Cranial and endocranial diversity in extant and fossil atelids (Platyrrhini: Atelidae): A geometric morphometric study.

OBJECTIVES: Platyrrhines constitute a diverse clade, with the modern Atelidae exhibiting the most variation in cranial and endocast morphology. The processes responsible for this diversification are not well understood. Here, we present a geometric morphometric study describing variation in cranial and endocranial shape of 14 species of Alouatta, Ateles, Brachyteles, and Lagothrix and two extinct taxa, Cartelles and Caipora. METHODS: We examined cranial and endocranial shape variation among species using images reconstructed from CT scans and geometric morphometric techniques based on three-dimensional landmarks and semilandmarks. Principal components analyses were used to explore variation, including the Procrustes shape coordinates, summing the logarithm of the Centroid Size, the common allometric component, and residual shape components. RESULTS: Differences in endocranial shape are related to a relative increase or decrease in the volume of the neocortex region with respect to brainstem and cerebellum regions. The relative position of the brainstem varies from a posterior position in Alouatta to a more ventral position in Ateles. The shape of both the cranium and endocast of Caipora is within the observed variation of Brachyteles. Cartelles occupies the most differentiated position relative to the extant taxa, especially in regards to its endocranial shape. CONCLUSIONS: The pattern of variation in the extant species in endocranial shape is similar to the variation observed in previous cranial studies, with Alouatta as an outlier. The similarities between Caipora and Brachyteles were unexpected and intriguing given the frugivorous adaptations inferred from the fossil's dentition. Our study shows the importance of considering both extant and fossil species when studying diversification of complex traits.

The Crystal Structure of Cancer Osaka Thyroid Kinase Reveals an Unexpected Kinase Domain Fold.

Macrophages are important cellular effectors in innate immune responses and play a major role in autoimmune diseases such as rheumatoid arthritis. Cancer Osaka thyroid (COT) kinase, also known as mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and tumor progression locus 2 (Tpl-2), is a serine-threonine (ST) kinase and is a key regulator in the production of pro-inflammatory cytokines in macrophages. Due to its pivotal role in immune biology, COT kinase has been identified as an attractive target for pharmaceutical research that is directed at the discovery of orally available, selective, and potent inhibitors for the treatment of autoimmune disorders and cancer. The production of monomeric, recombinant COT kinase has proven to be very difficult, and issues with solubility and stability of the enzyme have hampered the discovery and optimization of potent and selective inhibitors. We developed a protocol for the production of recombinant human COT kinase that yields pure and highly active enzyme in sufficient yields for biochemical and structural studies. The quality of the enzyme allowed us to establish a robust in vitro phosphorylation assay for the efficient biochemical characterization of COT kinase inhibitors and to determine the x-ray co-crystal structures of the COT kinase domain in complex with two ATP-binding site inhibitors. The structures presented in this study reveal two distinct ligand binding modes and a unique kinase domain architecture that has not been observed previously. The structurally versatile active site significantly impacts the design of potent, low molecular weight COT kinase inhibitors.

Comparative analysis of solar pasteurization versus solar disinfection for the treatment of harvested rainwater.

BACKGROUND: Numerous pathogens and opportunistic pathogens have been detected in harvested rainwater. Developing countries, in particular, require time- and cost-effective treatment strategies to improve the quality of this water source. The primary aim of the current study was thus to compare solar pasteurization (SOPAS; 70 to 79 °C; 80 to 89 °C; and ≥90 °C) to solar disinfection (SODIS; 6 and 8 hrs) for their efficiency in reducing the level of microbial contamination in harvested rainwater. The chemical quality (anions and cations) of the SOPAS and SODIS treated and untreated rainwater samples were also monitored. RESULTS: While the anion concentrations in all the samples were within drinking water guidelines, the concentrations of lead (Pb) and nickel (Ni) exceeded the guidelines in all the SOPAS samples. Additionally, the iron (Fe) concentrations in both the SODIS 6 and 8 hr samples were above the drinking water guidelines. A >99% reduction in Escherichia coli and heterotrophic bacteria counts was then obtained in the SOPAS and SODIS samples. Ethidium monoazide bromide quantitative polymerase chain reaction (EMA-qPCR) analysis revealed a 94.70% reduction in viable Legionella copy numbers in the SOPAS samples, while SODIS after 6 and 8 hrs yielded a 50.60% and 75.22% decrease, respectively. Similarly, a 99.61% reduction in viable Pseudomonas copy numbers was observed after SOPAS treatment, while SODIS after 6 and 8 hrs yielded a 47.27% and 58.31% decrease, respectively. CONCLUSION: While both the SOPAS and SODIS systems reduced the indicator counts to below the detection limit, EMA-qPCR analysis indicated that SOPAS treatment yielded a 2- and 3-log reduction in viable Legionella and Pseudomonas copy numbers, respectively. Additionally, SODIS after 8 hrs yielded a 2-log and 1-log reduction in Legionella and Pseudomonas copy numbers, respectively and could be considered as an alternative, cost-effective treatment method for harvested rainwater.

Targeting Bcr-Abl by combining allosteric with ATP-binding-site inhibitors.

In an effort to find new pharmacological modalities to overcome resistance to ATP-binding-site inhibitors of Bcr-Abl, we recently reported the discovery of GNF-2, a selective allosteric Bcr-Abl inhibitor. Here, using solution NMR, X-ray crystallography, mutagenesis and hydrogen exchange mass spectrometry, we show that GNF-2 binds to the myristate-binding site of Abl, leading to changes in the structural dynamics of the ATP-binding site. GNF-5, an analogue of GNF-2 with improved pharmacokinetic properties, when used in combination with the ATP-competitive inhibitors imatinib or nilotinib, suppressed the emergence of resistance mutations in vitro, displayed additive inhibitory activity in biochemical and cellular assays against T315I mutant human Bcr-Abl and displayed in vivo efficacy against this recalcitrant mutant in a murine bone-marrow transplantation model. These results show that therapeutically relevant inhibition of Bcr-Abl activity can be achieved with inhibitors that bind to the myristate-binding site and that combining allosteric and ATP-competitive inhibitors can overcome resistance to either agent alone.

The cranial morphology of the Botocudo Indians, Brazil.

The Botocudo Indians were hunter-gatherer groups that occupied the East-Central regions of Brazil decimated during the colonial period in the country. During the 19th century, craniometric studies suggested that the Botocudo resembled more the Paleoamerican population of Lagoa Santa than typical Native Americans groups. These results suggest that the Botocudo Indians might represent a population that retained the biological characteristics of early groups of the continent, remaining largely isolated from groups that gave origin to the modern Native South American variation. Moreover, recently, some of the Botocudo remains have been shown to have mitochondrial and autosomal DNA lineages currently found in Polynesian populations. Here, we explore the morphological affinities of Botocudo skulls within a worldwide context. Distinct multivariate analyses based on 32 craniometric variables show that 1) the two individuals with Polynesian DNA sequences have morphological characteristics that fall within the Polynesian and Botocudo variation, making their assignation as Native American specimens problematic, and 2) there are high morphological affinities between Botocudo, Early Americans, and the Polynesian series of Easter Island, which support the early observations that the Botocudo can be seen as retaining the Paleoamerican morphology, particularly when the neurocranium is considered. Although these results do not elucidate the origin of the Polynesian DNA lineages among the Botocudo, they support the hypothesis that the Botocudo represent a case of late survival of ancient Paleoamerican populations, retaining the morphological characteristics of ancestral Late Pleistocene populations from Asia.

Late shellmound occupation in southern Brazil: A multi-proxy study of the Galheta IV archaeological site.

Brazilian coastal archaeology is renowned for its numerous and large shellmounds (sambaquis), which had been continuously occupied from at least 8000 to 1000 years cal BP. However, changes in their structure and material culture in the late Holocene have led to different hypotheses concerning their ecological and cultural changes. The archaeological site Galheta IV (ca. 1300 to 500 years cal BP) offers new insights into the complexity of the late coastal occupation in southern Brazil. Our attempt was to determine whether Galheta IV can be classified as a sambaqui site, or if it belongs to a Southern proto-Jê settlement. Here, we reassessed Galheta's collections and applied a multi-proxy approach using: new 14C dates, zooarchaeology, δ13C and δ15N isotopes in bulk collagen and 87Sr/86Srenamel isotopic ratios from eight human individuals, ceramics analysis, and FTIR. The results indicate an intense exploitation of marine resources, with an area designated for processing animals located at the opposite side of the funerary areas. Bone tools and specific species of animals were found as burial accompaniments. No evidence of human cremations was detected. 87Sr/86Sr results indicate that the eight human individuals always lived on the coast, and did not come from the inland. The pottery analysis confirms the association with Itararé-Taquara, but contrary to what was assumed by previous studies, the pottery seems related to other coastal sites, and not to the highlands. In light of these findings, we propose that Galheta IV can be considered a funerary mound resulting from long and continuous interactions between shellmound and Southern proto-Jê populations. This study not only enhances our understanding of the late coastal occupation dynamics in southern Brazil but also underscores its importance in reshaping current interpretations of shellmound cultural changes over time.

Allosteric non-bisphosphonate FPPS inhibitors identified by fragment-based discovery.

Bisphosphonates are potent inhibitors of farnesyl pyrophosphate synthase (FPPS) and are highly efficacious in the treatment of bone diseases such as osteoporosis, Paget's disease and tumor-induced osteolysis. In addition, the potential for direct antitumor effects has been postulated on the basis of in vitro and in vivo studies and has recently been demonstrated clinically in early breast cancer patients treated with the potent bisphosphonate zoledronic acid. However, the high affinity of bisphosphonates for bone mineral seems suboptimal for the direct treatment of soft-tissue tumors. Here we report the discovery of the first potent non-bisphosphonate FPPS inhibitors. These new inhibitors bind to a previously unknown allosteric site on FPPS, which was identified by fragment-based approaches using NMR and X-ray crystallography. This allosteric and druggable pocket allows the development of a new generation of FPPS inhibitors that are optimized for direct antitumor effects in soft tissue.

Inhibitors of the Abl kinase directed at either the ATP- or myristate-binding site.

The ATP-competitive inhibitors dasatinib and nilotinib, which bind to catalytically different conformations of the Abl kinase domain, have recently been approved for the treatment of imatinib-resistant CML. These two new drugs, albeit very efficient against most of the imatinib-resistant mutants of Bcr-Abl, fail to effectively suppress the Bcr-Abl activity of the T315I (or gatekeeper) mutation. Generating new ATP site-binding drugs that target the T315I in Abl has been hampered, amongst others, by target selectivity, which is frequently an issue when developing ATP-competitive inhibitors. Recently, using an unbiased cellular screening approach, GNF-2, a non-ATP-competitive inhibitor, has been identified that demonstrates cellular activity against Bcr-Abl transformed cells. The exquisite selectivity of GNF-2 is due to the finding that it targets the myristate binding site located near the C-terminus of the Abl kinase domain, as demonstrated by genetic approaches, solution NMR and X-ray crystallography. GNF-2, like myristate, is able to induce and/or stabilize the clamped inactive conformation of Abl analogous to the SH2-Y527 interaction of Src. The molecular mechanism for allosteric inhibition by the GNF-2 inhibitor class, and the combined effects with ATP-competitive inhibitors such as nilotinib and imatinib on wild-type Abl and imatinib-resistant mutants, in particular the T315I gatekeeper mutant, are reviewed.

A simple protocol for amino acid type selective isotope labeling in insect cells with improved yields and high reproducibility.

An easy to use and robust approach for amino acid type selective isotope labeling in insect cells is presented. It relies on inexpensive commercial media and can be implemented in laboratories without sophisticated infrastructure. In contrast to previous protocols, where either high protein amounts or high incorporation ratios were obtained, here we achieve both at the same time. By supplementing media with a well considered amount of yeast extract, similar protein amounts as with full media are obtained, without compromising on isotope incorporation. In single and dual amino acid labeling experiments incorporation ratios are consistently ≥90% for all amino acids tested. This enables NMR studies of eukaryotic proteins and their interactions even for proteins with low expression levels. We show applications with human kinases, where protein-ligand interactions are characterized by 2D [(15)N, (1)H]- and [(13)C, (1)H]-HSQC spectra.

2,6-Naphthyridines as potent and selective inhibitors of the novel protein kinase C isozymes.

The present study describes a novel series of ATP-competitive PKC inhibitors based on the 2,6-naphthyridine template. Example compounds potently inhibit the novel Protein Kinase C (PKC) isotypes δ, ε, η, θ (in particular PKCε/η, and display a 10-100-fold selectivity over the classical PKC isotypes. The prototype compound 11 was found to inhibit PKCθ-dependent pathways in vitro and in vivo. In vitro, a-CD3/a-CD28-induced lymphocyte proliferation could be effectively blocked in 10% rat whole blood. In mice, 11 dose-dependently inhibited Staphylococcus aureus enterotoxin B-triggered IL-2 serum levels after oral dosing.

24.0 kyr cal BP stone artefact from Vale da Pedra Furada, Piauí, Brazil: Techno-functional analysis.

Current archaeological paradigm proposes that the first peopling of the Americas does not exceed the Last Glacial Maximum period. In this context, the acceptance of the anthropogenic character of the earliest stone artefacts generally rests on the presence of projectile points considered no more as typocentric but as typognomonic, since it allows, by itself, to certify the human character of the other associated artefacts. In other words, without this presence, nothing is certain. Archaeological research at Piauí (Brazil) attests to a Pleistocene human presence between 41 and 14 cal kyr BP, without any record of lithic projectile points. Here, we report the discovery and interpretation of an unusual stone artefact in the Vale da Pedra Furada site, in a context dating back to 24 cal kyr BP. The knapping stigmata and macroscopic use-wear traces reveal a conception centred on the configuration of double bevels and the production in the same specimen of at least two successive artefacts with probably different functions. This piece unambiguously presents an anthropic character and reveals a technical novelty during the Pleistocene occupation of South America.

Binding or bending: distinction of allosteric Abl kinase agonists from antagonists by an NMR-based conformational assay.

Allosteric inhibitors of Bcr-Abl have emerged as a novel therapeutic option for the treatment of CML. Using fragment-based screening, a search for novel Abl inhibitors that bind to the myristate pocket was carried out. Here we show that not all myristate ligands are functional inhibitors, but that the conformational state of C-terminal helix_I is a structural determinant for functional activity. We present an NMR-based conformational assay to monitor the conformation of this crucial helix_I and show that myristate ligands that bend helix_I are functional antagonists, whereas ligands that bind to the myristate pocket but do not induce this conformational change are kinase agonists. Activation of c-Abl by allosteric agonists has been confirmed in a biochemical assay.

Craniometric similarities within and between human populations in comparison with neutral genetic data.

The statement that pairs of individuals from different populations are often more genetically similar than pairs from the same population is a widespread idea inside and outside the scientific community. Witherspoon et al. ["Genetic similarities within and between human populations," Genetics 176:351-359 (2007)] proposed an index called the dissimilarity fraction (omega) to access in a quantitative way the validity of this statement for genetic systems. Witherspoon demonstrated that, as the number of loci increases, omega decreases to a point where, when enough sampling is available, the statement is false. In this study, we applied the dissimilarity fraction to Howells's craniometric database to establish whether or not similar results are obtained for cranial morphological traits. Although in genetic studies thousands of loci are available, Howells's database provides no more than 55 metric traits, making the contribution of each variable important. To cope with this limitation, we developed a routine that takes this effect into consideration when calculating omega. Contrary to what was observed for the genetic data, our results show that cranial morphology asymptotically approaches a mean omega of 0.3 and therefore supports the initial statement--that is, that individuals from the same geographic region do not form clear and discrete clusters--further questioning the idea of the existence of discrete biological clusters in the human species. Finally, by assuming that cranial morphology is under an additive polygenetic model, we can say that the population history signal of human craniometric traits presents the same resolution as a neutral genetic system dependent on no more than 20 loci.

Zinc isotope variations in archeological human teeth (Lapa do Santo, Brazil) reveal dietary transitions in childhood and no contamination from gloves.

Zinc (Zn) isotope ratios of dental enamel are a promising tracer for dietary reconstruction in archeology, but its use is still in its infancy. A recent study demonstrated a high risk of Zn contamination from nitrile, and latex gloves used during chemical sample preparation. Here we assess the potential impact of the use of such gloves during enamel sampling on the Zn isotope composition of teeth from a population of early Holocene hunter gatherers from Lapa do Santo, Lagoa Santa, Minas Gerais, Brazil. We first examined the amount of Zn and its isotopic composition released from the gloves used in this study by soaking them in weak nitric acid and water. We compared Zn isotope ratios obtained from teeth that were sampled wearing nitrile, latex or no gloves. Finally, we performed a linear mixed model (LMM) to investigate post hoc the relationship between the gloves used for sampling and the Zn isotope variability in dental enamel. We found that the gloves used in this study released a similar amount of Zn compared to previous work, but only in acidic solution. Zn isotope ratios of teeth and the LMM identified no sign of significant Zn coming from the gloves when teeth were handled for enamel sampling. We hypothesize that Zn in gloves is mostly released by contact with acids. We found that the main source of Zn isotope variability in the Lapa do Santo population was related to the developmental stage of the tooth tissues sampled. We report identical results for two individuals coming from a different archeological context. Tooth enamel formed in utero and/or during the two first years of life showed higher Zn isotope ratios than enamel formed after weaning. More work is required to systematically investigate if Zn isotopes can be used as a breastfeeding tracer.

A method for complete plant taxon and site inventories in large forest areas with the help of orienteering maps, as exemplified by target forests in Switzerland.

Complete plant inventories of large areas of forests in the moderate and boreal zone have thus far been infeasible and have also not been published. The use of orienteering maps (O-maps) for sampling for inventories was tested. In the sampling method presented herein, the "O-map/way method", O-maps were used for controlled and systematic inspection and sampling, making it possible to carry out successfully complete plant taxon and site inventories of large forest areas (1 to 100 ha). O-maps are much more suitable than the best national or similar topographic maps (NT-maps) for plant inventories in forests; O-maps have many advantages (smaller scale/better resolution, better legibility, internationally standardization, information on vegetation and accessibility), and they contain more small objects, ways (= tracks of any size; roads), and lines and thus have much smaller subareas that allow good orientation and systematic screening for plants. For the example of plant taxon inventories in 6 target areas of 25-85 ha of Swiss midland forests in the moderate/colline zone, the O-map/way method (all accessible areas are screened) was shown to be clearly superior to alternative sampling methods (partial areas screened), such as the NT-map/way method or a plot method, in which only 79.6±6.7% or 34.5±6.6%, respectively, of the taxa found by the O-map/way method were recorded. Taxa detected only by the O-map/way method were shown to be relatively rare at a local as well as at a national scale. The O-map/way method could also be successfully applied to the inventory of plant sites in large forest areas: As shown by the distribution of the sites of five plant species in a target area of 30.1 ha, the great majority of plant sites were detected only by the O-map/way method; but only a few sites were detected by the alternative methods. As O-maps for forests are widely available in many countries, the O-map/way method might allow for complete inventories and other studies in large forest areas.

NMR-Based Strategies to Elucidate Bioactive Conformations of Weakly Binding Ligands.

Key processes in molecular biology are regulated by interactions between biomolecules. Protein-proteinand protein-ligand interactions, e.g., in signal transduction pathways, rely on the subtle interactionsbetween atoms at the binding interface of the involved molecules. Because biomolecules often havemany interacting partners, these interactions are not necessarily strong. The study of molecularrecognition gives insight into the complex network of signaling in life and is the basis of structure-baseddrug design.In the situation where the interaction is weak, one of the traditional methods that can be appliedto obtain structural information (internuclear distances) of the bound ligand is the so-called transferredNOE (trNOE) method. Recently, it became possible to use transferred cross-correlated relaxation (trCCR)to directly measure dihedral angles. The combined use of these two techniques significantly improvesthe precision of the structure determination of ligands weakly bound to macromolecules.The application of these techniques will be discussed in detail for a peptide derived fromIKKß bound to the protein NEMO that plays an important rolein the NFκB pathway.

Covariation of the endocranium and splanchnocranium during great ape ontogeny.

That great ape endocranial shape development persists into adolescence indicates that the splanchnocranium succeeds brain growth in driving endocranial development. However, the extent of this splanchnocranial influence is unknown. We applied two-block partial least squares analyses of Procrustes shape variables on an ontogenetic series of great ape crania to explore the covariation of the endocranium (the internal braincase) and splanchnocranium (face, or viscerocranium). We hypothesized that a transition between brain growth and splanchnocranial development in the establishment of final endocranial form would be manifest as a change in the pattern of shape covariation between early and adolescent ontogeny. Our results revealed a strong pattern of covariation between endocranium and splanchnocranium, indicating that chimpanzees, gorillas, and orangutans share a common tempo and mode of morphological integration from the eruption of the deciduous dentition onwards to adulthood: a reflection of elongating endocranial shape and continuing splanchnocranial prognathism. Within this overarching pattern, we noted that species variation exists in magnitude and direction, and that the covariation between the splanchnocranium and endocranium is somewhat weaker in early infancy compared to successive age groups. When correcting our covariation analyses for allometry, we found that an ontogenetic signal remains, signifying that allometric variation alone is insufficient to account for all endocranial-splanchnocranial developmental integration. Finally, we assessed the influence of the cranial base, which acts as the interface between the face and endocranium, on the shape of the vault using thin-plate spline warping. We found that not all splanchnocranial shape changes during development are tightly integrated with endocranial shape. This suggests that while the developmental expansion of the brain is the main driver of endocranial shape during early ontogeny, endocranial development from infancy onwards is moulded by the splanchnocranium in conjunction with the neurocranium.

Functional characterization and phenotypic monitoring of human hematopoietic stem cell expansion and differentiation of monocytes and macrophages by whole-cell mass spectrometry.

The different facets of macrophages allow them to play distinct roles in tissue homeostasis, tissue repair and in response to infections. Individuals displaying dysregulated macrophage functions are proposed to be prone to inflammatory disorders or infections. However, this being a cause or a consequence of the pathology remains often unclear. In this context, we isolated and expanded CD34+ HSCs from healthy blood donors and derived them into CD14+ myeloid progenitors which were further enriched and differentiated into macrophages. Aiming for a comprehensive phenotypic profiling, we generated whole-cell mass spectrometry (WCMS) fingerprints of cell samples collected along the different stages of the differentiation process to build a predictive model using a linear discriminant analysis based on principal components. Through the capacity of the model to accurately predict sample's identity of a validation set, we demonstrate that WCMS profiles obtained from bona fide blood monocytes and respectively derived macrophages mirror profiles obtained from equivalent HSC derivatives. Finally, HSC-derived macrophage functionalities were assessed by quantifying cytokine and chemokine responses to a TLR agonist in a 34-plex luminex assay and by measuring their capacity to phagocytise mycobacteria. These functional read-outs could not discriminate blood monocytes-derived from HSC-derived macrophages. To conclude, we propose that this method opens new avenues to distinguish the impact of human genetics on the dysregulated biological properties of macrophages in pathological conditions.

Early human dispersals within the Americas.

Studies of the peopling of the Americas have focused on the timing and number of initial migrations. Less attention has been paid to the subsequent spread of people within the Americas. We sequenced 15 ancient human genomes spanning from Alaska to Patagonia; six are ≥10,000 years old (up to ~18× coverage). All are most closely related to Native Americans, including those from an Ancient Beringian individual and two morphologically distinct "Paleoamericans." We found evidence of rapid dispersal and early diversification that included previously unknown groups as people moved south. This resulted in multiple independent, geographically uneven migrations, including one that provides clues of a Late Pleistocene Australasian genetic signal, as well as a later Mesoamerican-related expansion. These led to complex and dynamic population histories from North to South America.

Evolutionary population history of early Paleoamerican cranial morphology.

The nature and timing of the peopling of the Americas is a subject of intense debate. In particular, it is unclear whether high levels of between-group craniometric diversity in South America result from multiple migrations or from local diversification processes. Previous attempts to explain this diversity have largely focused on testing alternative dispersal or gene flow models, reaching conflicting or inconclusive results. Here, a novel analytical framework is applied to three-dimensional geometric morphometric data to partition the effects of population divergence from geographically mediated gene flow to understand the ancestry of the early South Americans in the context of global human history. The results show that Paleoamericans share a last common ancestor with contemporary Native American groups outside, rather than inside, the Americas. Therefore, and in accordance with some recent genomic studies, craniometric data suggest that the New World was populated by multiple waves of dispersion from northeast Asia throughout the late Pleistocene and early Holocene.