The mechanisms of humic substances self-assembly with biological molecules: The case study of the prion protein.

Humic substances (HS) are the largest constituent of soil organic matter and are considered as a key component of the terrestrial ecosystem. HS may facilitate the transport of organic and inorganic molecules, as well as the sorption interactions with environmentally relevant proteins such as prions. Prions enter the environment through shedding from live hosts, facilitating a sustained incidence of animal prion diseases such as Chronic Wasting Disease and scrapie in cervid and ovine populations, respectively. Changes in prion structure upon environmental exposure may be significant as they can affect prion infectivity and disease pathology. Despite its relevance, the mechanisms of prion interaction with HS are still not completely understood. The goal of this work is to advance a structural-level picture of the encapsulation of recombinant, non-infectious, prion protein (PrP) into different natural HS. We observed that PrP precipitation upon addition of HS is mainly driven by a mechanism of "salting-out" whereby PrP molecules are rapidly removed from the solution and aggregate in insoluble adducts with humic molecules. Importantly, this process does not alter the protein folding since insoluble PrP retains its α-helical content when in complex with HS. The observed ability of HS to promote PrP insolubilization without altering its secondary structure may have potential relevance in the context of "prion ecology". These results suggest that soil organic matter interacts with prions possibly without altering the protein structures. This may facilitate prions preservation from biotic and abiotic degradation leading to their accumulation in the environment.

Prion protein interaction with soil humic substances: environmental implications.

Transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative disorders caused by prions. Animal TSE include scrapie in sheep and goats, and chronic wasting disease (CWD) in cervids. Effective management of scrapie in many parts of the world, and of CWD in North American deer population is complicated by the persistence of prions in the environment. After shedding from diseased animals, prions persist in soil, withstanding biotic and abiotic degradation. As soil is a complex, multi-component system of both mineral and organic components, it is important to understand which soil compounds may interact with prions and thus contribute to disease transmission. Several studies have investigated the role of different soil minerals in prion adsorption and infectivity; we focused our attention on the interaction of soil organic components, the humic substances (HS), with recombinant prion protein (recPrP) material. We evaluated the kinetics of recPrP adsorption, providing a structural and biochemical characterization of chemical adducts using different experimental approaches. Here we show that HS act as potent anti-prion agents in prion infected neuronal cells and in the amyloid seeding assays: HS adsorb both recPrP and prions, thus sequestering them from the prion replication process. We interpreted our findings as highly relevant from an environmental point of view, as the adsorption of prions in HS may affect their availability and consequently hinder the environmental transmission of prion diseases in ruminants.

Traceability of Italian garlic (Allium sativum L.) by means of HRMAS-NMR spectroscopy and multivariate data analysis.

(1)H High Resolution Magic Angle Spinning-Nuclear Magnetic Resonance (HRMAS-NMR) spectroscopy was used to analyse garlic (Allium sativum L.) belonging to red and white varieties and collected in different Italian regions, in order to address the traceability issue. 1D and 2D NMR spectra, performed directly on untreated small pieces of garlic, so without any sample manipulation, allowed the assignment of several compounds: organic acids, sugars, fatty acids, amino acids and the nutritionally important fructo-oligosaccharides and allyl-organosulphur compounds. Application of Partial Least Squares projections to latent structures-Discrimination Analysis provided an excellent model for the discrimination of both the variety and, most important, the place origin, allowing the identification of the metabolites contributing to such classifications. The presence of organosulphurs, allicin and some allyl-organosulphurs found by HRMAS-NMR, was confirmed also by SPME-GC-MS; 11 molecules were identified, containing from one up to three sulphur atoms and with and without allyl moieties.

The HRMAS-NMR tool in foodstuff characterisation.

High resolution magic angle spinning, that is, HRMAS, is a quite novel tool in NMR spectroscopy; it offers the almost unique opportunity of measuring intact tissues disguised as suspended or swollen in a deuterated solvent. The feasibility of (1)H-HRMAS-NMR in foodstuff characterisation has been exploited, but in spite of this, its applications are still limited. Metabolic profiling and biopolymer composition and aggregation are the topics investigated until now for raw vegetables, meat and processed foodstuff. Almost all known studies are reported in the next pages.

Soil humic acids may favour the persistence of hexavalent chromium in soil.

The interaction between hexavalent chromium Cr(VI), as K(2)CrO(4), and standard humic acids (HAs) in bulk solution was studied using three complementary analytical methods: UV-Visible spectroscopy, X-ray absorption spectroscopy and differential pulse stripping voltammetry. The observed UV-Vis and X-ray absorption spectra showed that, under our experimental conditions, HAs did not induce reduction of Cr(VI) to its trivalent chemical form. The interaction between Cr(VI) and HAs has rather led to the formation of Cr(VI)-HAs micelles via supramolecular chemical processes. The reported results could contribute towards explaining the relative persistence of ecotoxic hexavalent chromium in soils.

Organic polyanions act as complexants of prion protein in soil.

The persistence of prions, the causative agents of transmissible spongiform encephalopathies, in soil constitutes an environmental concern and substantial challenge. Experiments and theoretical modeling indicate that a particular class of natural polyanions diffused in soils and waters, generally referred to as humic substances (HSs), can participate in the adsorption of prions in soil in a non-specific way, mostly driven by electrostatic interactions and hydrogen bond networks among humic acid molecules and exposed polar protein residues. Adsorption of HSs on clay surface strongly raises the adsorption capacity vs proteins suggesting new experiments in order to verify if this raises or lowers the prion infectivity.

Direct detection of soil-bound prions.

Scrapie and chronic wasting disease are contagious prion diseases affecting sheep and cervids, respectively. Studies have indicated that horizontal transmission is important in sustaining these epidemics, and that environmental contamination plays an important role in this. In the perspective of detecting prions in soil samples from the field by more direct methods than animal-based bioassays, we have developed a novel immuno-based approach that visualises in situ the major component (PrP(Sc)) of prions sorbed onto agricultural soil particles. Importantly, the protocol needs no extraction of the protein from soil. Using a cell-based assay of infectivity, we also report that samples of agricultural soil, or quartz sand, acquire prion infectivity after exposure to whole brain homogenates from prion-infected mice. Our data provide further support to the notion that prion-exposed soils retain infectivity, as recently determined in Syrian hamsters intracerebrally or orally challenged with contaminated soils. The cell approach of the potential infectivity of contaminated soil is faster and cheaper than classical animal-based bioassays. Although it suffers from limitations, e.g. it can currently test only a few mouse prion strains, the cell model can nevertheless be applied in its present form to understand how soil composition influences infectivity, and to test prion-inactivating procedures.

Evaluation of extracted organic carbon and microbial biomass as stability parameters in ligno-cellulosic waste composts.

Extracted organic C and microbial biomass were evaluated as stability parameters in 3 different ligno-cellulosic waste composts. Organic C was extracted by both water and alkali and further separated in humic-like carbon (HLC) and nonhumic carbon (NHC). Conventional humification parameters, such as humification index and degree of humification were calculated from NHC and HLC. Microbial biomass carbon (B(C)) was determined as an indicator of the degree of biochemical transformation, whereas ninhydrin reactive N (B(NIN)) was measured to obtain the stability parameter B(NIN)/N(TOT) (N(TOT), total N). The water-extracted organic C did not provide reliable information on the transformations underwent by the ligno-cellulosic wastes during composting, since its content remained almost unaltered during the whole process. In contrast, parameters based on the alkali-extracted organic C and microbial biomass clearly reflected organic matter (OM) changes during the process. There was an increase in the net amount of HLC in the alkali extracts throughout composting, especially in the first 7 to 12 wk of the process, as well as a relative enrichment of HLC with respect to NHC. Values of humification index and degree of humification in end products were consistent with an adequate level of compost stability. The stability parameter B(NIN)/N(TOT) showed to be a reliable indicator of stability in ligno-cellulosic wastes. Parameters based on the alkali-extracted C and microbial biomass clearly reflected the transformation of the OM during composting and can be used as stability parameters in ligno-cellulosic waste composts.

An integrated chemical, thermal, and microbiological approach to compost stability evaluation.

The evaluation of compost stability is of the utmost importance for the reliability of composting as a recycling strategy. To date there is no single parameter that can give a sure indication of the stability of composts from different starting materials. This paper investigates different methods of evaluating the dynamics of transformation of materials and the stability level of the end products in a composting process. The following parameters were determined on compost samples of different ages from cotton (Gossypium herbaceum L.) cardings and yard wastes: humification index (HI), degree of humification (DH), thermogravimetry (TG) microbial biomass C (B(C)), and ninhydrin-reactive N (B(NIN)). Finally, from TG, derivative thermogravimetry (DTG) and differential scanning calorimetry (DSC) thermal stability parameters were deduced. Humification parameters in the end products (0.2 and 81% for HI and DH, respectively) showed the effective stability reached by the organic matter (OM). Thermal analysis evidenced the presence of two main organic pools with different thermal stability. During composting a relative increase in the more stable organic pool was indicated by the variation of the thermostability index R1 from 0.41 to 0.74. The parameter R1 was significantly correlated with both HI (r = -0.94; P < 0.05) and DH (r = 0.97; P < 0.05). Microbial biomass content dynamics reflected the availability of readily decomposable substrates. The ratio between B(NIN) and total N in the end product was 0.96%, indicating a good stability level. The simultaneous application of different approaches, considering different properties of composting materials, provides a more complete description of the stability and quality reached by the organic materials.