Regulatory guidelines and preclinical tools to study the biodistribution of RNA therapeutics.

The success of the messenger RNA-based COVID-19 vaccines of Moderna and Pfizer/BioNTech marks the beginning of a new chapter in modern medicine. However, the rapid rise of mRNA therapeutics has resulted in a regulatory framework that is somewhat lagging. The current guidelines either do not apply, do not mention RNA therapeutics, or do not have widely accepted definitions. This review describes the guidelines for preclinical biodistribution studies of mRNA/siRNA therapeutics and highlights the relevant differences for mRNA vaccines. We also discuss the role of in vivo RNA imaging techniques and other assays to fulfill and/or complement the regulatory requirements. Specifically, quantitative whole-body autoradiography, microautoradiography, mass spectrometry-based assays, hybridization techniques (FISH, bDNA), PCR-based methods, in vivo fluorescence imaging, and in vivo bioluminescence imaging, are discussed. We conclude that this new and rapidly evolving class of medicines demands a multi-layered approach to fully understand its biodistribution and in vivo characteristics.

Enhanced phytoextraction: I. Effect of EDTA and citric acid on heavy metal mobility in a calcareous soil.

Phytoextraction, the use of plants to extract heavy metals from contaminated soils, could be an interesting alternative to conventional remediation technologies. However, calcareous soils with relatively high total metal contents are difficult to phytoremediate due to low soluble metal concentrations. Soil amendments such as ethylene diaminetetraacetate (EDTA) have been suggested to increase heavy metal bioavailability and uptake in aboveground plant parts. Strong persistence of EDTA and risks of leaching of potentially toxic metals and essential nutrients have led to research on easily biodegradable soil amendments such as citric acid. In our research, EDTA is regarded as a scientific benchmark with which degradable alternatives are compared for enhanced phytoextraction purposes. The effects of increasing doses of EDTA (0.1,1,10 mmol kg(-1) dry soil) and citric acid (0.01, 0.05, 0.25, 0.442, 0.5 mol kg(-1) dry soil) on bioavailable fractions of Cu, Zn, Cd, and Pb were assessed in one part of our study and results are presented in this article. The evolution of labile soil fractions of heavy metals over time was evaluated using water paste saturation extraction (approximately soluble fraction), extraction with 1 M NH4OAc at pH 7 (approximately exchangeable fraction), and extraction with 0.5 M NH4OAc + 05 M HOAc + 0.02 M EDTA at pH 4.65 (approximately potentially bioavailable fraction). Both citric acid and EDTA produced a rapid initial increase in labile heavy metal fractions. Metal mobilization remained constant in time for soils treated with EDTA, but a strong exponential decrease of labile metal fractions was noted for soils treated with citric acid. The half life of heavy metal mobilization by citric acid varied between 1.5 and 5.7 d. In the following article, the effect of heavy metal mobilization on uptake by Helianthus annuus will be presented.

Enhanced phytoextraction: II. Effect of EDTA and citric acid on heavy metal uptake by Helianthus annuus from a calcareous soil.

High biomass producing plant species, such as Helianthus annuus, have potential for removing large amounts of trace metals by harvesting the aboveground biomass if sufficient metal concentrations in their biomass can be achieved However, the low bioavailability of heavy metals in soils and the limited translocation of heavy metals to the shoots by most high biomass producing plant species limit the efficiency of the phytoextraction process. Amendment of a contaminated soil with ethylene diamine tetraacetic acid (EDTA) or citric acid increases soluble heavy metal concentrations, potentially rendering them more available for plant uptake. This article discusses the effects of EDTA and citric acid on the uptake of heavy metals and translocation to aboveground harvestable plant parts in Helianthus annuus. EDTA was included in the research for comparison purposes in our quest for less persistent alternatives, suitable for enhanced phytoextraction. Plants were grown in a calcareous soil moderately contaminated with Cu, Pb, Zn, and Cd and treated with increasing concentrations of EDTA (0.1, 1, 3, 5, 7, and 10 mmol kg(-1) soil) or citric acid (0.01, 0.05, 0.25, 0.442, and 0.5 mol kg(-1) soil). Heavy metal concentrations in harvested shoots increased with EDTA concentration but the actual amount of phytoextracted heavy metals decreased at high EDTA concentrations, due to severe growth depression. Helianthus annuus suffered heavy metal stress due to the significantly increased bioavailable metal fraction in the soil. The rapid mineralization of citric acid and the high buffering capacity of the soil made citric acid inefficient in increasing the phytoextracted amounts of heavy metals. Treatments that did not exceed the buffering capacity of the soil (< 0.442 mol kg(-1) soil) did not result in any significant increase in shoot heavy metal concentrations. Treatments with high concentrations resulted in a dissolution of the carbonates and compaction of the soil. These physicochemical changes caused growth depression of Helianthus annuus. EDTA and citric acid added before sowing of Helianthus annuus did not appear to be efficient amendments when phytoextraction of heavy metals from calcareous soils is considered.

Availability of heavy metals for uptake by Salix viminalis on a moderately contaminated dredged sediment disposal site.

Extractability of Cd, Cr, Cu, Ni, Pb, and Zn in a dredged sediment disposal site was assessed using single extraction procedures (H2O; 0.01 M CaCl2; 1 M NH4OAc; NH4OAc-EDTA; CaCl2-TEA-DTPA). Only Cd and Zn were found to exceed statutory threshold values for total content. The field was planted with Salix viminalis "Orm" and accumulation of heavy metals in bark, leaves, roots, and wood was evaluated at seven sampling locations along an observed gradient in texture and pollution. Biomass production was high, ranging from 13.2 to 17.8 t ha(-1) y(-1) dry weight. Metal accumulation in aboveground plant parts was low, amounting to the following annually extracted mass of metals per ha: 5034 g Zn, 83 g Cd, 145 g Cu, 83 g Pb, 12 g Ni and 6 g Cr. The use of accumulating clones and the use of soil amendments might enhance extraction efficiency in future research.

Enhanced phytoextraction: in search of EDTA alternatives.

Enhanced phytoextraction proposes the use of soil amendments to increase the heavy-metal content of above-ground harvestable plant tissues. This study compares the effect of synthetic aminopolycarboxylic acids [ethylenediamine tetraacetatic acid (EDTA), nitriloacetic acid (NTA), and diethylenetriamine pentaacetic acid (DTPA)] with a number of biodegradable, low-molecular weight, organic acids (citric acid, ascorbic acid, oxalic acid, salicylic acid, and NH4 acetate) as potential soil amendments for enhancing phytoextraction of heavy metals (Cu, Zn, Cd, Pb, and Ni) by Zea mays. The treatments in this study were applied at a dose of 2 mmol/kg(-1) 1 d before sowing. To compare possible effects between presow and postgermination treatments, a second smaller experiment was conducted in which EDTA, citric acid, and NH4 acetate were added 10 d after germination as opposed to 1 d before sowing. The soil used in this screening was a moderately contaminated topsoil derived from a dredged sediment disposal site. This site has been in an oxidized state for more than 8 years before being used in this research. The high carbonate, high organic matter, and high clay content characteristic to this type of sediment are thought to suppress heavy-metal phytoavailability. Both EDTA and DTPA resulted in increased levels of heavy metals in the above-ground biomass. However, the observed increases in uptake were not as large as reported in the literature. Neither the NTA nor organic acid treatments had any significant effect on uptake when applied prior to sowing. This was attributed to the rapid mineralization of these substances and the relatively low doses applied. The generally low extraction observed in this experiment restricts the use of phytoextraction as an effective remediation alternative under the current conditions, with regard to amendments used, applied dose (2 mmol/kg(-1) soil), application time (presow), plant species (Zea mays), and sediment (calcareous clayey soil) under study.

Short- and longer-term effects of the willow root system on metal extractability in contaminated dredged sediment.

Willow (Salix spp.) stands are often proposed as vegetation covers for the restoration and stabilization of contaminated and derelict land. Planting willows on dredged sediment disposal sites for biomass production can be an alternative to traditional capping techniques. However, with the introduction of willow stands on dredged sediment disposal sites, the possibility of increased contaminant availability in the root zone must be acknowledged as it can increase the risk of leaching. Two trials investigated the availability of Cd, Zn, Cu, and Pb in the root zones of willows grown on contaminated sediment. To assess the effects of willow root growth on metal extractability and mobility, bulk and rhizosphere sediment samples were extracted with deionized water, ammonium acetate at pH 7, and ammonium acetate-EDTA at pH 4.65. A rhizobox experiment was used to investigate the short-term effect of willow roots on metal availability in oxic and anoxic sediment. Longer-term effects were assessed in a field trial. The rhizobox trial showed that Cd, Zn, and Cu extractability in the rhizosphere increased while the opposite was observed for Pb. This was attributed to the increased willow-induced oxidation rate in the root zone as a result of aeration and evapotranspiration, which masked the direct chemical and biological influences of the willow roots. The field trial showed that Cu and Pb, but not Cd, were more available in the root zone after water and ammonium acetate (pH 7) extraction compared with the bulk sediment. Sediment in the root zone was better structured and aggregated and thus more permeable for downward water flows, causing leaching of a fraction of the metals and significantly lower total contents of Cd, Cu, and Pb. These findings indicate that a vegetation cover strategy to stabilize sediments can increase metal availability in the root zone and that potential metal losses to the environment should be considered.

Phytoremediation prospects of willow stands on contaminated sediment: a field trial.

Establishing fast growing willow stands on land disposed contaminated dredged sediment can result in the revaluation of this material and opens possibilities for phytoremediation. A field trial was designed to assess the impact of planting a willow stand (Salix viminalis L. 'Orm') on the dissipation of organic contaminants (mineral oil and PAHs) in dredged sediment. In addition, the accumulation of heavy metals (Cd, Cu, Pb and Zn) in the biomass was determined. After 1.5 years, a significant decrease of 57% in the mineral oil concentration in the sediment planted with willow was observed. Degradation of mineral oil in sediment which was left fallow, was only 15%. The mineral oil degradation under willow was most pronounced (79%) in the root zone of the stand. In the sediment which was left fallow there was a significant reduction of the total PAH content by 32% compared with a 23% reduction in the planted sediment. The moderate and selective metal uptake, measured in this study, limits the prospects for phytoextraction of metals from dredged sediment.

Should foliar cadmium concentrations be expressed on a dry weight or dry ash weight basis?

Foliar analysis is a valuable tool for evaluating the pollution status of forests. However, the use of foliar diagnosis in large-scale surveys is a complicated process owing to the high variability within the crown. The method used to express foliar concentrations has often been found to diminish the variability. The effect of the method used to express element concentrations on the spatial variability of cadmium (Cd) in the leaves of crack willow (Salix fragilis L.) was investigated by sampling the leaves of one willow at 292 locations in the crown, each sampling location having a volume of 0.027 m3 (0.3 m x 0.3 m x 0.3 m). Cadmium showed a distinct spatial trend in the crown of the tree. Concentrations as low as 2.4 mg kg(-1) dry weight (DW) or 23.1 mg kg(-1) dry ash weight (DAW) were obtained in the top of the crown, and 10.6 mg kg(-1) DW or 73.0 mg kg(-1) DAW in the bottom of the crown. The lower relative standard deviation and weaker correlation with the sampling height support the use of DAW in large-scale surveys especially. The lower variability of the DAW Cd concentration makes this variable less sensitive to fluctuations caused by differences in growing conditions and sampling methodology. However, the majority of publications in this field report metal concentrations on a DW basis. Therefore, the restrictions set on the use of results expressed on a DAW basis in large-scale surveys of foliar metal concentrations have to be offset against the advantages offered by a reduction of the variability in metal concentrations.

Cd and Zn concentrations in small mammals and willow leaves on disposal facilities for dredged material.

Disposal sites for dredged material are often polluted with heavy metals. The uptake of Cd and Zn by small mammals and willow trees was assessed on three sites with a different pollution degree. Detailed soil sampling showed a huge variation in soil characteristics within the sites, typical for disposal sites for dredged material. This variation made risk assessment and interpretation of soil chemical analyses complicated. Small mammals were thought to give a more integral description of the contaminant level because they cover a large area. Cd concentrations in small mammals were elevated compared with background levels whereas Zn concentrations were rather low. There were no significant differences between sites in Cd or Zn levels in animals. Leaf analysis showed a clear response to the pollution degree of the sites. The calculations using the BIOMAG model indicated that the Cd of the soil caused low risk for predators. However, the elevated Cd and Zn concentrations in the willow leaves on the polluted sites and the high Cd concentrations in the small mammals on all sites indicated that ecosystem development should be carefully considered.