Correlated substitution analysis and the prediction of amino acid structural contacts

It has long been suspected that analysis of correlated amino acid substitutions should uncover pairs or clusters of sites that are spatially proximal in mature protein structures. Accordingly, methods based on different mathematical principles such as information theory, correlation coefficients and maximum likelihood have been developed to identify co-evolving amino acids from multiple sequence alignments. Sets of pairs of sites whose behaviour is identified by these methods as correlated are often significantly enriched in pairs of spatially proximal residues. However, relatively high levels of false-positive predictions typically render such methods, in isolation, of little use in the ab initio prediction of protein structure. Misleading signal (or problems with the estimation of significance levels) can be caused by phylogenetic correlations between homologous sequences and from correlation due to factors other than spatial proximity (for example, correlation of sites which are not spatially close but which are involved in common functional properties of the protein). In recent years, several workers have suggested that information from correlated substitutions should be combined with other sources of information (secondary structure, solvent accessibility, evolutionary rates) in an attempt to reduce the proportion of false-positive predictions. We review methods for the detection of correlated amino acid substitutions, compare their relative performance in contact prediction and predict future directions in the field.     

PERMANENT GENETIC RESOURCES: Isolation and characterization of microsatellite loci from the Arctic cisco (Coregonus autumnalis)

Eight polymorphic microsatellite loci were isolated and characterized for the Arctic cisco, Coregonus autumnalis. Loci were evaluated in 21 samples from the Colville River subsistence fishery. The number of alleles per locus ranged from two to 18. Observed heterozygosity of loci varied from 0.10 to 1.00, and expected heterozygosity ranged from 0.09 to 0.92. All eight microsatellite markers were in Hardy-Weinberg equilibrium. The loci presented here will be useful in describing population structure and exploring populations of origin for Arctic cisco.     

Comparative Genomics Reveals Early Emergence and Biased Spatiotemporal Distribution of SARS-CoV-2

Effective systems for the analysis of molecular data are fundamental for monitoring the spread of infectious diseases and studying pathogen evolution. The rapid identification of emerging viral strains, and/or genetic variants potentially associated with novel phenotypic features is one of the most important objectives of genomic surveillance of human pathogens and represents one of the first lines of defense for the control of their spread. During the COVID 19 pandemic, several taxonomic frameworks have been proposed for the classification of SARS-Cov-2 isolates. These systems, which are typically based on phylogenetic approaches, represent essential tools for epidemiological studies as well as contributing to the study of the origin of the outbreak. Here, we propose an alternative, reproducible, and transparent phenetic method to study changes in SARS-CoV-2 genomic diversity over time. We suggest that our approach can complement other systems and facilitate the identification of biologically relevant variants in the viral genome. To demonstrate the validity of our approach, we present comparative genomic analyses of more than 175,000 genomes. Our method delineates 22 distinct SARS-CoV-2 haplogroups, which, based on the distribution of high-frequency genetic variants, fall into four major macrohaplogroups. We highlight biased spatiotemporal distributions of SARS-CoV-2 genetic profiles and show that seven of the 22 haplogroups (and of all of the four haplogroup clusters) showed a broad geographic distribution within China by the time the outbreak was widely recognized—suggesting early emergence and widespread cryptic circulation of the virus well before its isolation in January 2020. General patterns of genomic variability are remarkably similar within all major SARS-CoV-2 haplogroups, with UTRs consistently exhibiting the greatest variability, with s2m, a conserved secondary structure element of unknown function in the 3′-UTR of the viral genome showing evidence of a functional shift. Although several polymorphic sites that are specific to one or more haplogroups were predicted to be under positive or negative selection, overall our analyses suggest that the emergence of novel types is unlikely to be driven by convergent evolution and independent fixation of advantageous substitutions, or by selection of recombined strains. In the absence of extensive clinical metadata for most available genome sequences, and in the context of extensive geographic and temporal biases in the sampling, many questions regarding the evolution and clinical characteristics of SARS-CoV-2 isolates remain open. However, our data indicate that the approach outlined here can be usefully employed in the identification of candidate SARS-CoV-2 genetic variants of clinical and epidemiological importance.     

Hydroxytyrosol from olive fruits prevents blue-light-induced damage in human keratinocytes and fibroblasts

Skin aging is a complex biological process influenced by a combination of endogenous or intrinsic and exogenous or extrinsic factors due to environmental damage. The primary environmental factor that causes human skin aging is the ultraviolet irradiation from the sun. Recently, it was established that the long-term exposure to light-emitting-diode-generated blue light (LED-BL) from electronic devices seems to have a relevant implication in the molecular mechanisms of premature photoaging. BL irradiation induces changes in the synthesis of various skin structures through DNA damage and overproduction of reactive oxygen species (ROS), matrix metalloproteinase-1 and -12, which are responsible for the loss of the main components of the extracellular matrix of skin like collagen type I and elastin. In the current study, using human keratinocytes and fibroblasts exposed to specific LED-BL radiation doses (45 and 15 J/cm ²), we produced an in vitro model of skin photoaging. We verified that, compared with untreated controls, the treatment with LED-BL irradiation results in the alteration of metalloprotease-1 (collagenase), metalloprotease-12 (elastase), 8-dihydroxy-2′-deoxyguanosine, proliferating cell nuclear antigen, and collagen type I. Moreover, we showed that the photoaging prevention is possible via the use of hydroxytyrosol extracted from olive fruits, well known for antioxidant properties. Our results demonstrated that hydroxytyrosol protects keratinocytes and fibroblasts from LED-BL-induced damage. Thus, hydroxytyrosol might be proposed as an encouraging candidate for the prevention of BL-induced premature photoaging.     

Contribution to the environmental ecology of Cryptogramma crispa (L.) R. Br. ex Hooker in the Alps

We studied the habitat characteristics in a number of Cryptogramma crispa stations throughout the Alps. Our objectives were: (1) to define the environmental ecology of C. crispa in the southern part of its distributional area in Europe; (2) to test the reliability of ecological indicator values of soil reaction, nutrient and dispersion status, light and temperature for this species. The habitats of C. crispa mostly consisted in coarse-grained stable screes (on rock slide deposits, talus cones, talus sheets, stone fields and moraines), within a broad elevation range with maximum frequency in the subalpine and lower alpine vegetation belts. At higher altitudes, C. crispa was mostly found on South-exposed slopes with high potential irradiance, whereas at low altitude it was most abundant on North-exposed rather gentle slopes formed of big boulders. The screes colonized by C. crispa were formed of acid metamorphic rocks or igneous rocks and had a coarse texture. The soil accumulated in the spaces among boulders had sandy texture and acidic reaction, with medium to high carbon and total nitrogen contents. Calcium lacks entirely in the soil. Our field data gave support to the Ellenberg–Landolt ecological indicator values of light and temperature, but not to those of soil reaction and soil nutrient content, for which some changes are proposed.     

Enhancing the immunogenicity of a permissive binding T cell epitope derived from the simian immunodeficiency virus-encoded negative regulatory factor.

Using the murine system we have analyzed an immunogenic T cell peptide epitope corresponding to amino acids 96-112 of the simian immunodeficiency virus-negative regulatory protein sequence. This epitope was unusual in that it was strongly immunogenic in mice of five of the six H-2 haplotypes tested. We generated a T cell hybridoma (SVNF) specific for this peptide in order to determine how manipulating the peptide might alter its immunogenicity. Substitution analysis showed that His 103, Pro 104, Val 106, and Pro 107 were important amino acids for stimulating SVNF because substitutions at these positions diminished the reactivity of SVNF. However, we also found that substituting an Ala for a Val at position 100 or a Val for an Ala at position 110 enhanced reactivity of SVNF. We were able to further enhance the immunogenicity of this epitope by extending the carboxyl terminus two amino acids and making the resulting carboxyl-terminal Lys an amide and by adding a Glu to the amino terminus. These modifications shifted the in vitro activity of SVNF at least two orders of magnitude. We also compared the ability of this modified peptide and the wild-type SIV nef 96-112 to prime a T cell response in vivo. We primed mice with various doses of either the wild-type or the modified peptide and looked at the ability of the draining lymph node cells to proliferate to wild-type peptide. We found that the modified peptide was 10- to 100-fold better at priming a T cell response than the wild-type peptide. Therefore, we were able to create a peptide that was more immunogenic than the wild-type peptide in vivo as well as in vitro. Manipulations such as these that enhance the immunogenicity of T cell epitopes must be considered in developing peptide vaccines against HIV or other infectious agents.     

Activation of Blood Coagulation in Two Prototypic Autoimmune Skin Diseases: A Possible Link with Thrombotic Risk

Coagulation activation has been demonstrated in two prototypic autoimmune skin diseases, chronic autoimmune urticaria and bullous pemphigoid, but only the latter is associated with increased thrombotic risk. Two markers of coagulation activation (prothrombin fragment F1+2 and fibrin fragment D-dimer) were measured by immunoenzymatic methods in plasma samples from 30 patients with active chronic autoimmune urticaria, positive for autologous serum skin test, 30 patients with active bullous pemphigoid and 30 healthy subjects. In skin biopsies, tissue factor expression was evaluated by both immunohistochemistry and in situ hybridization. F1+2 and D-dimer levels were higher in active chronic autoimmune urticaria (276.5±89.8 pmol/L and 5.56±4.40 nmol/L, respectively) than in controls (145.2±38.0 pmol/L and 1.06±0.25 nmol/L; P=0.029 and P=0.011) and were much higher in active bullous pemphigoid (691.7±318.7 pmol/L and 15.24±9.09 nmol/L, respectively) (P<0.0001). Tissue factor positivity was evident in skin biopsies of both disorders with higher intensity in bullous pemphigoid. F1+2 and D-dimer, during remission, were markedly reduced in both disorders. These findings support the involvement of coagulation activation in the pathophysiology of both diseases. The strong systemic activation of coagulation in bullous pemphigoid may contribute to increase the thrombotic risk and provides the rationale for clinical trials on anticoagulant treatments in this disease.