Myelin Basic Protein Fragmentation by Engineered Human Proteasomes with Different Catalytic Phenotypes Revealed Direct Peptide Ligands of MS-Associated and Protective HLA Class I Molecules.

Proteasomes exist in mammalian cells in multiple combinatorial variants due to the diverse regulatory particles and exchange of catalytic subunits. Here, using biotin carboxyl carrier domain of transcarboxylase from Propionibacterium shermanii fused with different proteasome subunits of catalytic and regulatory particles, we report comprehensive characterization of highly homogenous one-step purified human constitutive and immune 20S and 26S/30S proteasomes. Hydrolysis of a multiple sclerosis (MS) autoantigen, myelin basic protein (MBP), by engineered human proteasomes with different catalytic phenotypes, revealed that peptides which may be directly loaded on the HLA class I molecules are produced mainly by immunoproteasomes. We detected at least five MBP immunodominant core regions, namely, LPRHRDTGIL, SLPQKSHGR, QDENPVVHFF, KGRGLSLSRF and GYGGRASDY. All peptides, except QDENPVVHFF, which originates from the encephalitogenic MBP part, were associated with HLA I alleles considered to increase MS risk. Prediction of the affinity of HLA class I to this peptide demonstrated that MS-protective HLA-A*44 and -B*35 molecules are high-affinity binders, whereas MS-associated HLA-A*23, -A*24, -A*26 and -B*51 molecules tend to have moderate to low affinity. The HLA-A*44 molecules may bind QDENPVVHFF and its deamidated form in several registers with unprecedently high affinity, probably linking its distinct protective phenotype with thymic depletion of the repertoire of autoreactive cytotoxic T cells or induction of CD8+ regulatory T cells, specific to the encephalitogenic MBP peptide.

Chilean regulations on metal-polluted soils: The need to advance from adapting foreign laws towards developing sovereign legislation.

Chile as a major international Cu producer faces serious soil contamination issues in mining areas. Currently Chile does not have any specific law governing the maximum permissible concentrations of metals in soils to protect ecosystems and human health. Chile heavily relies on the use of environmental laws of 14 foreign countries; the choice of the country depends on the similarity of its environmental conditions with those in Chile. In this study, we used an online database to compare the similarity of Chilean rocks to those in foreign countries. Likewise, we performed soil sampling and determined the background concentrations of Cu, As, Pb, and Zn in soils of the Aconcagua basin, the largest river basin in the Valparaiso Region of central Chile. The results showed that geochemical patterns in Chile have the greatest resemblance to New Zealand, Mexico, and Italy. The background Cu concentration in the Aconcagua basin (134 mg kg-1) exceeded the legislated limits of New Zealand (100 mg kg-1) and Italy (120 mg kg-1), whereas the background Zn concentration (200 mg kg-1) exceeded the legislated limit of Italy (150 mg kg-1). Due to the elevated natural abundance of Cu and Zn in Chile, international laws should not be applied in Chile for the assessment of soil contamination. In addition, we assessed ecological risk using the results of our previous studies obtained by analyzing native field-contaminated soils of the Valparaiso region. In the Aconcagua basin, Cu posed high risk for plants in 11% of the samples, whereas As posed high risk for earthworms in 48% of the samples. We suggest that future studies are required to search for other organisms that can serve as biomarkers of metal toxicity because our previous studies were limited to plants and earthworms. Importantly, As posed high risk to human health in 25% of the samples in our study. There is a need for future studies to demonstrate empirically an association between soil As and children's blood As in order to establish the national threshold values of soil As to protect human health. We conclude that there is an urgent need in Chile to advance from the current approach of adapting foreign laws to developing Chilean sovereign environmental legislation.

Key energy metabolisms in modern living microbialites from hypersaline Andean lagoons of the Salar de Atacama, Chile.

Microbialites are organosedimentary structures formed mainly due to the precipitation of carbonate minerals, although they can also incorporate siliceous, phosphate, ferric, and sulfate minerals. The minerals' precipitation occurs because of local chemical changes triggered by changes in pH and redox transformations catalyzed by the microbial energy metabolisms. Here, geochemistry, metagenomics, and bioinformatics tools reveal the key energy metabolisms of microbial mats, stromatolites and an endoevaporite distributed across four hypersaline lagoons from the Salar de Atacama. Chemoautotrophic and chemoheterotrophic microorganisms seem to coexist and influence microbialite formation. The microbialite types of each lagoon host unique microbial communities and metabolisms that influence their geochemistry. Among them, photosynthetic, carbon- and nitrogen- fixing and sulfate-reducing microorganisms appear to control the main biogeochemical cycles. Genes associated with non-conventional energy pathways identified in MAGs, such as hydrogen production/consumption, arsenic oxidation/reduction, manganese oxidation and selenium reduction, also contribute to support life in microbialites. The presence of genes encoding for enzymes associated with ureolytic processes in the Cyanobacteria phylum and Gammaproteobacteria class might induce carbonate precipitation in hypersaline environments, contributing to the microbialites formation. To the best of our knowledge, this is the first study characterizing metagenomically microbialites enriched in manganese and identifying metabolic pathways associated with manganese oxidation, selenium reduction, and ureolysis in this ecosystem, which suggests that the geochemistry and bioavailability of energy sources (As, Mn and Se) shapes the microbial metabolisms in the microbialites.

Global issues in setting legal limits on soil metal contamination: A case study of Chile.

The establishment of legal limits for soil contamination with trace elements is a global issue that has not yet been resolved. However, the resolution of any global problem begins at the national level. In this vein, we present the case of Chile, the world's leading copper producer, where soil contamination by trace elements in mining areas has been severe. We evaluated the magnitude of the ecological and human health risks from exposure to arsenic (As), copper (Cu), zinc (Zn), and lead (Pb) in soils of the La Ligua and Petorca basins, two important mining areas in Chile. Contrary to what might be expected in soils affected by Cu mining activities, As was identified as the most hazardous element in the studied soils, both in terms of ecological and human health risks. On the other hand, Chile does not currently have specific legislation establishing legal limits on soil contamination with trace elements. Since Chile is geochemically similar to New Zealand, Mexico, and Italy, we used the limits of these three countries as benchmarks. We determined the background concentrations of As, Cu, Zn, and Pb in the soils of the two river basins under study and found that they tend to exceed the limits established by foreign laws. We also found that the differences in background elemental concentrations in the studied soils were primarily due to the varied lithology of soil-forming rocks. This means that absolute "one-limit-fits-all" values of element concentrations may not be adequate to regulate the level of soil contamination in areas affected by mining. As a fundamental first step, it is necessary to establish background soil concentrations of trace elements in each river basin in Chile. It is clear that Chile urgently needs to move from rubber-stamping foreign laws to the development of national legislation on soil metal contamination.

Corrigendum: In Vivo Isotopic Labeling of Symbiotic Bacteria Involved in Cellulose Degradation and Nitrogen Recycling within the Gut of the Forest Cockchafer (Melolontha hippocastani).

[This corrects the article on p. 1970 in vol. 8, PMID: 29075241.].

Publisher Correction: Geochemical constraints on the Hadean environment from mineral fingerprints of prokaryotes.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

In Vivo Isotopic Labeling of Symbiotic Bacteria Involved in Cellulose Degradation and Nitrogen Recycling within the Gut of the Forest Cockchafer (Melolontha hippocastani).

The guts of insects harbor symbiotic bacterial communities. However, due to their complexity, it is challenging to relate a specific symbiotic phylotype to its corresponding function. In the present study, we focused on the forest cockchafer (Melolontha hippocastani), a phytophagous insect with a dual life cycle, consisting of a root-feeding larval stage and a leaf-feeding adult stage. By combining in vivo stable isotope probing (SIP) with 13C cellulose and 15N urea as trophic links, with Illumina MiSeq (Illumina-SIP), we unraveled bacterial networks processing recalcitrant dietary components and recycling nitrogenous waste. The bacterial communities behind these processes change between larval and adult stages. In 13C cellulose-fed insects, the bacterial families Lachnospiraceae and Enterobacteriaceae were isotopically labeled in larvae and adults, respectively. In 15N urea-fed insects, the genera Burkholderia and Parabacteroides were isotopically labeled in larvae and adults, respectively. Additionally, the PICRUSt-predicted metagenome suggested a possible ability to degrade hemicellulose and to produce amino acids of, respectively, 13C cellulose- and 15N urea labeled bacteria. The incorporation of 15N from ingested urea back into the insect body was confirmed, in larvae and adults, by isotope ratio mass spectrometry (IRMS). Besides highlighting key bacterial symbionts of the gut of M. hippocastani, this study provides example on how Illumina-SIP with multiple trophic links can be used to target microorganisms embracing different roles within an environment.

Geochemical constraints on the Hadean environment from mineral fingerprints of prokaryotes.

The environmental conditions on the Earth before 4 billion years ago are highly uncertain, largely because of the lack of a substantial rock record from this period. During this time interval, known as the Hadean, the young planet transformed from an uninhabited world to the one capable of supporting, and inhabited by the first living cells. These cells formed in a fluid environment they could not at first control, with homeostatic mechanisms developing only later. It is therefore possible that present-day organisms retain some record of the primordial fluid in which the first cells formed. Here we present new data on the elemental compositions and mineral fingerprints of both Bacteria and Archaea, using these data to constrain the environment in which life formed. The cradle solution that produced this elemental signature was saturated in barite, sphene, chalcedony, apatite, and clay minerals. The presence of these minerals, as well as other chemical features, suggests that the cradle environment of life may have been a weathering fluid interacting with dry-land silicate rocks. The specific mineral assemblage provides evidence for a moderate Hadean climate with dry and wet seasons and a lower atmospheric abundance of CO2 than is present today.

Bacterial Community and PHB-Accumulating Bacteria Associated with the Wall and Specialized Niches of the Hindgut of the Forest Cockchafer (Melolontha hippocastani).

A characterization of the bacterial community of the hindgut wall of two larval and the adult stages of the forest cockchafer (Melolontha hippocastani) was carried out using amplicon sequencing of the 16S rRNA gene fragment. We found that, in second-instar larvae, Caulobacteraceae and Pseudomonadaceae showed the highest relative abundances, while in third-instar larvae, the dominant families were Porphyromonadaceae and Bacteroidales-related. In adults, an increase of the relative abundance of Bacteroidetes, Proteobacteria (γ- and δ- classes) and the family Enterococcaceae (Firmicutes) was observed. This suggests that the composition of the hindgut wall community may depend on the insect's life stage. Additionally, specialized bacterial niches hitherto very poorly described in the literature were spotted at both sides of the distal part of the hindgut chamber. We named these structures "pockets." Amplicon sequencing of the 16S rRNA gene fragment revealed that the pockets contained a different bacterial community than the surrounding hindgut wall, dominated by Alcaligenaceae and Micrococcaceae-related families. Poly-ß-hydroxybutyrate (PHB) accumulation in the pocket was suggested in isolated Achromobacter sp. by Nile Blue staining, and confirmed by gas chromatography-mass spectrometry analysis (GC-MS) on cultured bacterial mass and whole pocket tissue. Raman micro-spectroscopy allowed to visualize the spatial distribution of PHB accumulating bacteria within the pocket tissue. The presence of this polymer might play a role in the colonization of these specialized niches.

Spodoptera littoralis detoxifies neurotoxic 3-nitropropanoic acid by conjugation with amino acids.

Spodoptera littoralis is a phytophagous generalist. Its host range includes more than 40 plant species, some of which produce 3-nitropropanoic acid (3-NPA), an irreversible inhibitor of mitochondrial succinate dehydrogenase. Growth in larvae fed an artificial diet with a sublethal admixture of 3-NPA (4.2 µmol per g) was slowed significantly, but larvae experienced no increase in mortality. In contrast, larvae injected with 25.2 µmol/g (bodyweight) 3-NPA experienced acute toxicity and death. To study the detoxification mechanism of 3-NPA in S. littoralis, the insect frass was analyzed by HPLC-MS. Comparative analysis of 3-NPA-treated and -untreated control samples using HR-MS(2) revealed a group of differential signals that were identified as amino acid amides of 3-NPA with glycine, alanine, serine, and threonine. When sublethal amounts of stable isotope-labeled 3-NPA were injected into a larva's hemolymph, 3-NPA amino acid conjugates were identified as putative detoxification products. Bioassays with synthetic standards confirmed that the toxicity of the amides was negligible in comparison to the toxicity of free 3-NPA, demonstrating that amino acid conjugation in S. littoralis represents an efficient way to detoxify 3-NPA. Furthermore, biosynthetic studies using crude fractions of the gut tissue indicated that conjugation of 3-NPA with amino acids occurs in epithelial cells of the insect's gut. Taken together, these results suggest that the detoxification of 3-NPA in S. littoralis proceeds via conjugation to specific amino acids within the epithelial cells followed by export of the nontoxic amino acid conjugates to the hemolymph via as yet uncharacterized mechanisms, most likely involving the Malpighian tubules.

From cytoplasm to environment: the inorganic ingredients for the origin of life.

Early in its history, Earth's surface developed from an uninhabitable magma ocean to a place where life could emerge. The first organisms, lacking ion transporters, fixed the composition of their cradle environment in their intracellular fluid. Later, though life adapted and spread, it preserved some qualities of its initial environment within. Modern prokaryotes could thus provide insights into the conditions of early Earth and the requirements for the emergence of life. In this work, we constrain Earth's life-forming environment through detailed analysis of prokaryotic intracellular fluid. Rigorous assessment of the constraints placed on the early Earth environment by intracellular liquid will provide insight into the conditions of abiogenesis, with implications not only for our understanding of early Earth but also the formation of life elsewhere in the Universe.

A single amino acid substitution controls DAF-dependent phenotype of echovirus 11 in rhabdomyosarcoma cells.

Decay accelerating factor (DAF, CD55) is used by DAF-dependent (Daf+) variants of echovirus 11 (EV11) as a primary cellular receptor. The interaction of EV11 with DAF is completely reversible, therefore DAF-dependent variants require an unidentified coreceptor to initiate uncoating. Daf- variants of EV11, which do not interact with DAF, use an alternative primary cellular receptor. The aim of this study was to test the hypothesis whether the coreceptor, which is necessary for the uncoating of DAF-dependent variants, may act as an alternative primary receptor for the Daf- variants of EV11. By using the model of the two closely related daf+ and daf- clones of EV11 in rhabdomyosarcoma (RD) cell line, it was shown that a single amino acid substitution in the capsid protein VP2 could control the expression of the DAF-dependent phenotype. Anti-DAF monoclonal antibody has blocked the infection of RD cells by the DAF-dependent daf+ clone, but not by the daf- clone of EV11. Since the structural proteins of the two clones differed only in the receptor binding site for DAF, the unidentified non-DAF primary receptor for the daf- clone might have the same conformation as the uncoating coreceptor required for the daf+ clone. Despite the difference in primary receptors, both daf+ and daf- clones were equally inhibited by a monoclonal antibody to beta2-microglobulin. The monoclonal antibody B9.12.1 to class I human leukocyte antigen molecules showed no inhibitory effect in regards to either clone. The hypothesis of convergent intracellular traffic of Daf+ and Daf- variants of EV11 is discussed.

Two clusters of mutations map distinct receptor-binding sites of echovirus 11 for the decay-accelerating factor (CD55) and for canyon-binding receptors.

In this study we present the comparative sequence analysis of the parental haemagglutinating (daf+) and mutant non-haemagglutinating (daf-) clones of echovirus 11 (EV11) isolated from the prototype strain Gregory. The sequence comparison revealed only a single amino acid substitution in the capsid protein VP2 of each mutant clone. These substitutions were located in the area of viral receptor-binding site for DAF. Since daf- mutants of EV11 did not interact with DAF, they used an alternative receptor for the cell entry. To elucidate the nature of the alternative receptor we used subvariant clones of EV11 adapted to human rhabdomyosarcoma (RD), human carcinoma (HEp-2) and African Green monkey kidney (BGM) cell lines. The usage of the subvariant clones with altered host range and the cell cultures of human and simian origin allowed us to map the amino acid substitutions associated with the adaptation of EV11 to the alternative cellular receptors. These amino acid substitutions were located on the surface of the virion in the canyon area. Hence the virus canyon may serve as the receptor-binding site for the alternative (in respect to DAF) cellular receptor(s).

The composition, structural properties and binding of very-low-density and low-density lipoproteins to the LDL receptor in normo- and hypertriglyceridemia: relation to the apolipoprotein E phenotype.

The composition, apolipoprotein structure and lipoprotein binding to the LDL receptor were studied for very-low-density (VLDL) and low-density lipoprotein (LDL) particles isolated from subjects with apoE phenotype E3/3 (E3), E2/2 or E2/3 (E2+) and E3/4 or E4/4 (E4+) and a wide range of plasma triglyceride (TG) contents. The data combined for all three phenotype groups can be summarized as follows. (i) A decrease in accessibility of VLDL tryptophan residues to I- anions with a decrease in tryptophan surface density, concomitant with an increase in VLDL dimensions, reflects the increased efficiency of protein-protein interactions. (ii) A gradual increase in the quenching constant for LDL apoB fluorescence with an increase in TG/cholesterol (Chol) ratio reflects the 'freezing' effect of Chol molecules on apoB dynamics. (iii) Different mechanisms specific for a particular lipoprotein from E3/3 or E2/3 subjects are responsible for apoE-mediated VLDL binding and apoB-mediated LDL binding to the LDL receptor in a solid-phase binding assay. (iv) The 'spacing' effect of apoC-III molecules on apoE-mediated VLDL binding results in a decrease in the number of binding sites. (v) The maximum of the dependence of the LDL binding affinity constant on relative tryptophan density corresponds to LDL intermediate size. VLDL particles from hypertriglyceridemic E2/3 heterozygotic individuals had remnant-like properties (increased cholesterol, apoE and decreased apoC-III content) while their binding efficiency was unchanged. Based on the affinity constant value and LDL-Chol content, increased competition between VLDL and LDL for the binding to the LDL receptor upon increase in plasma TG is suggested, and LDL from hypertriglyceridemic E3/3 homozygotic individuals is the most efficient competitor.