Impact of Degree of Ionization and PEGylation on the Stability of Nanoparticles of Chitosan Derivatives at Physiological Conditions

Nowadays, the therapeutic efficiency of small interfering RNAs (siRNA) is still limited by the efficiency of gene therapy vectors capable of carrying them inside the target cells. In this study, siRNA nanocarriers based on low molecular weight chitosan grafted with increasing proportions (5 to 55%) of diisopropylethylamine (DIPEA) groups were developed, which allowed precise control of the degree of ionization of the polycations at pH 7.4. This approach made obtaining siRNA nanocarriers with small sizes (100–200 nm), positive surface charge and enhanced colloidal stability (up to 24 h) at physiological conditions of pH (7.4) and ionic strength (150 mmol L⁻¹) possible. Moreover, the PEGylation improved the stability of the nanoparticles, which maintained their colloidal stability and nanometric sizes even in an albumin-containing medium. The chitosan-derivatives displayed non-cytotoxic effects in both fibroblasts (NIH/3T3) and macrophages (RAW 264.7) at high N/P ratios and polymer concentrations (up to 0.5 g L⁻¹). Confocal microscopy showed a successful uptake of nanocarriers by RAW 264.7 macrophages and a promising ability to silence green fluorescent protein (GFP) in HeLa cells. These results were confirmed by a high level of tumor necrosis factor-α (TNFα) knockdown (higher than 60%) in LPS-stimulated macrophages treated with the siRNA-loaded nanoparticles even in the FBS-containing medium, findings that reveal a good correlation between the degree of ionization of the polycations and the physicochemical properties of nanocarriers. Overall, this study provides an approach to enhance siRNA condensation by chitosan-based carriers and highlights the potential of these nanocarriers for in vivo studies.     

Potential CO2 emissions mitigation through forest prescribed burning: A case study in Patagonia, Argentina

Wildland fire is a natural force that has shaped most vegetation types of the world. However, its inappropriate management during the last century has led to more frequent and catastrophic fires. Wildland fires are also recognized as one of the sources of CO₂ and other greenhouse gases (GHG) that influence global climate change. As one of the techniques used to reduce the risk of destructive wildfires, prescribed burning has the potential of mitigating carbon emissions, and effectively contributes to the efforts proposed as part of the Clean Development Mechanism within the Kyoto protocol. In order to apply this concept to a real case, a simulation study was conducted in pine afforestation in the Andean region of Patagonia, Argentina, with the objective of evaluating the potential of prescribed burning for reducing GHG emissions. The scenario was established for a ten year period, in which simulated prescribed burning was compared to the traditional management scheme, which included the probability of annual average of wildfire occurrence based on available wildfire statistics. The two contrasting scenarios were: (1) managed afforestation, affected by the annual average rate of wildfires occurred in the same type of afforestation in the region, without prescribed burning, and (2) same as (1) but with the application of simulated prescribed burning. In order to estimate carbon stocks, and CO₂ removals and emissions, we followed the guidelines given for GHG inventories on the Agriculture, Forestry and Other Land Uses (AFOLU) sector of the International Panel on Climate Change (IPCC), while the terminology used was the established by IPCC (2003). Data of afforested area, thinnings, and biomass growth were taken from previous surveys in the study area. Downed dead wood and litter (forest fuel load, FFL) was estimated adjusting equations fitted to those fuels, based on field data. Results show that comparing the two scenarios, prescribed burning reduced CO₂ emissions by 44% compared to the situation without prescribed burning. The prescribed burning scenario represented about 12% of the total emissions (prescribed burning plus wildfires). Furthermore, avoided wildfires by simulated prescribed burning allowed an additional 78% GHG emissions mitigation due to extra biomass growth. Simulated prescribed burning in commercial afforestation of Patagonia appears to be an effective management practice not only to prevent wildfires, but also an efficient tool to mitigate GHG emissions. However, more studies in different scenarios would be needed to generalize these benefits to other ecosystems.     

Genetic diversity of papaya (Carica papaya L.) F5 recombinant inbred lines using the Ward-MLM strategy

Genetic Resources and Crop Evolution - The development of segregating populations in papaya allows the development of new high fruit yield and excellent fruit quality pure line or hybrid cultivars....     

Immobilization of angiotensin-converting enzyme on glyoxyl-agarose

The assay of angiotensin-converting enzyme (ACE) inhibition by food-derived peptides is usually carried out by using soluble ACE in a batch process. The purification of this enzyme from tissues is not an easy task, and the resulting preparation loses activity very fast. In addition, ACE commercial preparations are very expensive. In this work the immobilization of ACE, through lysine amino groups, to 4% beads cross-linked (4 BCL) glyoxyl-agarose is described. The amount of immobilized enzyme increased with increasing concentrations of enzyme and with incubation time until a saturation point was reached at 50 mg protein/mL gel and 3.5 hours, respectively. The IC50 values for a noncompetitive sunflower peptide inhibitor were similar for the soluble (30.56 microM) and immobilized (32.7 microM) enzymes. An immobilized derivative was obtained that was 60 times more stable than the soluble enzyme at 60 degrees C. This procedure yields a derivative that can be reused and has increased thermal stability compared to that of the soluble enzyme. Thus, ACE immobilization is a good alternative to using soluble freshly prepared or commercial preparations because of economical and practical reasons.     

Peanut and Amaranthus leucocarpus lectins discriminate between memory and naive/quiescent porcine lymphocytes

Lectins are relevant tools to isolate and characterize different cellular sub-populations. In this work, we used the lectins Arachis hypogaea (Peanut agglutinin, PNA) and Amaranthus leucocarpus (ALL), specific for Galss1, 3GalNAc, to characterize naive and memory lymphocytes from pigs, experimentally infected with the porcine rubulavirus (RvP). Our results showed that both lectins recognized preferentially lymphocytes with the CD4(+)CD8(+) phenotype (P<0.05). The phenotypic analysis of the cells recognized by these lectins indicated that PNA(+) lymphocytes showed higher rate of the CD29 antigen (PNA(+)CD29(high)) than ALL(+) (ALL(+)CD29(low)). The number of PNA(+)CD29(high) lymphocytes increased after 8 weeks of experimental infection with RvP, and most of the ALL(+)CD29(low) cells became CD29(middle). PNA(+) lymphocytes isolated from infected pigs proliferated after stimulation with the RvP, whereas ALL(+) cells did not. In vitro assays indicated that the ALL(+) cells from previously infected pigs diminished from 7.5 +/- 2 to 0.5 +/- 0.3% after RvP stimulation; whereas PNA(+) cells increased from 4 +/- 1 to 42 +/- 2%, whereas no modification in ALL(+) or PNA(+) cellular population was identified in lymphocytes from naive animals after RvP stimulation. Our results suggest that the cellular distribution/organization of the O-glycosydically linked glycans on lymphocytes may correlate with biological functions, and that PNA could be a tool to isolate specifically porcine memory T cell subsets, whereas ALL could be useful to isolate naive/quiescent T lymphocytes.     

Succession and management of tropical dry forests in the Americas: Review and new perspectives

Understanding tropical forest succession is critical for the development of tropical forest conservation strategies worldwide, given that tropical secondary forests can be considered the forests of the future. Tropical dry forests (TDF) are among the most threatened tropical ecosystems, there are more secondary forests and forest restoration efforts that require a better understanding of successional processes. The main goal of this synthesis for this special issue on the ecology and management of tropical dry forests in the Americas is to present a summarized review of the current knowledge of the ecology and management implications associated to TDF succession. We explore specific issues associated to tropical dry forest succession with emphasis on the use of chronosequences, plant diversity and composition, plant phenology and remote sensing, pollination, and animal–plant interactions; all under the integrating umbrella of ecosystem succession. We also emphasize the need to conduct socio-ecological research to understand changes in land-use history and its effects on succession and forest regeneration of TDF. We close this paper with some thoughts and ideas associated with the strong need for an integrating dimension not considered until today: the role of cyberinfrastructure and eco-informatics as a tool to support sound conservation, management and understanding of TDF in the Americas.     

Effects of thinning and canopy type on growth dynamics of Pinus sylvestris: inter-annual variations and intra-annual interactions with microclimate

We assessed the effects of thinning (0, 20 and 30 % extraction of basal area) and canopy type (pine–beech vs. pine plots, beech accounting for 12 % of total basal area) on radial growth of dominant and codominant Scots pine at inter-annual scale and on microclimatic conditions, radial growth and xylogenesis 9 years after thinning at intra-annual scale. Thinning weakly affected pine growth, which was enhanced 3 years after harvesting. Over time, a gradual reduction in pine growth in mixed canopy relative to pure canopy occurred only in unthinned plots apparently due to beech expansion. Indeed, 9 years after thinning, a higher seasonal radial increment and a greater number of tracheids were produced under pine canopy in the unthinned plots, whereas no differences between canopy types were observed in the thinned plots. Radial increment and tracheid production were mainly affected by tree water status (air and soil humidity, throughfall). The differences of tree water status caused by treatments, and plausibly disparities in tree size and tree-to-tree competition, were the main drivers explaining the patterns observed for radial increment and xylogenesis. Our results suggest that the negative effects of beech competition on Scots pine growth in similar mixed forest may be controlled to some extent by thinning.     

Towards standardised crown condition assessment in poplar plantations

? This work aims at developing new tools for rapid assessment of forest health indicators in poplar plantations.

? Crown transparency and discoloration were visually evaluated in all trees of four 15 m-radius sub-plots in 32 poplar clonal plantations, which were chosen according to a factorial scheme with three factors: tree age, site quality and understorey vegetation management. A subset of trees was assessed using digital photos processed with a semi-automatic image analysis system (the CROCO software) in order to compare visual and digital crown transparency estimates.

? Poplar crown conditions were better in young stands and rich sites. Harrowing understorey vegetation improved tree health in poor sites. Samples of 20 trees per stand provided the same information about crown transparency and discoloration as 60 trees. Calibration curves of digital crown transparency estimates were successfully fitted against visual crown transparency estimates. The same effects of stand age and site quality could be detected with digital crown transparency as response variable.

? The use of digital photos processed with CROCO in ca. twenty trees per stand is therefore recommended to accurately and objectively monitor crown condition in clonal poplar plantations.

     

Growth Response and Resistance to Streptococcus iniae of Nile Tilapia, Oreochromis niloticus, Fed Diets Containing Distiller's Dried Grains with Solubles

This study was conducted to evaluate the effect of dietary levels of distiller’s dried grains with solubles (DDGS) on growth, body composition, hematology, and resistance of Nile tilapia, Oreochromis niloticus, to Streptococcus iniae challenge. Five isocaloric diets containing DDGS at levels of 0, 10, 20, and 40%, and 40% DDGS + lysine (Diets 1–5) as partial replacements of a combination of soybean meal (SBM) and corn meal (CM) on an equal protein basis were fed to juvenile Nile tilapia (9.41 ± 0.14 g) for 10 wk. Fish fed Diet 4 had the lowest weight gain (WG), feed efficiency ratio, protein efficiency ratio (PER), and whole‐body protein. Supplementation of lysine to the 40% DDGS diet (Diet 5) improved WG and PER. Hematological and immunological parameters were not affected by dietary treatment. There were no significant differences among the average number of days to first mortality after S. iniae challenge and cumulative mortality 14 d postchallenge among fish in various treatments. DDGS can be incorporated in tilapia diet at a level of 20% as a substitute for a combination of SBM and CM without affecting their growth performance, body composition, hematological parameters, immune response, and resistance to S. iniae infection.     

Bound malondialdehyde in foods: bioavailability of N,N'-di-(4-methyl-1,4-dihydropyridine-3,5-dicarbaldehyde)lysine

Reactions between lipid peroxidation products and proteins in foods have detrimental nutritional effects, most importantly, losses of essential amino acids. One of the major products of the reaction of malondialdehyde and alkanals with amino groups are 4-substituted 1,4-dihydropyridine-3,5-dicarbaldehyde derivatives. The product of the reaction of lysine with malondialdehyde and acetaldehyde, N,N'-di-(4-methyl-1,4-dihydropyridine-3,5-dicarbaldehyde)lysine (MDDL), has been synthesized and used for in vitro and in vivo bioavailability studies. Release of free lysine did not occur in incubations of MDDL with tissue homogenates. After oral administration of radioactively labeled MDDL, radioactivity was only recovered in feces. Radioactivity was not incorporated into hepatic microsomes after intraperitoneal administration, which would have indicated release of available lysine. These results show that MDDL is a form of unavailable lysine, because it is not metabolized to free lysine and cannot be absorbed from the gut. Thus, formation of this derivative in foods would result in loss of available lysine.