMV-R8.2 plasmid was kindly provided by Dr. Trono (27). The p4cis plasmid that encodes a luciferase reporter, VP40, GP and VP24, the pCAGGS-NP, pCAGGS-VP35, pCAGGS-VP30, pCAGGS-L, pCAGGS-T7, and pCAGGS-Tim1 plasmids were produced as explained previously (28). Viruses Pseudotyped viruses were produced by the co-transfection of pHIV-luciferase, pCMV-R8.2, and different envelope plasmids into HEK293T cells that were 90% confluent in a 10-cm plate with Lipofectamine 2000 by following the manufacturer’s instructions (Invitrogen). The amounts of plasmids were listed as follows: HIV-luc/Zaire EBOV-GP(2014) pseudotyped viruses: 4.5 g of pHIV-luciferase, 4.5 g of pCMV-R8.2, and 7.65 g of pcDNA3.1-Zaire EBOV-GP(2014); HIV-luc/VSV-G pseudotyped viruses: 4.5 g of pHIV-luciferase, 4.5 g of pCMV-R8.2, and 2.7 g of pCMV-VSV-G; HIV-luc/SARS-CoV-S pseudotyped viruses: 6.5 g of pHIV-luciferase, 8 g of pCMV-R8.2, and 20 g of pcDNA3.1-SARS-CoV-S; and HIV-luc/MERS-CoV-S.Teicoplanin derivatives such as dalbavancin, oritavancin, and telavancin can also inhibit the access of Ebola, MERS, and SARS viruses. Teicoplanin derivatives such as dalbavancin, oritavancin, and telavancin can also inhibit the entry of Ebola, MERS, and SARS viruses. Mechanistic studies showed that teicoplanin blocks Ebola virus entry by specifically inhibiting the activity of cathepsin L, opening a novel avenue for the development of additional glycopeptides as potential inhibitors of cathepsin L-dependent viruses. Notably, given that teicoplanin has routinely been used in the clinic with low toxicity, our work provides a promising prospect for the prophylaxis and treatment of Ebola, MERS, and SARS virus infection. genus have been identified, including (2,C6). Ebola virus infection leads to severe viral hemorrhagic fever in humans and non-human primates. In March 2014, outbreaks of Ebola viruses began in Guinea and caused over 28,000 cases of infection and over 11,000 deaths, which posed a severe threat to public health worldwide. The Ebola virus genome contains seven genes that encode the NP, VP35, VP40, glycoprotein (GP), VP30, VP24, and RNA-dependent RNA polymerase (L) virus proteins. To infect host cells, the GPs of Ebola viruses first bind to attachment molecules such as 1 integrins, DC-SIGNs, L-SIGNs, lectins, TIM-1s, Tyro3 family proteins, heparan sulfates, or folate receptor- (7,C13). Ebola viruses are then internalized by macropinocytosis and subsequently transported through the early and late endosomes and the endo/lysosomes (14,C16), where the Ebola virus GPs are cleaved by cathepsin L and subsequently cathepsin B to expose the receptor-binding domains (17). After binding the specific receptor NPC1, Ebola viruses release their genomes into the cytoplasm of the host cells (16, 18). Anti-EBOV vaccines and drugs are under extensive development. Two promising vaccines, rVSVG-EBOV-GP and cAd3-EBOV, have been shown to render non-human primates resistant to Ebola virus infections and are currently in clinical trials (19, 20). In addition, the anti-EBOV monoclonal antibody Zmapp, siRNAs, and other compounds that can inhibit Ebola virus infections have been developed (21,C24). Furthermore, several clinically approved drugs were also reported to inhibit Ebola virus infections (25, 26). However, because the IC50 values of those drugs were relatively high, more anti-EBOV drugs with potent inhibitory activity are urgently needed. To facilitate their identification, the method of high throughput screening of clinically approved drugs, which could be applied immediately in the clinic, is a reasonable approach. In this study, we identified teicoplanin and several other glycopeptide antibiotics as Ebola virus entry inhibitors with high effectiveness and low cytotoxicity, providing a encouraging means to effect the prophylaxis and treatment of Ebola disease infection. Experimental Methods Cell Tradition HEK293T, A549, HeLa, Huh7.5.1, and Madin-Darby canine kidney cell lines were taken care of in Dulbecco’s modified Eagle’s medium (Gibco) with 10% fetal calf serum (Gibco), 100 devices/ml penicillin, and 100 g/ml streptomycin (Gibco) at 37 C and 5% CO2. THP-1 cell lines were managed in RPMI1640 medium (Gibco) with 10% fetal calf serum, 100 devices/ml penicillin, and 100 g/ml streptomycin at 37 C and 5% CO2. Main human being umbilical vein endothelial cells were maintained in human being endothelial-SFM (Gibco) with 30 ng/ml endothelial cell growth product (Merck Millipore), 20 ng/ml recombinant human being FGF fundamental (146 amino acids) protein (R&D Systems), 20% fetal calf serum, 100 devices/ml penicillin, and 100 g/ml streptomycin at 37 C and 5% CO2. Plasmids GP sequence of Zaire EBOV-2014 was chemically synthesized and put into pcDNA3.1 plasmid. The pHIV-luciferase and pCMV-VSV-G plasmids were from Addgene, and the pCMV-R8.2 plasmid was kindly provided by Dr. Trono (27). The p4cis plasmid that encodes a luciferase reporter, VP40, GP and VP24, the pCAGGS-NP, pCAGGS-VP35, pCAGGS-VP30, pCAGGS-L, pCAGGS-T7, and Naspm trihydrochloride pCAGGS-Tim1 plasmids were produced as explained previously (28). Viruses Pseudotyped viruses were produced by the co-transfection of pHIV-luciferase, pCMV-R8.2, and different envelope plasmids into HEK293T cells that were 90% confluent inside a 10-cm plate with Lipofectamine 2000 by following a manufacturer’s instructions (Invitrogen). The amounts of plasmids were listed as follows: HIV-luc/Zaire EBOV-GP(2014) pseudotyped viruses: 4.5 g of pHIV-luciferase, 4.5 g of pCMV-R8.2, and 7.65 g of pcDNA3.1-Zaire EBOV-GP(2014); HIV-luc/VSV-G pseudotyped viruses: 4.5 g of pHIV-luciferase, 4.5 g of pCMV-R8.2, and 2.7 g of pCMV-VSV-G; HIV-luc/SARS-CoV-S pseudotyped viruses: 6.5 g of pHIV-luciferase, 8 g of pCMV-R8.2, and.THP-1 cell lines were maintained in RPMI1640 medium (Gibco) with 10% fetal calf serum, 100 devices/ml penicillin, and 100 g/ml streptomycin at 37 C and 5% CO2. of additional glycopeptides as potential inhibitors of cathepsin L-dependent viruses. Notably, given that teicoplanin offers routinely been used in the medical center with low toxicity, our work provides a encouraging prospect for the prophylaxis and treatment of Ebola, MERS, and SARS disease infection. genus have been recognized, including (2,C6). Ebola disease infection prospects to severe viral hemorrhagic fever in humans and non-human primates. In March 2014, outbreaks of Ebola viruses began in Guinea and caused over 28,000 instances of illness and over 11,000 deaths, which posed a severe threat to general public health worldwide. The Ebola disease genome consists of seven genes that encode the NP, VP35, VP40, glycoprotein (GP), VP30, VP24, and RNA-dependent RNA polymerase (L) disease proteins. To infect sponsor cells, the GPs of Ebola viruses 1st bind to attachment molecules such as 1 integrins, DC-SIGNs, L-SIGNs, lectins, TIM-1s, Tyro3 family proteins, heparan sulfates, or folate receptor- (7,C13). Ebola viruses are then internalized by macropinocytosis and consequently transported through the early and late endosomes and the endo/lysosomes (14,C16), where the Ebola virus GPs are cleaved by cathepsin L and consequently cathepsin B to expose the receptor-binding domains (17). After binding the specific receptor NPC1, Ebola viruses launch their genomes into the cytoplasm of the sponsor cells (16, 18). Anti-EBOV vaccines and medicines are under considerable development. Two encouraging vaccines, rVSVG-EBOV-GP and cAd3-EBOV, have been shown to render non-human primates resistant to Ebola disease infections and are currently in clinical tests (19, 20). In addition, the anti-EBOV monoclonal antibody Zmapp, siRNAs, and additional compounds that can inhibit Ebola disease infections have been developed (21,C24). Furthermore, several clinically approved medicines were also reported to inhibit Ebola disease infections (25, 26). However, because the IC50 ideals of those medicines were relatively high, more anti-EBOV medicines with potent inhibitory activity are urgently needed. To facilitate their recognition, the method of high throughput screening of clinically authorized drugs, which could be applied immediately in the medical center, is a reasonable approach. With this study, we recognized teicoplanin and several additional glycopeptide antibiotics as Ebola disease access inhibitors with high effectiveness and low cytotoxicity, providing a encouraging means to effect the prophylaxis and treatment of Ebola disease infection. Experimental Methods Cell Tradition HEK293T, A549, HeLa, Huh7.5.1, and Madin-Darby canine kidney cell lines were taken care of in Dulbecco’s modified Eagle’s medium (Gibco) with 10% fetal calf serum (Gibco), 100 devices/ml penicillin, and 100 g/ml streptomycin (Gibco) at 37 C and 5% CO2. THP-1 cell lines were managed in RPMI1640 medium (Gibco) with 10% fetal calf serum, 100 systems/ml penicillin, and 100 g/ml streptomycin at 37 C and 5% CO2. Principal individual umbilical vein endothelial cells had been maintained in individual endothelial-SFM (Gibco) with 30 ng/ml endothelial cell development dietary supplement (Merck Millipore), 20 ng/ml recombinant individual FGF simple (146 proteins) proteins (R&D Systems), 20% fetal leg serum, 100 systems/ml penicillin, and 100 g/ml streptomycin at 37 C and 5% CO2. Plasmids GP series of Zaire EBOV-2014 was chemically synthesized and placed into pcDNA3.1 plasmid. The pHIV-luciferase and pCMV-VSV-G plasmids had been extracted from Addgene, as well as the pCMV-R8.2 plasmid was kindly supplied by Dr. Trono (27). The p4cis plasmid that encodes a luciferase reporter, VP40, GP and VP24, the pCAGGS-NP, pCAGGS-VP35, pCAGGS-VP30, pCAGGS-L, pCAGGS-T7, and pCAGGS-Tim1 plasmids had been produced as defined previously (28). Infections Pseudotyped infections had been made by the co-transfection of pHIV-luciferase, pCMV-R8.2, and various envelope plasmids into HEK293T cells which were 90% confluent within a 10-cm dish with Lipofectamine 2000 by following manufacturer’s guidelines (Invitrogen). The levels of plasmids had been listed the following: HIV-luc/Zaire EBOV-GP(2014) pseudotyped infections: 4.5 g of pHIV-luciferase, 4.5 g of pCMV-R8.2, and 7.65 g of pcDNA3.1-Zaire EBOV-GP(2014); HIV-luc/VSV-G pseudotyped infections: 4.5 g of pHIV-luciferase, 4.5 g of pCMV-R8.2, and 2.7 g of pCMV-VSV-G; HIV-luc/SARS-CoV-S pseudotyped infections: 6.5 g of pHIV-luciferase, 8 g of pCMV-R8.2, and 20 g of pcDNA3.1-SARS-CoV-S; and HIV-luc/MERS-CoV-S pseudotyped infections: 4.5 g of pHIV-luciferase, 4.5 g of pCMV-R8.2, and 10 g of pcDNA3.1-MERS-CoV-S. After 48 h, the supernatants which contain the pseudotyped infections had been gathered and filtered through a 0.45-m pore-size filter (Pall) and stored at ?80 C until make use of. Ebola transcription- and replication-competent virus-like contaminants (trVLPs) had been made by the co-transfection of 250 ng.