From Discrete to Continuum Models of Three-Dimensional Deformations in Epithelial Sheets

Metamorphic proteins, including proteins with high levels of sequence identity but different folds, are exceptions to the long-standing rule-of-thumb that proteins with as little as 30% sequence identity adopt the same fold. Which topologies can be bridged by these highly identical sequences remains an open question. Here we bridge two 3-α-helix bundle proteins with two radically different folds. Using a straightforward approach, we engineered the sequences of one subdomain within maltose binding protein (MBP, α/β/α-sandwich) and another within outer surface protein A (OspA, β-sheet) to have high sequence identity (80 and 77%, respectively) with engineered variants of protein G (G_A, 3-α-helix bundle). Circular dichroism and nuclear magnetic resonance spectra of all engineered variants demonstrate that they maintain their native conformations despite substantial sequence modification. Furthermore, the MBP variant (80% identical to G_A) remained active. Thermodynamic analysis of numerous G_A and MBP variants suggests that the key to our approach involved stabilizing the modified MBP and OspA subdomains via external interactions with neighboring substructures, indicating that subdomain interactions can stabilize alternative folds over a broad range of sequence variation. These findings suggest that it is possible to bridge one fold with many other topologies, which has implications for protein folding, evolution, and misfolding diseases.     

Mating-type genes and the genetic structure of a world-wide collection of the tomato pathogen Cladosporium fulvum

Two mating-type genes, designated MAT1-1-1 and MAT1-2-1, were cloned and sequenced from the presumed asexual ascomycete Cladosporium fulvum (syn. Passalora fulva). The encoded products are highly homologous to mating-type proteins from members of the Mycosphaerellaceae, such as Mycosphaerella graminicola and Cercospora beticola. In addition, the two MAT idiomorphs of C. fulvum showed regions of homology and each contained one additional putative ORF without significant similarity to known sequences. The distribution of the two mating-type genes in a world-wide collection of 86 C. fulvum strains showed a departure from a 1:1 ratio (chi(2)=4.81, df=1). AFLP analysis revealed a high level of genotypic diversity, while strains of the fungus were identified with similar virulence spectra but distinct AFLP patterns and opposite mating-types. These features could suggest the occurrence of recombination in C. fulvum.     

Classification and gradient analysis of the beech forest vegetation of the southern Rodopi (northeast Greece)

Pure and mixed beech forest vegetation of the southern Rodopi range (northeast Greece) was studied using 614 relevés and multivariate analyses (TWINSPAN and DCA). Classification of the relevés resulted in 12 vegetation units, 8 of which were ranked as associations or communities and the rest as subcommunities and variants. DCA diagrams of relevés and taxa indicated that floristic differentiation was attributed mainly to factors such as altitude (affecting temperature and humidity), soil nutrient content and substrate type (affecting physical and chemical soil properties). Differential taxa of vegetation units were chosen based on their phi coefficient values, which were calculated from three different percentage synoptic tables that corresponded to three ranks (ecological groups, associations and communities, and subcommunities and variants) of floristic differentiation. The calculation of phi coefficient on the basis of relative constancy of taxa helps to overcome the problem of the dependence of fidelity values on the number of relevés per vegetation unit and to facilitate the better investigation of the floristic differentiation even of rare vegetation units represented by a small number of relevés. Furthermore, the calculation of fidelity values for different hierarchical levels enables a more detailed and thorough investigation of the floristic differentiation of the vegetation units.     

Non‐lethal proteasome inhibition activates pro‐tumorigenic pathways in multiple myeloma cells

Multiple myeloma (MM) is a haematological malignancy being characterized by clonal plasma cell proliferation in the bone marrow. Targeting the proteasome with specific inhibitors (PIs) has been proven a promising therapeutic strategy and PIs have been approved for the treatment of MM and mantle‐cell lymphoma; yet, while outcome has improved, most patients inevitably relapse. As relapse refers to MM cells that survive therapy, we sought to identify the molecular responses induced in MM cells after non‐lethal proteasome inhibition. By using bortezomib (BTZ), epoxomicin (EPOX; a carfilzomib‐like PI) and three PIs, namely Rub999, PR671A and Rub1024 that target each of the three proteasome peptidases, we found that only BTZ and EPOX are toxic in MM cells at low concentrations. Phosphoproteomic profiling after treatment of MM cells with non‐lethal (IC₁₀) doses of the PIs revealed inhibitor‐ and cell type‐specific readouts, being marked by the activation of tumorigenic STAT3 and STAT6. Consistently, cytokine/chemokine profiling revealed the increased secretion of immunosuppressive pro‐tumorigenic cytokines (IL6 and IL8), along with the inhibition of potent T cell chemoattractant chemokines (CXCL10). These findings indicate that MM cells that survive treatment with therapeutic PIs shape a pro‐tumorigenic immunosuppressive cellular and secretory bone marrow microenvironment that enables malignancy to relapse.     

Stabilization of Oxidic Tailings and Contaminated Soils by Calcium Oxyphosphate Addition: The Case of Montevecchio (Sardinia,Italy)

The effectiveness of an in situ heavy metals fixation technique aimed at converting contaminants to low solubility and low bioavail-ability forms, eliminating the risk posed by oxidic tailings and contaminated soils, was investigated. Calcium oxyphosphate salt (Ca(H₂PO₄)₂·H₂O) was used as a stabilizing agent for oxidic tailings and contaminated soils originating from Montevecchio, Sardinia, Italy. Stabilization was effected by mixing the contaminated soil or oxidic tailing sample with calcium oxyphosphate salt at various doses. The effectiveness of stabilization was evaluated by USEPA TCLP standard toxicity testing. Complementary EDTA extraction tests and biological tests using beans Phaseolous vulgaris as plant indicator were carried out. The toxicity of Pb and Cd was reduced below TCLP regulatory limits at calcium oxyphosphate doses higher than 0.7 and 0.2% w/w for soils and tailings, respectively. Lead solubility according to the EDTA test decreased with phosphate dose for both materials tested. Lead uptake by plant leaves and roots from the soil sample decreased with the phosphate addition, while Cd uptake remained almost constant. An adverse effect on plant growth and Zn uptake was observed for calcium oxyphosphate dose up to 1.1% w/w. Based on the results, a remediation scheme for oxidic tailings and contaminated soils is proposed.     

Allotypic variation in antigen processing controls antigenic peptide generation from SARS-CoV-2 S1 spike glycoprotein

Population genetic variability in immune system genes can often underlie variability in immune responses to pathogens. Cytotoxic T-lymphocytes are emerging as critical determinants of both severe acute respiratory syndrome coronavirus 2 infection severity and long-term immunity, after either recovery or vaccination. A hallmark of coronavirus disease 2019 is its highly variable severity and breadth of immune responses between individuals. To address the underlying mechanisms behind this phenomenon, we analyzed the proteolytic processing of S1 spike glycoprotein precursor antigenic peptides across ten common allotypes of endoplasmic reticulum aminopeptidase 1 (ERAP1), a polymorphic intracellular enzyme that can regulate cytotoxic T-lymphocyte responses by generating or destroying antigenic peptides. We utilized a systematic proteomic approach that allows the concurrent analysis of hundreds of trimming reactions in parallel, thus better emulating antigen processing in the cell. While all ERAP1 allotypes were capable of producing optimal ligands for major histocompatibility complex class I molecules, including known severe acute respiratory syndrome coronavirus 2 epitopes, they presented significant differences in peptide sequences produced, suggesting allotype-dependent sequence biases. Allotype 10, previously suggested to be enzymatically deficient, was rather found to be functionally distinct from other allotypes. Our findings suggest that common ERAP1 allotypes can be a major source of heterogeneity in antigen processing and through this mechanism contribute to variable immune responses in coronavirus disease 2019.