Hepatitis C virus(HCV)infection represents an important public health problem worldwide.Reduction of HCV morbidity and mortality is a current challenge owned to several viral and host factors.Virus molecular evolution...Hepatitis C virus(HCV)infection represents an important public health problem worldwide.Reduction of HCV morbidity and mortality is a current challenge owned to several viral and host factors.Virus molecular evolution plays an important role in HCV transmission,disease progression and therapy outcome.The high degree of genetic heterogeneity characteristic of HCV is a key element for the rapid adaptation of the intrahost viral population to different selection pressures(e.g.,host immune responses and antiviral therapy).HCV molecular evolution is shaped by different mechanisms including a high mutation rate,genetic bottlenecks,genetic drift,recombination,temporal variations and compartmentalization.These evolutionary processes constantly rearrange the composition of the HCV intrahost population in a staging manner.Remarkable advances in the understanding of the molecular mechanism controlling HCV replication have facilitated the development of a plethora of direct-acting antiviral agents against HCV.As a result,superior sustained viral responses have been attained.The rapidly evolving field of anti-HCV therapy is expected to broad its landscape even further with newer,more potent antivirals,bringing us one step closer to the interferon-free era.展开更多
Agave guiengola Gentry is an endemic plant from a very small locality in Oaxaca,Mexico.Its conservation status is fragile and can rapidly worsen.Because of its scarcity,this agave has been used solely for ornamental p...Agave guiengola Gentry is an endemic plant from a very small locality in Oaxaca,Mexico.Its conservation status is fragile and can rapidly worsen.Because of its scarcity,this agave has been used solely for ornamental purposes,but it could have other uses if more plants were available.In vitro propagation by enhanced axillary sprouting from stem segments was attained using Murashige and Skoog Basal Medium(MS)as well as basal medium supplemented with cytokinins 6-Benzylaminopurine(BA)or 6-(γ,γ-Dimethylallylamino)purine(2iP).The best treatment for shoot induction in semisolid medium consisted in MS supplemented with 2 mg l^(–1) BA,obtaining a mean of 3.7 shoots per explant.Other interesting responses were observed,such as nodular callus induction using combinations of BA and 2,4-Dichlorophenoxyacetic acid(2,4-D);root induction without Plant Growth Regulators(PGR);and generation of shoot clusters.These clusters constituted an excellent explant for micropropagation in temporary immersion bioreactors,obtaining a propagation rate of 43 shoots per explant with 1 min immersion and 6 h immersion frequencies.All new plants rooted and survived the transfer to soil.This study developed an in vitro propagation scheme to produce individuals that can be used either for reforestation,economical purposes,or to carry out studies in this species to assess its full potential,avoiding exploitation from wild plants.展开更多
BACKGROUND Identifying a potential single monogenetic disorder in healthy couples is costly due to the Assisted Reproduction facilities'current methodology for screening,which focuses on the detecting multiple gen...BACKGROUND Identifying a potential single monogenetic disorder in healthy couples is costly due to the Assisted Reproduction facilities'current methodology for screening,which focuses on the detecting multiple genetic disorders at once.Here,we report the successful application of a low-cost and fast preimplantation genetic testing for monogenic/single gene defects(PGT-M)approach for detecting propionic acidemia(PA)in embryos obtained from a confirmed heterozygous propionyl-CoA carboxylase alpha subunit(PCCA)couple.CASE SUMMARY A fertile 32-years old Mexican couple with denied consanguinity sought antenatal genetic counseling.They were suspected obligate PA carriers due to a previous deceased PA male newborn with an unknown PCCA/propionyl-CoA carboxylase beta subunit(PCCB)genotype.Next-Generation Sequencing revealed a heterozygous genotype for a pathogenic PCCA variant(c.2041-1G>T,ClinVar:RCV-000802701.1;dbSNP:rs1367867218)in both parents.The couple requested in vitro fertilization(IVF)and PGT-M for PA.From IVF,12 oocytes were collected and fertilized,of which two resulted in high-quality embryos.Trophectoderm biopsies and Whole Genome Amplification by a fragmentation/amplification-based method were performed and revealed that the two embryos were euploid.Endpoint polymerase chain reaction and further Sanger sequencing of the exon-intron borders revealed a wild-type PCCA male embryo and a heterozygous c.2041-1G>T female embryo.Both embryos were transferred,resulting in a clinical pregnancy and the delivery of a healthy male newborn(38 wk,weight:4080 g,length:49 cm,APGAR 9/9).The absence of PA was confirmed by expanded newborn screening.CONCLUSION We show that using PGT-M with Whole Genome Amplification templates,coupled with IVF,can reduce the transmission of a pathogenic variant of the PCCA gene.展开更多
To increase protein stability and test protein function, three double-cysteine mutations were individually introduced by protein engineering into the cysteine-free Cry3Aa δ-endotoxin from Bacillus thuringiensis. Thes...To increase protein stability and test protein function, three double-cysteine mutations were individually introduced by protein engineering into the cysteine-free Cry3Aa δ-endotoxin from Bacillus thuringiensis. These mutations were designed to create disulfide bonds between α-helices 2 and 5 (positions 110 - 193), and α-helices 5 and 7 (positions 195 - 276 and 198 - 276). Comparison of the CD spectra of the wild-type and the double-cysteine mutant proteins indicates a tighter helical packing consistent with formation of at least two of the disulfide bonds between the central and the outer helices. Thermal stability analysis indi-cates that potential covalent linkages between the central α-helix 5 and the other helices increase resistance to thermal denaturation by 10?C to 14?C com-pared to the thermal stability of the wild-type protein. Spectroscopic analysis of the disulfide-specific absorbance band indicates that the double mutant proteins are more stable to temperature and denaturant (guanidine hydrochloride) than the wild-type protein, as a result of the formation of two of the disulfide bridges. These results indicate that the double muta-tions M110C/F193C and A198C/V276C successfully established disulfide bonds, resulting in a more stable structure of the entire toxin. Despite the increase in stability and structural changes introduced by the disulfide bonds, no effect on toxicity was observed. A possible mechanism involving the insertion of all of domain I of Cry3Aa toxin into the target membrane accounts for these observations.展开更多
The probative value of animal forensic genetic evidence relies on laboratory accuracy and reliability.Inter-laboratory comparisons allow laboratories to evaluate their performance on specific tests and analyses and to...The probative value of animal forensic genetic evidence relies on laboratory accuracy and reliability.Inter-laboratory comparisons allow laboratories to evaluate their performance on specific tests and analyses and to continue to monitor their output.The International Society for Animal Genetics(ISAG)administered animal forensic comparison tests(AFCTs)in 2016 and 2018 to assess the limitations and capabilities of laboratories offering forensic identification,parentage and species determination services.The AFCTs revealed that analyses of low DNA template concentrations(≤300 pg/μL)constitute a significant challenge that has prevented many laboratories from reporting correct identification and parentage results.Moreover,a lack of familiarity with species testing protocols,interpretation guidelines and representative databases prevented over a quarter of the participating laboratories from submitting correct species determination results.Several laboratories showed improvement in their genotyping accuracy over time.However,the use of forensically validated standards,such as a standard forensic short tandem repeat(STR)kit,preferably with an allelic ladder,and stricter guidelines for STR typing,may have prevented some common issues from occurring,such as genotyping inaccuracies,missing data,elevated stutter products and loading errors.The AFCTs underscore the importance of conducting routine forensic comparison tests to allow laboratories to compare results from each other.Laboratories should keep improving their scientific and technical capabilities and continuously evaluate their personnel’s proficiency in critical techniques such as low copy number(LCN)analysis and species testing.Although this is the first time that the ISAG has conducted comparison tests for forensic testing,findings from these AFCTs may serve as the foundation for continuous improvements of the overall quality of animal forensic genetic testing.展开更多
基金Supported by Project Salud 2012-C01-181585,CONACYT and PAPIIT TA200112,Dirección General de Asuntos del Personal Academico,Universidad Nacional Autónoma de México(in part)Argentine National Agency for Scientific and Technology Promotion(PICT 2012 No804)National Research Council(CONICET,PIP 2010 No51)
文摘Hepatitis C virus(HCV)infection represents an important public health problem worldwide.Reduction of HCV morbidity and mortality is a current challenge owned to several viral and host factors.Virus molecular evolution plays an important role in HCV transmission,disease progression and therapy outcome.The high degree of genetic heterogeneity characteristic of HCV is a key element for the rapid adaptation of the intrahost viral population to different selection pressures(e.g.,host immune responses and antiviral therapy).HCV molecular evolution is shaped by different mechanisms including a high mutation rate,genetic bottlenecks,genetic drift,recombination,temporal variations and compartmentalization.These evolutionary processes constantly rearrange the composition of the HCV intrahost population in a staging manner.Remarkable advances in the understanding of the molecular mechanism controlling HCV replication have facilitated the development of a plethora of direct-acting antiviral agents against HCV.As a result,superior sustained viral responses have been attained.The rapidly evolving field of anti-HCV therapy is expected to broad its landscape even further with newer,more potent antivirals,bringing us one step closer to the interferon-free era.
基金This work was supported financially by project AGS-2015-02-01-267656,Fondo Mixto CONACYT-Gobierno del Estado de Aguascalientes,and project PIBT-18-2,Universidad Autónoma de Aguascalientes,México.
文摘Agave guiengola Gentry is an endemic plant from a very small locality in Oaxaca,Mexico.Its conservation status is fragile and can rapidly worsen.Because of its scarcity,this agave has been used solely for ornamental purposes,but it could have other uses if more plants were available.In vitro propagation by enhanced axillary sprouting from stem segments was attained using Murashige and Skoog Basal Medium(MS)as well as basal medium supplemented with cytokinins 6-Benzylaminopurine(BA)or 6-(γ,γ-Dimethylallylamino)purine(2iP).The best treatment for shoot induction in semisolid medium consisted in MS supplemented with 2 mg l^(–1) BA,obtaining a mean of 3.7 shoots per explant.Other interesting responses were observed,such as nodular callus induction using combinations of BA and 2,4-Dichlorophenoxyacetic acid(2,4-D);root induction without Plant Growth Regulators(PGR);and generation of shoot clusters.These clusters constituted an excellent explant for micropropagation in temporary immersion bioreactors,obtaining a propagation rate of 43 shoots per explant with 1 min immersion and 6 h immersion frequencies.All new plants rooted and survived the transfer to soil.This study developed an in vitro propagation scheme to produce individuals that can be used either for reforestation,economical purposes,or to carry out studies in this species to assess its full potential,avoiding exploitation from wild plants.
文摘BACKGROUND Identifying a potential single monogenetic disorder in healthy couples is costly due to the Assisted Reproduction facilities'current methodology for screening,which focuses on the detecting multiple genetic disorders at once.Here,we report the successful application of a low-cost and fast preimplantation genetic testing for monogenic/single gene defects(PGT-M)approach for detecting propionic acidemia(PA)in embryos obtained from a confirmed heterozygous propionyl-CoA carboxylase alpha subunit(PCCA)couple.CASE SUMMARY A fertile 32-years old Mexican couple with denied consanguinity sought antenatal genetic counseling.They were suspected obligate PA carriers due to a previous deceased PA male newborn with an unknown PCCA/propionyl-CoA carboxylase beta subunit(PCCB)genotype.Next-Generation Sequencing revealed a heterozygous genotype for a pathogenic PCCA variant(c.2041-1G>T,ClinVar:RCV-000802701.1;dbSNP:rs1367867218)in both parents.The couple requested in vitro fertilization(IVF)and PGT-M for PA.From IVF,12 oocytes were collected and fertilized,of which two resulted in high-quality embryos.Trophectoderm biopsies and Whole Genome Amplification by a fragmentation/amplification-based method were performed and revealed that the two embryos were euploid.Endpoint polymerase chain reaction and further Sanger sequencing of the exon-intron borders revealed a wild-type PCCA male embryo and a heterozygous c.2041-1G>T female embryo.Both embryos were transferred,resulting in a clinical pregnancy and the delivery of a healthy male newborn(38 wk,weight:4080 g,length:49 cm,APGAR 9/9).The absence of PA was confirmed by expanded newborn screening.CONCLUSION We show that using PGT-M with Whole Genome Amplification templates,coupled with IVF,can reduce the transmission of a pathogenic variant of the PCCA gene.
文摘To increase protein stability and test protein function, three double-cysteine mutations were individually introduced by protein engineering into the cysteine-free Cry3Aa δ-endotoxin from Bacillus thuringiensis. These mutations were designed to create disulfide bonds between α-helices 2 and 5 (positions 110 - 193), and α-helices 5 and 7 (positions 195 - 276 and 198 - 276). Comparison of the CD spectra of the wild-type and the double-cysteine mutant proteins indicates a tighter helical packing consistent with formation of at least two of the disulfide bonds between the central and the outer helices. Thermal stability analysis indi-cates that potential covalent linkages between the central α-helix 5 and the other helices increase resistance to thermal denaturation by 10?C to 14?C com-pared to the thermal stability of the wild-type protein. Spectroscopic analysis of the disulfide-specific absorbance band indicates that the double mutant proteins are more stable to temperature and denaturant (guanidine hydrochloride) than the wild-type protein, as a result of the formation of two of the disulfide bridges. These results indicate that the double muta-tions M110C/F193C and A198C/V276C successfully established disulfide bonds, resulting in a more stable structure of the entire toxin. Despite the increase in stability and structural changes introduced by the disulfide bonds, no effect on toxicity was observed. A possible mechanism involving the insertion of all of domain I of Cry3Aa toxin into the target membrane accounts for these observations.
文摘The probative value of animal forensic genetic evidence relies on laboratory accuracy and reliability.Inter-laboratory comparisons allow laboratories to evaluate their performance on specific tests and analyses and to continue to monitor their output.The International Society for Animal Genetics(ISAG)administered animal forensic comparison tests(AFCTs)in 2016 and 2018 to assess the limitations and capabilities of laboratories offering forensic identification,parentage and species determination services.The AFCTs revealed that analyses of low DNA template concentrations(≤300 pg/μL)constitute a significant challenge that has prevented many laboratories from reporting correct identification and parentage results.Moreover,a lack of familiarity with species testing protocols,interpretation guidelines and representative databases prevented over a quarter of the participating laboratories from submitting correct species determination results.Several laboratories showed improvement in their genotyping accuracy over time.However,the use of forensically validated standards,such as a standard forensic short tandem repeat(STR)kit,preferably with an allelic ladder,and stricter guidelines for STR typing,may have prevented some common issues from occurring,such as genotyping inaccuracies,missing data,elevated stutter products and loading errors.The AFCTs underscore the importance of conducting routine forensic comparison tests to allow laboratories to compare results from each other.Laboratories should keep improving their scientific and technical capabilities and continuously evaluate their personnel’s proficiency in critical techniques such as low copy number(LCN)analysis and species testing.Although this is the first time that the ISAG has conducted comparison tests for forensic testing,findings from these AFCTs may serve as the foundation for continuous improvements of the overall quality of animal forensic genetic testing.
