Engineering of papaya mosaic virus (PapMV) nanoparticles through fusion of the HA11 peptide to several putative surface-exposed sites

Papaya mosaic virus has been shown to be an efficient adjuvant and vaccine platform in the design and improvement of innovative flu vaccines. So far, all fusions based on the PapMV platform have been located at the C-terminus of the PapMV coat protein. Considering that some epitopes might interfere with the self-assembly of PapMV CP when fused at the C-terminus, we evaluated other possible sites of fusion using the influenza HA11 peptide antigen. Two out of the six new fusion sites tested led to the production of recombinant proteins capable of self assembly into PapMV nanoparticles; the two functional sites are located after amino acids 12 and 187. Immunoprecipitation of each of the successful fusions demonstrated that the HA11 epitope was located at the surface of the nanoparticles. The stability and immunogenicity of the PapMV-HA11 nanoparticles were evaluated, and we could show that there is a direct correlation between the stability of the nanoparticles at 37°C (mammalian body temperature) and the ability of the nanoparticles to trigger an efficient immune response directed towards the HA11 epitope. This strong correlation between nanoparticle stability and immunogenicity in animals suggests that the stability of any nanoparticle harbouring the fusion of a new peptide should be an important criterion in the design of a new vaccine.     

Effect of methylmalonate on ketone body metabolism in developing rat brain

The oxidation of 3-hydroxy[3-¹⁴C]butyrate to CO₂ and its incorporation into cerebral lipids by cortex slices from one-week old rats were markedly inhibited by methylmalonate. However, methylmalonate had no effect on the metabolism of labelled aceto- acetate, glucose and acetate by brain slices. Addition of propionate in the incubation medium reduced cerebral lipogenesis from labelled 3-hydroxybutyrate and acetate. Acute methylmalonic acidemia induced in one-week old pups by injecting 3% methylmalonate solution caused a reduction in the incorporation of labelled 3-hydroxybutyrate into cerebral lipids. However, acute methylmalonic acidemia had no effect on cerebral lipogensis $\text{in vivo}$ from labelled acetate. These findings show (i) the conversion of 3-hydroxybutyrate to acetoacetate by 3-hydroxybutyrate dehydrogenase in the brain is inhibited by methylmalonate, and (ii) an inhibition of cerebral lipid synthesis by propionate, which also accumulates in patients with methylmalonic aciduria.     

Quantifying multiple pressure interactions affecting populations of a recreationally and commercially important freshwater fish

The expanding human global footprint and growing demand for freshwater have placed tremendous stress on inland aquatic ecosystems. Aichi Target 10 of the Convention on Biological Diversity aims to minimize anthropogenic pressures affecting vulnerable ecosystems, and pressure interactions are increasingly being incorporated into environmental management and climate change adaptation strategies. In this study, we explore how climate change, overfishing, forest disturbance, and invasive species pressures interact to affect inland lake walleye (Sander vitreus) populations. Walleye support subsistence, recreational, and commercial fisheries and are one of most sought‐after freshwater fish species in North America. Using data from 444 lakes situated across an area of 475 000 km² in Ontario, Canada, we apply a novel statistical tool, R‐INLA, to determine how walleye biomass deficit (carrying capacity—observed biomass) is impacted by multiple pressures. Individually, angling activity and the presence of invasive zebra mussels (Dreissena polymorpha) were positively related to biomass deficits. In combination, zebra mussel presence interacted negatively and antagonistically with angling activity and percentage decrease in watershed mature forest cover. Velocity of climate change in growing degree days above 5°C and decrease in mature forest cover interacted to negatively affect walleye populations. Our study demonstrates how multiple pressure evaluations can be conducted for hundreds of populations to identify influential pressures and vulnerable ecosystems. Understanding pressure interactions is necessary to guide management and climate change adaptation strategies, and achieve global biodiversity targets.     

Ecology of Microbial Saprophytes and Pathogens in Tissue Culture and Field-Grown Plants: Reasons for Contamination Problems In Vitro

This review compares published surveys of microbial populations in plant tissue and cell cultures with the microbial saprophytes and pathogens found on field grown plants and microbial populations in the laboratory environment. From this comparison and the measured reduction in contamination after improvements in working practices in the laboratory, conclusions can be drawn about the importance of the explant and the laboratory as sources of contamination.

Mechanisms of pathogenicity in vitro are described to explain why bacteria, fungi, and yeasts that are not pathogenic to plants in the field become pathogens in plant tissue cultures. Conversely, plant metabolism and its effect on the tissue culture environment are described to explain why prokaryotes, viruses, and viroids that cause disease in the field can stay latent in vitro.

Detection methods for latent contaminants in plant tissue cultures are summarized, and the strategies and methods for prevention or treatment of contamination are discussed.     

Liposomal Delivery Systems for Encapsulation of Ferrous Sulfate: Preparation and Characterization

Liposomal delivery systems for water-soluble bioactives were prepared using the pro-liposome and the microfluidization technologies. Iron, an essential micronutrient as ferrous sulfate and ascorbic acid, as an antioxidant for iron were encapsulated in the liposomes. Liposomes prepared by the microfluidization technology using 6% (w/w) concentration of the lipid encapsulated with ferrous sulfate and ascorbic acid had particle size distributions around 150 to 200 nm, whereas liposomes from the pro-liposome technology resulted in particle sizes of about 5 μm. The encapsulation efficiency of ferrous sulfate was 58% for the liposomes prepared by the microfluidization using 6% (w/w) lipid and 7.5% of ferrous sulfate concentrations, and it was 11% for the liposomes from pro-liposome technology using 1.5% (w/v) lipid and 15% of ferrous-sulfate concentration. Both the liposomes exhibited similar levels of oxidative stability, demonstrating the feasibility of microfluidization-based liposomal delivery systems for large-scale food/nutraceutical applications.     

The elastic properties of valve interstitial cells undergoing pathological differentiation

Increasing evidence indicates that the progression of calcific aortic valve disease (CAVD) is influenced by the mechanical forces experienced by valvular interstitial cells (VICs) embedded within the valve matrix. The ability of VICs to sense and respond to tissue-level mechanical stimuli depends in part on cellular-level biomechanical properties, which may change with disease. In this study, we used micropipette aspiration to measure the instantaneous elastic modulus of normal VICs and of VICs induced to undergo pathological differentiation in vitro to osteoblast or myofibroblast lineages on compliant and stiff collagen gels, respectively. We found that VIC elastic modulus increased after subculturing on stiff tissue culture-treated polystyrene and with pathological differentiation on the collagen gels. Fibroblast, osteoblast, and myofibroblast VICs had distinct cellular-level elastic properties that were not fully explained by substrate stiffness, but were correlated with α-smooth muscle actin expression levels. C-type natriuretic peptide, a peptide expressed in aortic valves in vivo, prevented VIC stiffening in vitro, consistent with its ability to inhibit α-smooth muscle actin expression and VIC pathological differentiation. These data demonstrate that VIC phenotypic plasticity and mechanical adaptability are linked and regulated both biomechanically and biochemically, with the potential to influence the progression of CAVD.     

The role of the soil seed and seedling bank in the regeneration of diverse plant communities in the subtropical Ailao Mountains,Southwest China

We compared species composition and diversity of the soil seed and seedling banks in three secondary vegetation types (shrubland, Populus bonatii forest, Lithocarpus regrowth forest) and a primary old-growth forest in the subtropical Ailao Mountains of southwestern China to clarify the importance of seed and seedling banks for forest dynamics. The average species richness was the highest in soil samples from the shrubland (26.80 ± 1.98), and the lowest from the primary forest (9.93 ± 0.50). The density of germinable tree seeds increased from the secondary vegetation to the primary forest, and the density of shrub, forb, and graminoid seeds decreased significantly. The most abundant seedlings recorded in soil samples were light-demanding species in the shrubland and Populus bonatii forest. For ground flora, the number of shrub seedlings strongly decreased with the increase in stand age, and shade-tolerant tree seedlings tended to increase. The species similarity between the seed bank and the aboveground vegetation in all sites was low (Sørensen’s index = 0.11–0.33), however, the shrubland had higher similarity compared with the other three plant communities. In the primary forest, light-demanding woody species dominated in soil seed banks, while shade-tolerant species dominated in the overstory and the forest floor. In the primary forest, seedlings of dominant tree species were rare in the understory, and no seeds of the dominant species were found in the soil. Results indicated that the early stages of vegetation recovery should take into account the possibility of recovering soil seed bank processes. However, colonization and establishment of tree seedlings will be difficult once a primary forest is destroyed.     

Inhibition of In Vivo HIV Infection in Humanized Mice by Gene Therapy of Human Hematopoietic Stem Cells with a Lentiviral Vector Encoding a Broadly Neutralizing Anti-HIV Antibody

Due to the inherent immune evasion properties of the HIV envelope, broadly neutralizing HIV-specific antibodies capable of suppressing HIV infection are rarely produced by infected individuals. We examined the feasibility of utilizing genetic engineering to circumvent the restricted capacity of individuals to endogenously produce broadly neutralizing HIV-specific antibodies. We constructed a single lentiviral vector that encoded the heavy and light chains of 2G12, a broadly neutralizing anti-HIV human antibody, and that efficiently transduced and directed primary human B cells to secrete 2G12. To evaluate the capacity of this approach to provide protection from in vivo HIV infection, we used the humanized NOD/SCID/γ_c^null mouse model, which becomes populated with human B cells, T cells, and macrophages after transplantation with human hematopoietic stem cells (hu-HSC) and develops in vivo infection after inoculation with HIV. The plasma of the irradiated NOD/SCID/γ_c^null mice transplanted with hu-HSC transduced with the 2G12-encoding lentivirus contained 2G12 antibody, likely secreted by progeny human lymphoid and/or myeloid cells. After intraperitoneal inoculation with high-titer HIV-1_JR-CSF, mice engrafted with 2G12-transduced hu-HSC displayed marked inhibition of in vivo HIV infection as manifested by a profound 70-fold reduction in plasma HIV RNA levels and an almost 200-fold reduction in HIV-infected human cell numbers in mouse spleens, compared to control hu-HSC-transplanted NOD/SCID/γ_c^null mice inoculated with equivalent high-titer HIV-1_JR-CSF. These results support the potential efficacy of this new gene therapy approach of using lentiviral vectors encoding a mixture of broadly neutralizing HIV antibodies for the treatment of HIV infection, particularly infection with multiple-drug-resistant isolates.While broadly neutralizing human immunodeficiency virus (HIV)-specific antibodies have the capacity to prevent or suppress HIV infection, they are rarely produced by infected individuals, thereby markedly compromising the ability of the humoral response to control HIV infection (reviewed in reference 28). The high degree of sequence variability in the gp120 structure limits the number of highly conserved epitopes available for targeting by neutralizing antibodies (40). In addition, HIV utilizes several mechanisms to shield the limited number of conserved neutralizing epitopes from the potentially potent antiviral effects of HIV envelope-specific antibodies (14). First, the envelope protein is heavily glycosylated, and the linkage of the most immunoreactive envelope peptide structures to poorly immunogenic glycans shields them from antibody binding (37). Second, exposure of neutralizing epitopes not protected from antibody binding by glycosylation is greatly reduced by trimerization of the gp120-gp41 structure (5). Third, susceptibility of other neutralizing epitopes to antibodies is greatly reduced by limiting their accessibility to antibody binding to the brief transient phase of conformational changes that occur only during binding of the envelope protein to its cellular receptors, CD4 and CCR5 or CXCR4 (41). These intrinsic structural features of gp120 greatly reduce the capacity of natural HIV infection or vaccination to generate broadly neutralizing antibodies able to prevent or control infection. Despite these constraints, rare human antibodies with broad anti-HIV neutralizing activity, i.e., 2G12, b12, 2F5, and 4E10, have been isolated (2).The capacity of passive immunization with neutralizing antibodies to prevent infection was suggested by challenge studies demonstrating that transferred neutralizing antibodies protected monkeys from infection by simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus (SHIV) (15). These studies were extended to humans, including several studies that examined the effect of passive immunotherapy using 2G12, 2F5, and 4E10 on inhibition of HIV replication in infected individuals (20). Passive immunotherapy with a triple combination of 2G12, 2F5, and 4E10 delayed viral rebound after the cessation of highly active antiretroviral therapy (HAART), and activity of 2G12 was critical for inhibitory activity by this antibody combination (18). The key role of 2G12 in suppressing HIV replication was supported by the development of viral rebound in parallel with the emergence of HIV isolates resistant to neutralization by 2G12 (19).While HIV infection may be controlled by the lifelong treatment of HIV-infected individuals with periodic infusions of neutralizing-antibody cocktails every few weeks, this is not a practical or cost-effective therapeutic approach. Eliciting these antibodies by vaccination has not been successful. Therefore, we investigated whether we could circumvent the mechanisms that limit the endogenous production of broadly neutralizing HIV-specific antibodies using a molecular genetic approach to generate B cells that secrete these protective antibodies. In a proof-of-concept study, we examined the capacity of a single lentiviral vector to express the heavy and light chains of the 2G12 antibody, a well-studied anti-HIV human antibody that has broad neutralizing activity both against T cell line-adapted and primary HIV isolates (31). The 2G12 antibody was generated by applying murine/human xenohybridoma technology to establish human hybridoma cell lines from B cells isolated from HIV-infected individuals (16), and it targets the high-mannose and/or hybrid glycans of residues 295, 332, and 392 and peripheral glycans from residues 386 and 448 on gp120. In the current study we demonstrated that a lentiviral vector encoding the heavy and light chains of the 2G12 antibody reprogrammed B cells in vitro to secrete 2G12 with functional neutralizing activity. Furthermore, we demonstrated that the 2G12 lentiviral vector genetically modified human hematopoietic stem cells (hu-HSC), enabling them to differentiate in vivo into progeny cells that secreted 2G12 antibody that inhibited the development of in vivo HIV infection in humanized mice.