The Efficient Extraction Method of Collagen from Deteriorated Leather Artifacts.

Collagen is the most crucial component of leather artifacts and analyzing collagen can provide vital information for studying and conserving such artifacts. However, collagen in leather artifacts often faces challenges such as degradation, denaturation, and contamination, which make it difficult to achieve an ideal protein extract using traditional extraction methods. This study aimed to find an efficient collagen extraction strategy for aging leather by comparing and improving commonly used methods. The results of comparing different extraction methods indicated that a NaOH solution was highly effective in extracting collagen from aged leather. To determine the optimal conditions for collagen extraction from the NaOH solution, we conducted orthogonal experiments. The results revealed that a NaOH concentration of 0.05 mol/L, a dissolution temperature of 80 °C, and a dissolution time of 12 h were the most favorable conditions. To validate the effectiveness of this method, we performed SDS-PAGE and biological mass spectrometry tests on collagen extracts from leather samples with varying degrees of aging. All collagen extracts exhibited distinct bands in the gel, and the molecular weight of collagen in each sample exceeded 20 kDa. Furthermore, even with a reduced sample mass of 1 mg (micro-destructive sampling), biological mass spectrometry identified 124 peptides in the protein extract. Notably, four of these peptides were unique to cattle hide collagen and were not present in the collagen of pig, sheep, horse, deer, or human skins. These experimental findings confirm the efficacy of the NaOH solution for extracting collagen from aging leather, suggesting that it can serve as a significant method for collagen identification and analysis in leather artifacts.

Phylogenetic Articulation of Uric Acid Evolution in Mammals and How It Informs a Therapeutic Uricase.

The role of uric acid during primate evolution has remained elusive ever since it was discovered over 100 years ago that humans have unusually high levels of the small molecule in our serum. It has been difficult to generate a neutral or adaptive explanation in part because the uricase enzyme evolved to become a pseudogene in apes thus masking typical signals of sequence evolution. Adding to the difficulty is a lack of clarity on the functional role of uric acid in apes. One popular hypothesis proposes that uric acid is a potent antioxidant that increased in concentration to compensate for the lack of vitamin C synthesis in primate species ∼65 Ma. Here, we have expanded on our previous work with resurrected ancient uricase proteins to better resolve the reshaping of uricase enzymatic activity prior to ape evolution. Our results suggest that the pivotal death-knell to uricase activity occurred between 20 and 30 Ma despite small sequential modifications to its catalytic efficiency for the tens of millions of years since primates lost their ability to synthesize vitamin C, and thus the two appear uncorrelated. We also use this opportunity to demonstrate how molecular evolution can contribute to biomedicine by presenting ancient uricases to human immune cells that assay for innate reactivity against foreign antigens. A highly stable and highly catalytic ancient uricase is shown to elicit a lower immune response in more human haplotypes than other uricases currently in therapeutic development.

Contributions of substitutions and indels to the structural variations in ancient protein superfamilies.

BACKGROUND: Quantitative evaluation of protein structural evolution is important for our understanding of protein biological functions and their evolutionary adaptation, and is useful in guiding protein engineering. However, compared to the models for sequence evolution, the quantitative models for protein structural evolution received less attention. Ancient protein superfamilies are often considered versatile, allowing genetic and functional diversifications during long-term evolution. In this study, we investigated the quantitative impacts of sequence variations on the structural evolution of homologues in 68 ancient protein superfamilies that exist widely in sequenced eukaryotic, bacterial and archaeal genomes. RESULTS: We found that the accumulated structural variations within ancient superfamilies could be explained largely by a bilinear model that simultaneously considers amino acid substitution and insertion/deletion (indel). Both substitutions and indels are essential for explaining the structural variations within ancient superfamilies. For those ancient superfamilies with high bilinear multiple correlation coefficients, the influence of each unit of substitution or indel on structural variations is almost constant within each superfamily, but varies greatly among different superfamilies. The influence of each unit indel on structural variations is always larger than that of each unit substitution within each superfamily, but the accumulated contributions of indels to structural variations are lower than those of substitutions in most superfamilies. The total contributions of sequence indels and substitutions (46% and 54%, respectively) to the structural variations that result from sequence variations are slightly different in ancient superfamilies. CONCLUSIONS: Structural variations within ancient protein superfamilies accumulated under the significantly bilinear influence of amino acid substitutions and indels in sequences. Both substitutions and indels are essential for explaining the structural variations within ancient superfamilies. For those structural variations resulting from sequence variations, the total contribution of indels is slightly lower than that of amino acid substitutions. The regular clock exists not only in protein sequences, but also probably in protein structures.

The biochemical signatures of stress: A preliminary analysis of osteocalcin concentrations and macroscopic skeletal changes associated with stress in the 13th - 17th centuries black friars population.

OBJECTIVE: As a chemical precursor to the hard tissue changes well-studied in bioarchaeological research, osteocalcin provides a unique opportunity to assess stress via fluctuations in bone metabolism. The main objectives of this research were 1) to successfully extract osteocalcin from the Black Friars skeletal population; 2) to assess the diagenetic change between individual bone samples; and 3) to compare osteocalcin concentrations across sex, age, time period and macroscopic indicators of stress. METHODS: Twenty adult individuals were selected from the 13th-17th centuries Black Friars skeletal population with bone samples taken from the clavicle and femur. Total protein was assessed through a MicroBCA analysis with osteocalcin quantified using a Human Quantikine ELISA kit. Diagenetic change was assessed using Fourier transform infrared spectroscopy and the attenuated total reflectance method. RESULTS: Osteocalcin concentrations showed no significant differences between sex or age groups; however, between time period the post-medieval individuals showed a significant reduction of osteocalcin in both the clavicle and the femur. There were no significant differences in osteocalcin concentrations between those with and without past stress indicators and only one significant difference among the chronic indicators. The diagenetic results demonstrated a similar degree of crystallinity between all samples. CONCLUSIONS: While preliminary in nature, this study was successful in demonstrating the potential use of osteocalcin in future health-related research and how the study of osteocalcin may contribute to a better understanding of how and when stress begins to affect the skeletal tissues.

Experimental evidence for the thermophilicity of ancestral life.

Theoretical studies have focused on the environmental temperature of the universal common ancestor of life with conflicting conclusions. Here we provide experimental support for the existence of a thermophilic universal common ancestor. We present the thermal stabilities and catalytic efficiencies of nucleoside diphosphate kinases (NDK), designed using the information contained in predictive phylogenetic trees, that seem to represent the last common ancestors of Archaea and of Bacteria. These enzymes display extreme thermal stabilities, suggesting thermophilic ancestries for Archaea and Bacteria. The results are robust to the uncertainties associated with the sequence predictions and to the tree topologies used to infer the ancestral sequences. Moreover, mutagenesis experiments suggest that the universal ancestor also possessed a very thermostable NDK. Because, as we show, the stability of an NDK is directly related to the environmental temperature of its host organism, our results indicate that the last common ancestor of extant life was a thermophile that flourished at a very high temperature.