Generation, Characterization and Epitope Mapping of Two Neutralizing and Protective Human Recombinant Antibodies against Influenza A H5N1 Viruses

The development of new therapeutic targets and strategies to control highly pathogenic avian influenza (HPAI) H5N1 virus infection in humans is urgently needed. Broadly cross-neutralizing recombinant human antibodies obtained from the survivors of H5N1 avian influenza provide an important role in immunotherapy for human H5N1 virus infection and definition of the critical epitopes for vaccine development.We have characterized two recombinant baculovirus-expressed human antibodies (rhAbs), AVFluIgG01 and AVFluIgG03, generated by screening a Fab antibody phage library derived from a patient recovered from infection with a highly pathogenic avian influenza A H5N1 clade 2.3 virus. AVFluIgG01 cross-neutralized the most of clade 0, clade 1, and clade 2 viruses tested, in contrast, AVFluIgG03 only neutralized clade 2 viruses. Passive immunization of mice with either AVFluIgG01 or AVFluIgG03 antibody resulted in protection from a lethal H5N1 clade 2.3 virus infection. Furthermore, through epitope mapping, we identify two distinct epitopes on H5 HA molecule recognized by these rhAbs and demonstrate their potential to protect against a lethal H5N1 virus infection in a mouse model.Importantly, localization of the epitopes recognized by these two neutralizing and protective antibodies has provided, for the first time, insight into the human antibody responses to H5N1 viruses which contribute to the H5 immunity in the recovered patient. These results highlight the potential of a rhAbs treatment strategy for human H5N1 virus infection and provide new insight for the development of effective H5N1 pandemic vaccines

Development of nuclear microsatellite markers to facilitate germplasm conservation and population genetics studies of five groups of tropical perennial plants with edible fruits and shoots: rambutan (Nephelium lappaceum L.), sapodilla (Manilkara zapota (L.) P. Royen), lychee (Litchi chinensis Sonn.), mangosteen (Garcinia mangostana Linn. and Garcinia cochinchinensis (Lour.) Choisy) and bamboo (Bambusa vulgaris Schrad. ex J.C. Wendl and Guadua angustifolia Kunth)

Simple sequence repeat (SSR) enriched libraries for five groups of tropical perennial plants with edible fruits and shoots were prepared and sequenced in a GS-FLX Roche 454: sapodilla (Manilkara zapota (L.) P. Royen), lychee (Litchi chinensis Sonn.), mangosteen (Garcinia mangostana Linn. and G. cochinchinensis (Lour.) Choisy), rambutan (Nephelium lappaceum L.), and bamboo (Bambusa vulgaris Schrad. ex J.C. Wendl and Guadua angustifolia Kunth). For SSR development, these species were organized by their common names in five groups. A total of 3870 SSR primer sets were designed, using capillary electrophoresis 1872 nuclear SSRs were tested on 4 to 10 DNA samples within each plant group, that is 384 loci for each of the four groups of fruit trees and 336 loci for the bamboo group. Only 7.9% of the primers tested did not result in amplifi- cation. All 1872 SSRs are provided, we highlight 178 SSRs (between 26 and 47 per group) considered top-quality polymorphic SSRs that amplified all the samples, had strong fluorescence signal, presented no stutters and showed minimum non-specific amplification or background fluorescence. A total of 66,057 contig sequences were submitted to GenBank Database. Markers presented here will be useful not only for conservation efforts in banks of germplasm, but also for in-depth analysis of population genetics which usually requires evaluation of large number of loci

Development of nuclear microsatellite markers to facilitate germplasm conservation and population genetics studies of five groups of tropical perennial plants with edible fruits and shoots: rambutan (Nephelium lappaceum L.), sapodilla (Manilkara zapota (L.) P. Royen), lychee (Litchi chinensis Sonn.), mangosteen (Garcinia mangostana Linn. and Garcinia cochinchinensis (Lour.) Choisy) and bamboo (Bambusa vulgaris Schrad. ex J.C. Wendl and Guadua angustifolia Kunth)

Simple sequence repeat (SSR) enriched libraries for five groups of tropical perennial plants with edible fruits and shoots were prepared and sequenced in a GS-FLX Roche 454: sapodilla (Manilkara zapota (L.) P. Royen), lychee (Litchi chinensis Sonn.), mangosteen (Garcinia mangostana Linn. and G. cochinchinensis (Lour.) Choisy), rambutan (Nephelium lappaceum L.), and bamboo (Bambusa vulgaris Schrad. ex J.C. Wendl and Guadua angustifolia Kunth). For SSR development, these species were organized by their common names in five groups. A total of 3870 SSR primer sets were designed, using capillary electrophoresis 1872 nuclear SSRs were tested on 4 to 10 DNA samples within each plant group, that is 384 loci for each of the four groups of fruit trees and 336 loci for the bamboo group. Only 7.9% of the primers tested did not result in amplification. All 1872 SSRs are provided, we highlight 178 SSRs (between 26 and 47 per group) considered topquality polymorphic SSRs that amplified all the samples, had strong fluorescence signal, presented no stutters and showed minimum non-specific amplification or background fluorescence. A total of 66,057 contig sequences were submitted to GenBank Database. Markers presented here will be useful not only for conservation efforts in banks of germplasm, but also for in-depth analysis of population genetics which usually requires evaluation of large number of loci

Development of a Large Set of Microsatellite Markers in Zapote Mamey (\u3ci\u3ePouteria sapota\u3c/i\u3e (Jacq.) H.E. Moore & Stearn) and Their Potential Use in the Study of the Species

Pouteria sapota is known for its edible fruits that contain unique carotenoids, as well as for its fungitoxic, anti-inflammatory and anti-oxidant activity. However, its genetics is mostly unknown, including aspects about its genetic diversity and domestication process. We did high-throughput sequencing of microsatellite-enriched libraries of P. sapota, generated 5223 contig DNA sequences, 1.8 Mbp, developed 368 microsatellites markers and tested them on 29 individuals from 10 populations (seven wild, three cultivated) from Mexico, its putative domestication center. Gene ontology BLAST analysis of the DNA sequences containing microsatellites showed potential association to physiological functions. Genetic diversity was slightly higher in cultivated than in the wild gene pool (HE = 0.41 and HE = 0.35, respectively), although modified Garza–Williamson Index and Bottleneck software showed evidence for a reduction in genetic diversity for the cultivated one. Neighbor Joining, 3D Principal Coordinates Analysis and assignment tests grouped most individuals according to their geographic origin but no clear separation was observed between wild or cultivated gene pools due to, perhaps, the existence of several admixed populations. The developed microsatellites have a great potential in genetic population and domestication studies of P. sapota but additional sampling will be necessary to better understand how the domestication process has impacted the genetic diversity of this fruit crop

Involvement of human and canine MRP1 and MRP4 in benzylpenicillin transport.

The blood-brain barrier (BBB) is a dynamic and complex interface between blood and the central nervous system (CNS). It protects the brain by preventing toxic substances from entering the brain but also limits the entry of therapeutic agents. ATP-binding cassette (ABC) efflux transporters are critical for the functional barrier and present a formidable impediment to brain delivery of therapeutic agents including antibiotics. The aim of this study was to investigate the possible involvement of multidrug resistance-associated protein 1 and 4 (MRP1 and MRP4), two ABC transporters, in benzylpenicillin efflux transport using wild-type (WT) MDCKII cells and cells overexpressing those human transporters, as well as non-selective and selective inhibitors. We found that inhibiting MRP1 or MRP4 significantly increased [3H]benzylpenicillin uptake in MDCKII-WT, -MRP1 or -MRP4 cells. Similar results were also found in HepG2 cells, which highly express MRP1 and MRP4, and hCMEC/D3 cells which express MRP1. The results indicate that human and canine MRP1 and MRP4 are involved in benzylpenicillin efflux transport. They could be potential therapeutic targets for improving the efficacy of benzylpenicillin for treating CNS infections since both MRP1 and MRP4 express at human blood-brain barrier

Role of Human Breast Cancer Related Protein versus P-Glycoprotein as an Efflux Transporter for Benzylpenicillin: Potential Importance at the Blood-Brain Barrier.

While the blood-brain barrier (BBB) protects the brain by controlling the access of solutes and toxic substances to brain, it also limits drug entry to treat central nervous system disorders. Many drugs are substrates for ATP-binding cassette (ABC) transporters at the BBB that limit their entry into the brain. The role of those transporters in limiting the entry of the widely prescribed therapeutic, benzylpenicillin, has produced conflicting results. This study investigated the possible potential involvement of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), two ABC transporters, in benzylpenicillin transport at BBB in human using MDCKII cells overexpressing those transporters as well as pharmacological inhibition. MDCKII cells overexpressing human BCRP (MDCKII-BCRP) but not those overexpressing human P-gp (MDCKII-MDR cells) had reduced [3H]benzylpenicillin uptake. Similarly, inhibiting BCRP increased [3H]benzylpenicillin uptake in MDCKII-BCRP cells, while inhibiting P-gp in MDCKII-MDR cells had no effect on uptake although there was evidence that benzylpenicillin is a substrate for canine P-gp. While inhibiting BCRP affected [3H]benzylpenicillin cell concentrations it did not affect transepithelial flux in MDCKII-BCRP cells. In summary, the results indicate that human BCRP and not human P-gp is involved in benzylpenicillin transport. However, targeting BCRP alone was not sufficient to alter transepithelial flux in MDCKII cells. Whether it would be sufficient to alter blood-to-brain flux at the human BBB remains to be investigated

Development of nuclear microsatellite markers to facilitate germplasm conservation and population genetics studies of five groups of tropical perennial plants with edible fruits and shoots: rambutan (Nephelium lappaceum L.), sapodilla (Manilkara zapota (L.) P. Royen), lychee (Litchi chinensis Sonn.), mangosteen (Garcinia mangostana Linn. and Garcinia cochinchinensis (Lour.) Choisy) and bamboo (Bambusa vulgaris Schrad. ex J.C. Wendl and Guadua angustifolia Kunth)

Simple sequence repeat (SSR) enriched libraries for five groups of tropical perennial plants with edible fruits and shoots were prepared and sequenced in a GS-FLX Roche 454: sapodilla (Manilkara zapota (L.) P. Royen), lychee (Litchi chinensis Sonn.), mangosteen (Garcinia mangostana Linn. and G. cochinchinensis (Lour.) Choisy), rambutan (Nephelium lappaceum L.), and bamboo (Bambusa vulgaris Schrad. ex J.C. Wendl and Guadua angustifolia Kunth). For SSR development, these species were organized by their common names in five groups. A total of 3870 SSR primer sets were designed, using capillary electrophoresis 1872 nuclear SSRs were tested on 4 to 10 DNA samples within each plant group, that is 384 loci for each of the four groups of fruit trees and 336 loci for the bamboo group. Only 7.9% of the primers tested did not result in amplification. All 1872 SSRs are provided, we highlight 178 SSRs (between 26 and 47 per group) considered topquality polymorphic SSRs that amplified all the samples, had strong fluorescence signal, presented no stutters and showed minimum non-specific amplification or background fluorescence. A total of 66,057 contig sequences were submitted to GenBank Database. Markers presented here will be useful not only for conservation efforts in banks of germplasm, but also for in-depth analysis of population genetics which usually requires evaluation of large number of loci

Development of nuclear microsatellite markers to facilitate germplasm conservation and population genetics studies of five groups of tropical perennial plants with edible fruits and shoots: rambutan (Nephelium lappaceum L.), sapodilla (Manilkara zapota (L.) P. Royen), lychee (Litchi chinensis Sonn.), mangosteen (Garcinia mangostana Linn. and Garcinia cochinchinensis (Lour.) Choisy) and bamboo (Bambusa vulgaris Schrad. ex J.C. Wendl and Guadua angustifolia Kunth)

Simple sequence repeat (SSR) enriched libraries for five groups of tropical perennial plants with edible fruits and shoots were prepared and sequenced in a GS-FLX Roche 454: sapodilla (Manilkara zapota (L.) P. Royen), lychee (Litchi chinensis Sonn.), mangosteen (Garcinia mangostana Linn. and G. cochinchinensis (Lour.) Choisy), rambutan (Nephelium lappaceum L.), and bamboo (Bambusa vulgaris Schrad. ex J.C. Wendl and Guadua angustifolia Kunth). For SSR development, these species were organized by their common names in five groups. A total of 3870 SSR primer sets were designed, using capillary electrophoresis 1872 nuclear SSRs were tested on 4 to 10 DNA samples within each plant group, that is 384 loci for each of the four groups of fruit trees and 336 loci for the bamboo group. Only 7.9% of the primers tested did not result in amplifi- cation. All 1872 SSRs are provided, we highlight 178 SSRs (between 26 and 47 per group) considered top-quality polymorphic SSRs that amplified all the samples, had strong fluorescence signal, presented no stutters and showed minimum non-specific amplification or background fluorescence. A total of 66,057 contig sequences were submitted to GenBank Database. Markers presented here will be useful not only for conservation efforts in banks of germplasm, but also for in-depth analysis of population genetics which usually requires evaluation of large number of loci