Sexual dimorphism and biomechanical loading in occipital bone morphological variation.

We explore the contribution of biological sex and biomechanical activity from subsistence to occipital bone variation. Previous studies have used occipital bone traits to determine biological sex and identify ancestry to differing degrees of success. Biomechanical stress from variation in subsistence and gender-based divisions of labor could perhaps explain some of the noise in the signal for these grouping variables. To explore this possibility, we used metric (foramen magnum length and breadth, external occipital protuberance depth, lambda-inion length, bicondylar breadth) and nonmetric traits (general occipital form, presence of a nuchal crest, and nuchal line count). We collected original data and mined published data for our analysis using skeletal collections of Native American hunter-gatherers and horticulturalists, a historic military site, and contemporary study collections. We find that the foramen magnum area exhibits sexual dimorphism and is not influenced by subsistence, but the accuracy of sex estimation is only 71%, suggesting the chance of being correct at slightly more than two-thirds. All traits exhibited sex-based variation but only bicondylar breadth and lambda-inion metrics exhibited subsistence-based variation. Given the limited amount of variance explained by either sex or sex and subsistence, biomechanics may still play a role but not from the influence of subsistence practices in these datasets. Additional data from a wider array of skeletal samples, perhaps with known occupation, is warranted if we are to understand how occipital variation is shaped.

Parallel evolution of multiple mechanisms for demethylase inhibitor fungicide resistance in the barley pathogen Pyrenophora teres f. sp. maculata.

The fungal pathogen Pyrenophora teres f. sp. maculata (Ptm), responsible for spot-form of net blotch (SFNB), is currently the most significant disease of barley in Australia and a major disease worldwide. Management of SFNB relies heavily on fungicides and in Australia the demethylase inhibitors (DMIs) predominate. There have been sporadic reports of resistance to DMIs in Ptm but the mechanisms remain obscure. Ptm isolates collected from 1996 to 2019 in Western Australia were tested for fungicide sensitivity levels. Decreased sensitivity to DMIs was observed in isolates collected after 2015. Resistance factors to tebuconazole fell into two classes; moderate resistance (MR; RF 6-11) and high resistance (HR; RFs 30-65). Mutations linked to resistance were detected in the promoter region and coding sequence of the DMI target gene Cyp51A. Solo-LTR insertion elements were found at 5 different locations in the promoter region. Three different non-synonymous mutations encoded an altered protein with a phenylalanine to leucine substitution at position 489, F489L (F495I in the archetype CYP51A of Aspergillus fumigatus). F489L mutations have also been found in DMI-resistant strains of P. teres f. sp. teres. Ptm isolates carrying either a LTR insertion element or a F489L allele displayed the MR1 or MR2 phenotypes, respectively. Isolates carrying both an insertion element and a F489L mutation displayed the HR phenotype. Multiple mechanisms acting both alone and in concert were found to contribute to DMI resistance in Ptm. Moreover, these mutations have emerged repeatedly in Western Australian Ptm populations by a process of parallel evolution.

Spider-silk inspired polymeric networks by harnessing the mechanical potential of ß-sheets through network guided assembly.

The high toughness of natural spider-silk is attributed to their unique ß-sheet secondary structures. However, the preparation of mechanically strong ß-sheet rich materials remains a significant challenge due to challenges involved in processing the polymers/proteins, and managing the assembly of the hydrophobic residues. Inspired by spider-silk, our approach effectively utilizes the superior mechanical toughness and stability afforded by localised ß-sheet domains within an amorphous network. Using a grafting-from polymerisation approach within an amorphous hydrophilic network allows for spatially controlled growth of poly(valine) and poly(valine-r-glycine) as ß-sheet forming polypeptides via N-carboxyanhydride ring opening polymerisation. The resulting continuous ß-sheet nanocrystal network exhibits improved compressive strength and stiffness over the initial network lacking ß-sheets of up to 30 MPa (300 times greater than the initial network) and 6 MPa (100 times greater than the initial network) respectively. The network demonstrates improved resistance to strong acid, base and protein denaturants over 28 days.

Morphometric analysis of shape differences in Windover and Point Hope archaic human mandibles.

OBJECTIVES: The mandible can provide valuable information on both the life history and genetic makeup of Archaic human populations. The following analysis tests two hypotheses: (a) that there are significant differences in morphology in mandibular shape between the genders amongst Archaic North American Homo sapiens and (b) that there is a significant difference in variance in mandibular shape between Archaic Windover and Point Hope. MATERIALS AND METHODS: A sample made from mandible specimens taken from both populations is subjected to Principal Component Analyses (PCA). The component scores from the PCAs are subjected to both a Multivariate Analysis of Covariance (mancova) and a general Multivariate Analysis of Variance (manova) to determine whether significant differences in variance exist between the sexes and the populations. RESULTS: The mancova found that there are no significant interactions between the PC scores in population, sex, or size. Significant differences in variance were found between males and females and between the Windover and Point Hope populations. CONCLUSIONS: Differences in variance observed between the populations are suspected to be due to differences in subsistence strategies and possibly non-masticatory utilizations of teeth. Differences in variance between the genders are suspected to be genetic in origin.

A Protein-Capsid-Based System for Cell Delivery of Selenocysteine.

Selenocysteine (Sec) has received a lot of attention as a potential anticancer drug. However, its broad cytotoxicity limits its therapeutic usefulness. Thus, Sec is an attractive candidate for targeted drug delivery. Here, we demonstrate for the first time that an engineered version of the capsid formed by Aquifex aeolicus lumazine synthase (AaLS) can act as a nanocarrier for delivery of Sec to cells. Specifically, a previously reported variant of AaLS (AaLS-IC), which contains a single cysteine per subunit that projects into the capsid interior, was modified by reaction with the diselenide dimer of Sec (Sec2) to generate a selenenylsulfide conjugate between the capsid and Sec (AaLS-IC-Sec). Importantly, it was determined that the structural context of the reactive cysteine was important for efficient capsid loading. Further, the encapsulated Sec could be quantitatively released from AaLS-IC-Sec by reducing agents such as glutathione or dithiothreitol. To assess cellular penetrance capabilities of AaLS-IC-Sec and subsequent cytotoxic response, six different cells line models were examined. Across the cell lines analyzed, cytotoxic sensitivity correlated with cellular uptake and intracellular trafficking patterns. Together these findings suggest that the engineered AaLS-IC capsid is a promising vehicle for targeted cell delivery of Sec.

An 8000-year-old case of thalassemia from the Windover, Florida skeletal population.

Thalassemia is a congenital blood disorder which destroys red blood cells at a faster rate than can be produced, resulting in anemia. Historically, this disease is found more often in Old World populations, such as Middle Eastern and Southeast Asian. The earliest reported skeletal evidence of thalassemia comes from the eastern Mediterranean (Atlit-Yam) and is correlated with early agriculturalists' exposure to malarial parasites. While there have been virtually no skeletal reports of thalassemia in prehistoric Native American populations, among the individuals from the 8000-year-old hunter-gatherer site of Windover, Florida there is a single potential case of the disease. A female in her early 20's exhibits bilateral foreshortening of the humeri with indications of premature epiphyseal fusion. Both proximal humeri are medio-laterally compressed, the gleno-humeral joint surfaces exhibit medial deformation, and bones show expansion of the medullary cavity with increased cancellous bone growth. These characteristics have been reported as indicators of thalassemia in both clinical and archaeological contexts. Alternate diagnoses such as congenital dislocation or injuries during child birth are considered but fail to account for the full set of characteristics shown. Individual #76 may, therefore, represent the oldest reported case of thalassemia from a native North American skeletal population.

In vivo encapsulation of nucleic acids using an engineered nonviral protein capsid.

In Nature, protein capsids function as molecular containers for a wide variety of molecular cargoes. Such containers have great potential for applications in nanotechnology, which often require encapsulation of non-native guest molecules. Charge complementarity represents a potentially powerful strategy for engineering novel encapsulation systems. In an effort to explore the generality of this approach, we engineered a nonviral, 60-subunit capsid, lumazine synthase from Aquifex aeolicus (AaLS), to act as a container for nucleic acid. Four mutations were introduced per subunit to increase the positive charge at the inner surface of the capsid. Characterization of the mutant (AaLS-pos) revealed that the positive charges lead to the uptake of cellular RNA during production and assembly of the capsid in vivo. Surprisingly, AaLS-pos capsids were found to be enriched with RNA molecules approximately 200-350 bases in length, suggesting that this simple charge complementarity approach to RNA encapsulation leads to both high affinity and a degree of selectivity. The ability to control loading of RNA by tuning the charge at the inner surface of a protein capsid could illuminate aspects of genome recognition by viruses and pave the way for the development of improved RNA delivery systems.

Crystal structures of a halophilic archaeal malate synthase from Haloferax volcanii and comparisons with isoforms A and G.

BACKGROUND: Malate synthase, one of the two enzymes unique to the glyoxylate cycle, is found in all three domains of life, and is crucial to the utilization of two-carbon compounds for net biosynthetic pathways such as gluconeogenesis. In addition to the main isoforms A and G, so named because of their differential expression in E. coli grown on either acetate or glycolate respectively, a third distinct isoform has been identified. These three isoforms differ considerably in size and sequence conservation. The A isoform (MSA) comprises ~530 residues, the G isoform (MSG) is ~730 residues, and this third isoform (MSH-halophilic) is ~430 residues in length. Both isoforms A and G have been structurally characterized in detail, but no structures have been reported for the H isoform which has been found thus far only in members of the halophilic Archaea. RESULTS: We have solved the structure of a malate synthase H (MSH) isoform member from Haloferax volcanii in complex with glyoxylate at 2.51 Å resolution, and also as a ternary complex with acetyl-coenzyme A and pyruvate at 1.95 Å. Like the A and G isoforms, MSH is based on a ß8/α8 (TIM) barrel. Unlike previously solved malate synthase structures which are all monomeric, this enzyme is found in the native state as a trimer/hexamer equilibrium. Compared to isoforms A and G, MSH displays deletion of an N-terminal domain and a smaller deletion at the C-terminus. The MSH active site is closely superimposable with those of MSA and MSG, with the ternary complex indicating a nucleophilic attack on pyruvate by the enolate intermediate of acetyl-coenzyme A. CONCLUSIONS: The reported structures of MSH from Haloferax volcanii allow a detailed analysis and comparison with previously solved structures of isoforms A and G. These structural comparisons provide insight into evolutionary relationships among these isoforms, and also indicate that despite the size and sequence variation, and the truncated C-terminal domain of the H isoform, the catalytic mechanism is conserved. Sequence analysis in light of the structure indicates that additional members of isoform H likely exist in the databases but have been misannotated.