Profiling the differentially expressed genes in two rice varieties during rapid grain-filling stages

Grain filling is an important agronomic trait, which directly affects the final yield of rice. Partially filled and empty rice grains are among the factors that limit the yield of MR219, one of the highest yielding rice varieties in Malaysia. In this study, the NSF 20 K rice oligonucleotide array, which contains 20,000 70-mer oligonucleotide probes, was used for direct comparison of the transcriptomes of MR219 and MR84 (a rice variety that has higher percentage of filled grains compared to MR219), during rapid grain-filling period at 5 and 10 days after fertilization (DAF). A total of 155 and 233 genes were differentially expressed in MR219 compared to MR84 at 5 and 10 DAF, respectively; and 9 of these expression ratios were tested using quantitative real-time RT PCR. Among the differentially expressed genes identified were those encoding hexokinase, various sugar transporters, GSDL-like lipase/acylhydrolase, brassinosteroid-insensitive 1-associated receptor kinase 1 precursor and homeobox protein GLABRA2, which were analyzed by real-time RT PCR in this study. The differences demonstrated by these genes in their transcript levels and profiles, between the two rice varieties understudied at different stages of grain filling may contribute to the formulation of hypotheses toward the understanding of poor percentage of filled grains in MR219

Optimization of multimeric human papillomavirus L2 vaccines.

We sought to define the protective epitopes within the amino terminus of human papillomavirus (HPV) type 16 minor capsid protein L2. Passive transfer of mice with rabbit antisera to HPV16 L2 peptides 17-36, 32-51 and 65-81 provided significant protection against vaginal HPV16 challenge, whereas antisera to 47-66, 108-120 or 373-392 did not. Vaccination with L1 virus-like particles induces a high titer, but generally type-restricted neutralizing antibody response. Conversely, vaccination with L2 11-88, especially multimers thereof, induces antibodies that neutralize a broad range of papillomavirus types, albeit at lower titers than for L1 VLP. With the intent of enhancing the immunogenicity and the breadth of protection by focusing the immune response to the key protective epitopes, we designed L2 fusion proteins consisting of residues ∼11-88 of eight divergent mucosal HPV types 6, 16, 18, 31, 39, 51, 56, 73 (11-88×8) or residues ∼13-47 of fifteen HPV types (13-47×15). The 11-88×8 was significantly more immunogenic than 13-47×15 in Balb/c mice regardless of the adjuvant used, suggesting the value of including the 65-81 protective epitope in the vaccine. Since the L2 47-66 peptide antiserum failed to elicit significant protection, we generated an 11-88×8 construct deleted for this region in each subunit (11-88×8Δ). Mice were vaccinated with 11-88×8 and 11-88×8Δ to determine if deletion of this non-protective epitope enhanced the neutralizing antibody response. However, 11-88×8Δ was significantly less immunogenic than 11-88×8, and even the addition of a known T helper epitope, PADRE, to the construct (11-88×8ΔPADRE) failed to recover the immunogenicity of 11-88×8 in C57BL/6 mice, suggesting that while L2 47-66 is not a critical protective or T helper epitope, it nevertheless contributes to the immunogenicity of the L2 11-88×8 multimer vaccine

Vaginal Challenge of Balb/c and C57BL/6 mice vaccinated with 11–88×8, 11–88×8Δ and 11–88×8ΔPADRE (11–88×8ΔP) in Alum+MPL.

<p>Balb/c or C57BL/6 mice were vaccinated three times at 2 week intervals with the indicated 11–88×8, 11–88×8Δ and 11–88×8ΔPADRE using Alum+MPL as adjuvant. The mice were vaginally challenged one month later using HPV16 pseudovirions carrying a luciferase reporter. Infection is measured as bioluminescence.</p

Passive transfer of HPV16 L2 peptide antisera protects mice against vaginal challenge with HPV16.

<p>Mice were injected i.p. with 0.1 ml buffer or rabbit antiserum to KLH-coupled HPV16 L2 peptides encompassing residues 17–36, residues 32–51, residues 47–66, residues 65–81, 108–120, residues 373–392, or antiserum to HPV16 L1 VLP. One day later the mice were challenged intra-vaginally with HPV16 pseudovirion encoding luciferase.</p

Peptide blockade of 11–88×8 antiserum.

<p>A. Sera of Balb/c mice immunized three times with 11–88×8 or 11–88×8Δ using Alum+MPL as adjuvant were harvested two weeks after the final boost. The antisera diluted 1∶100 were reacted with microtiter plates coated with 1 µg of two HPV16 L2 peptides encompassing residues 43–62 and 48–67. After washing, specific reactivity was measured by ELISA using peroxidase-linked anti-mouse IgG. B. Pooled sera of Balb/c mice immunized three times with 11–88×8 using Alum+MPL as adjuvant were harvested either pre-immunization (Pre-bleed) or two weeks after the final boost (×8 Sera). The antiserum to 11–88×8 was diluted 1∶50 in a total volume of 200 µl and incubated with 7 µg of HPV16 L2 peptide encompassing residues 13–32, 18–37, 23–42, 28–47, 33–52, 38–57, 43–62, 48–67, 53–72, 58–77, 63–82 or 71–90 for an hour prior to mixing with HPV16 pseudovirions carrying a SEAP reporter for a further hour at ambient temperature and subsequent infection of 293TT cells. Infection was measured as optical density, and only HPV16 L2 pepitdes 13–32 and 18–37 substantially blocked neutralization by 1∶3200 dilution of antiserum to 11–88×8. C. Sera of Balb/c mice immunized three times with 11–88×8 using Alum+MPL as adjuvant were harvested two weeks after the final boost and pooled (×8). The 11–88×8 antiserum was mixed with two HPV16 L2 peptides encompassing residues 43–62 and 48–67 (+Peptide) in the same ratios as in B. The 11–88×8 antiserum was administered i.p. either alone or pre-mixed with peptide in volumes of 20 µl, 5 µl or 2 µl to naïve mice (in groups of 5). Separate groups of mice received 200 µg each of antibody affinity purified with protein G columns from the sera of rabbits hyper-immunized with HPV16 L2 peptides 17–36, 47–66 or 373–392. All groups of mice (except the Background group) were subsequently challenged intra-vaginally with HPV16 pseudovirions carrying a luciferase reporter. Infection was assessed by measuring bioluminescence three days later.</p

HPV in vitro neutralization titers of sera of mice vaccinated with 13–47×15 and 11–88×8 in different adjuvants.

<p>Balb/c mice were vaccinated three times at 2 week intervals with the indicated 13–47×15 and 11–88×8 proteins formulated in alum, alum+MPL, alum+CpG or GPI-0100. Sera were harvested two weeks later for testing in vitro neutralization titers against HPV16 (A), HPV18 (B), HPV45 (C) and HPV58 (D) pseudovirions, or HPV16 at 3 months after the final vaccination (E).</p