The population history of extant and extinct hyenas

We have analyzed partial DNA sequences of the mitochondrialcytochrome b gene from extant striped, brown, and spotted hyenasas well as from Pleistocene cave hyenas. Sequences of the Pleistocenecave hyenas from Eurasia and modern spotted hyenas from Africaare intermixed in phylogenetic analyses, questioning any taxonomicdelineation between the two groups. Contrary to cave hyenasin Eurasia, spotted hyenas in Africa show a phylogeographicpattern with little geographical overlap between two mitochondrialDNA (mtDNA) clades, suggesting two Pleistocene refugia in thenorth and south of Africa. Our results, furthermore, suggestthree waves of migration from Africa to Eurasia for spottedhyenas, around 3, 1, and 0.3 MYA. A recent emigration of stripedhyenas from Africa to Eurasia took place less than 0.1 MYA,resulting in a dramatic expansion of the geographical rangeof striped hyenas. In striped hyenas and within the geographicalrange of mtDNA clades in spotted hyenas, we found identicalsequences several thousand kilometers apart, indicating a highrate of migration during the Pleistocene as well as the Holocene.Both striped and brown hyenas show low amounts of genetic diversity,with the latter ones displaying just a single haplotype.     

In vitro characterization of the presenilin-dependent gamma-secretase complex using a novel affinity ligand

Gamma-secretase is the enzyme activity releasing the amyloid-beta peptide from membrane-bound processing intermediates derived from the beta-amyloid precursor protein. Cellular release and subsequent aggregation of the amyloid-beta peptide is thought to be causative for the pathogenesis of Alzheimer's disease. Gamma-secretase performs an unusual intramembranous cleavage and has been closely linked to a macromolecular complex containing presenilins. To generate a molecular probe for gamma-secretase, we have developed a novel biotinylated affinity ligand which is based on a specific inhibitor containing a hydroxyethylene dipeptide isostere, known to serve as a transition state analogue for aspartic proteinases. Using this probe we confirmed the presence of the presenilin heterodimer and mature nicastrin in the active enzyme complex and, furthermore, that substrate binding site(s) and active center(s) are spatially separated. Affinity precipitations suggest that only a discrete fraction of cellular presenilin is present in the active gamma-secretase complex and that both gamma(40)- and gamma(42)-activities are mediated by the same molecular entity. This was also reflected by a co-distribution of both enzyme activities in subcellular fractions enriched for trans-Golgi network membranes.     

A rapid loss of stripes: the evolutionary history of the extinct quagga

Twenty years ago, the field of ancient DNA was launched with the publication of two short mitochondrial (mt) DNA sequences from a single quagga (Equus quagga) museum skin, an extinct South African equid (Higuchi et al. 1984 Nature312, 282-284). This was the first extinct species from which genetic information was retrieved. The DNA sequences of the quagga showed that it was more closely related to zebras than to horses. However, quagga evolutionary history is far from clear. We have isolated DNA from eight quaggas and a plains zebra (subspecies or phenotype Equus burchelli burchelli). We show that the quagga displayed little genetic diversity and very recently diverged from the plains zebra, probably during the penultimate glacial maximum. This emphasizes the importance of Pleistocene climate changes for phylogeographic patterns in African as well as Holarctic fauna.     

Conserved residues within the putative active site of gamma-secretase differentially influence enzyme activity and inhibitor binding

Gamma-secretase performs the final processing step in the generation of amyloid-beta (Abeta) peptides, which are believed to be causative for Alzheimer's disease. Presenilins (PS) are required for gamma-secretase activity and the presence of two essential intramembranous aspartates (D257 and D385) has implicated this region as the putative catalytic centre of an aspartyl protease. The presence of several key hydrogen-bonding residues around the active site of classical aspartyl proteases led us to investigate the role of both the critical aspartates and two nearby conserved hydrogen bond donors in PS1. Generation of cell lines stably overexpressing the D257E, D385E, Y256F and Y389F engineered mutations has enabled us to determine their role in enzyme catalysis and binding of a transition state analogue gamma-secretase inhibitor. Here we report that replacement of either tyrosine residue alters gamma-secretase cleavage specificity, resulting in an increase in the production of the more pathogenic Abeta42 peptide in both cells and membranous enzyme preparations, without affecting inhibitor binding. In contrast, replacement of either of the aspartate residues precludes inhibitor binding in addition to inactivation of the enzyme. Together, these data further incriminate the region around the intramembranous aspartates as the active site of the enzyme, targeted by transition state analogue inhibitors, and highlight the roles of individual residues.     

DNA damage induced by paclitaxel and DNA repair capability of peripheral blood lymphocytes as evaluated by the alkaline comet assay

This study was designed to assess whether the chemotherapeutic drug paclitaxel can induce DNA damage in peripheral blood lymphocytes of human healthy donors, and to evaluate if such damage could be repaired. Venous blood was collected by routine venipuncture, the lymphocytes were isolated and cultured and then treated with 100nM, 500nM, 10microM, and 30microM of taxol for 4h. The alkaline comet assay technique was used to quantify the level of DNA damage and the DNA repair in lymphocytes. A significant increase in DNA damage was achieved when the cells were incubated with paclitaxel concentrations of 10microM or above. To test the DNA repair capability, the lymphocytes were allowed to recover for 2, 4, 6, and 24h. The DNA damage was almost completely repaired after 24h of incubation demonstrating a time-dependent repair capability. In conclusion, we demonstrate that paclitaxel induces DNA damage in peripheral blood lymphocytes and that this damage can be repaired.     

What,how, and how much do herbivores eat? The Continuous Bite Monitoring method for assessing forage intake of grazing animals

Determining herbage intake is pivotal for studies on grazing ecology. Direct observation of animals allows describing the interactions of animals with the pastoral environment along the complex grazing process. The objectives of the study were to evaluate the reliability of the continuous bite monitoring (CBM) method in determining herbage intake in grazing sheep compared to the standard double‐weighing technique method during 45‐min feeding bouts; evaluate the degree of agreement between the two techniques; and to test the effect of different potential sources of variation on the reliability of the CBM. The CBM method has been used to describe the intake behavior of grazing herbivores. In this study, we evaluated a new approach to this method, that is, whether it is a good proxy for determining the intake of grazing animals. Three experiments with grazing sheep were carried out in which we tested for different sources of variations, such as the number of observers, level of detail of bite coding grid, forage species, forage allowance, sward surface height heterogeneity, experiment site, and animal weight, to determine the short‐term intake rate (45 min). Observer (P_exp1  = 0.018, P_exp2  = 0.078, and P_exp3  = 0.006), sward surface height (P_exp2  < 0.001), total number of bites observed per grazing session (P_exp2  < 0.001 and P_exp3  < 0.001), and sward depletion (P_exp3  < 0.001) were found to affect the absolute error of intake estimation. The results showed a high correlation and agreement between the two methods in the three experiments, although intake was overestimation by CBM on experiments 2 and 3 (181.38 and 214.24 units, respectively). This outcome indicates the potential of CBM to determining forage intake with the benefit of a greater level of detail on foraging patterns and components of the diet. Furthermore, direct observation is not invasive nor disrupts natural animal behavior.     

Ancient Admixture in Human History

Population mixture is an important process in biology. We present a suite of methods for learning about population mixtures, implemented in a software package called ADMIXTOOLS, that support formal tests for whether mixture occurred and make it possible to infer proportions and dates of mixture. We also describe the development of a new single nucleotide polymorphism (SNP) array consisting of 629,433 sites with clearly documented ascertainment that was specifically designed for population genetic analyses and that we genotyped in 934 individuals from 53 diverse populations. To illustrate the methods, we give a number of examples that provide new insights about the history of human admixture. The most striking finding is a clear signal of admixture into northern Europe, with one ancestral population related to present-day Basques and Sardinians and the other related to present-day populations of northeast Asia and the Americas. This likely reflects a history of admixture between Neolithic migrants and the indigenous Mesolithic population of Europe, consistent with recent analyses of ancient bones from Sweden and the sequencing of the genome of the Tyrolean “Iceman.”     

System-wide perturbation analysis with nearly complete coverage of the yeast proteome by single-shot ultra HPLC runs on a bench top Orbitrap

Yeast remains an important model for systems biology and for evaluating proteomics strategies. In-depth shotgun proteomics studies have reached nearly comprehensive coverage, and rapid, targeted approaches have been developed for this organism. Recently, we demonstrated that single LC-MS/MS analysis using long columns and gradients coupled to a linear ion trap Orbitrap instrument had an unexpectedly large dynamic range of protein identification (Thakur, S. S., Geiger, T., Chatterjee, B., Bandilla, P., Frohlich, F., Cox, J., and Mann, M. (2011) Deep and highly sensitive proteome coverage by LC-MS/MS without prefractionation. Mol. Cell Proteomics 10, 10.1074/mcp.M110.003699). Here we couple an ultra high pressure liquid chromatography system to a novel bench top Orbitrap mass spectrometer (Q Exactive) with the goal of nearly complete, rapid, and robust analysis of the yeast proteome. Single runs of filter-aided sample preparation (FASP)-prepared and LysC-digested yeast cell lysates identified an average of 3923 proteins. Combined analysis of six single runs improved these values to more than 4000 identified proteins/run, close to the total number of proteins expressed under standard conditions, with median sequence coverage of 23%. Because of the absence of fractionation steps, only minuscule amounts of sample are required. Thus the yeast model proteome can now largely be covered within a few hours of measurement time and at high sensitivity. Median coverage of proteins in Kyoto Encyclopedia of Genes and Genomes pathways with at least 10 members was 88%, and pathways not covered were not expected to be active under the conditions used. To study perturbations of the yeast proteome, we developed an external, heavy lysine-labeled SILAC yeast standard representing different proteome states. This spike-in standard was employed to measure the heat shock response of the yeast proteome. Bioinformatic analysis of the heat shock response revealed that translation-related functions were down-regulated prominently, including nucleolar processes. Conversely, stress-related pathways were up-regulated. The proteomic technology described here is straightforward, rapid, and robust, potentially enabling widespread use in the yeast and other biological research communities.     

Lymphotoxin β receptor activation on macrophages induces cross-tolerance to TLR4 and TLR9 ligands

Our previous studies indicated that lymphotoxin β receptor (LTβR) activation controls and downregulates inflammatory reactions. In this study, we report that LTβR activation on primary mouse macrophages results in induction of tripartite motif containing (TRIM) 30α, which negatively regulates NF-κB activation induced by TLR signaling. LTβR activation results in a downregulation of proinflammatory cytokine and mediator expression upon TLR restimulation, demonstrating that LTβR signaling is involved in the induction of TLR cross-tolerance. Specific knockdown experiments using TRIM30α-specific small interfering RNA abolished the LTβR-dependent induction of TRIM30α and LTβR-mediated TLR cross-tolerance. Concordantly, LTβR activation on bone marrow-derived macrophages induced cross-tolerance to TLR4 and TLR9 ligands in vitro. Furthermore, we have generated cell type-specific LTβR-deficient mice with ablation of LTβR expression on macrophages/neutrophils (LTβR(flox/flox) × LysM-Cre). In bone marrow-derived macrophages derived from these mice LTβR-induced cross-tolerance to TLR4 and TLR9 ligands was impaired. Additionally, mice with a conditional ablation of LTβR expression on macrophages (LTβR(flox/flox) × LysM-Cre) are resistant to LTβR-induced TLR4 tolerance in vivo. Collectively, our data indicate that LTβR activation on macrophages by T cell-derived lymphotoxin α(1)β(2) controls proinflammatory responses by activation of a TRIM30α-controlled, counterregulatory signaling pathway to protect against exacerbating inflammatory reactions.     

Indirect co-culture of lung carcinoma cells with hyperthermia-treated mesenchymal stem cells influences tumor spheroid growth in a collagen-based 3-dimensional microfluidic model

BackgroundMesenchymal stem cells (MSCs) have paradoxically been reported to exert either pro- or anti-tumor effects in vitro. Hyperthermia, in combination with chemotherapy, has tumor-inhibiting effects; however, its role, together with MSCs, so far is not well understood. Furthermore, a lot of research is conducted using conventional 2-dimensional in vitro models that do not mimic the actual tumor microenvironment.AimIn light of this fact, an indirect method of co-culturing human amniotic membrane-derived MSCs (AMMSCs) with collagen-encapsulated human lung carcinoma cells (A549) was performed using a 3-dimensional (3D) tumor-on-chip device.MethodsThe conditioned medium of AMMSCs (AMMSC-CM) or heat-treated AMMSCs (heat-AMMSC-CM) was utilized to create indirect co-culture conditions. Tumor spheroid growth characterization, immunocytochemistry and cytotoxicity assays, and anti-cancer peptide (P1) screening were performed to determine the effects of the conditioned medium.ResultsThe A549 cells cultured inside the 3D microfluidic chip developed into multicellular tumor spheroids over five days of culture. The AMMSC-CM, contrary to previous reports claiming its tumor-inhibiting potential, led to significant proliferation of tumor spheroids. Heat-AMMSC-CM led to reductions in both spheroid diameter and cell proliferation. The medium containing the P1 peptide was found to be the least cytotoxic to tumor spheroids in co-culture compared with the monoculture and heat-co-culture groups.ConclusionsHyperthermia, in combination with the anticancer peptide, exhibited highest cytotoxic effects. This study highlights the growing importance of 3D microfluidic tumor models for testing stem-cell-based and other anti-cancer therapies.