The use of Quantitative Agarose Gel Electrophoresis for rapid analysis of the integrity of protein-DNA complexes

Recent biochemical studies evaluated the affinity of histones to DNA in the context of nucleosome core particle (NCP). These have indicated a concentration-dependence for nucleosome stability. However, when studying chromatin the preferred templates are nucleosome arrays (NA) and not the NCP. Biochemical methods are poorly suited for structural analysis of chromatin. To overcome that technical hindrance, and investigate the effect of concentration on stability of the histone-DNA interactions, we have applied the multigel Quantitative Agarose Gel Electrophoresis (QAGE) method to in vitro-assembled nucleosomal arrays. The results demonstrated the method to be extremely valuable for the evaluation of the effect of low concentration on NA. However, QAGE is a fairly time-demanding and complex method. To maximize the efficiency of use of this technology, we devised a protocol that allowed for multiple sets of templates to be analyzed simultaneously. Briefly, samples can be loaded at regular intervals and analyzed individually for their molecular composition. The technique presented in this study describes the calibration steps and proof of concept necessary to validate the use of multiple loading of multigel to evaluate the composition of nucleosomal arrays as a function of concentration.     

A primary assembly of a bovine haplotype block map based on a 15,036-single-nucleotide polymorphism panel genotyped in holstein-friesian cattle

Analysis of data on 1000 Holstein-Friesian bulls genotyped for 15,036 single-nucleotide polymorphisms (SNPs) has enabled genomewide identification of haplotype blocks and tag SNPs. A final subset of 9195 SNPs in Hardy-Weinberg equilibrium and mapped on autosomes on the bovine sequence assembly (release Btau 3.1) was used in this study. The average intermarker spacing was 251.8 kb. The average minor allele frequency (MAF) was 0.29 (0.05-0.5). Following recent precedents in human HapMap studies, a haplotype block was defined where 95% of combinations of SNPs within a region are in very high linkage disequilibrium. A total of 727 haplotype blocks consisting of > or =3 SNPs were identified. The average block length was 69.7 +/- 7.7 kb, which is approximately 5-10 times larger than in humans. These blocks comprised a total of 2964 SNPs and covered 50,638 kb of the sequence map, which constitutes 2.18% of the length of all autosomes. A set of tag SNPs, which will be useful for further fine-mapping studies, has been identified. Overall, the results suggest that as many as 75,000-100,000 tag SNPs would be needed to track all important haplotype blocks in the bovine genome. This would require approximately 250,000 SNPs in the discovery phase.     

Biocidal activity of three wood essential oils against Ixodes scapularis (Acari: Ixodidae), Xenopsylla cheopis (Siphonaptera: Pulicidae), and Aedes aegypti (Diptera: Culicidae)

The biocidal activity of three steam distilled wood essential oils-incense cedar, Calocedrus decurrens (Torr.) Florin; Port-Orford-cedar, Chamaecyparis lawsoniana (A. Murr.) Parl.; and western juniper, Juniperus occidentalis (Hook)--were evaluated against adult Aedes aegypti (L.) (Diptera: Culicidae) and Xenopsylla cheopis (Rothchild) (Siphonaptera: Pulicidae) and nymphal Ixodes scapularis Say (Acari: Ixodidae). In vitro laboratory bioassays were conducted to establish baseline dose-mortality data through 24 h. Incense cedar heartwood was the most toxic to all three vector species followed in order of activity by western juniper and Port-Orford-cedar based on LC50 and LC90 values. Ae. aegypti were substantially more susceptible to the oils than either I. scapularis or X. cheopis.     

Protein kinase D interaction with TLR5 is required for inflammatory signaling in response to bacterial flagellin

Protein kinase D (PKD), also called protein kinase C (PKC)mu, is a serine-threonine kinase that is involved in diverse areas of cellular function such as lymphocyte signaling, oxidative stress, and protein secretion. After identifying a putative PKD phosphorylation site in the Toll/IL-1R domain of TLR5, we explored the role of this kinase in the interaction between human TLR5 and enteroaggregative Escherichia coli flagellin in human epithelial cell lines. We report several lines of evidence that implicate PKD in TLR5 signaling. First, PKD phosphorylated the TLR5-derived target peptide in vitro, and phosphorylation of the putative target serine 805 in HEK 293T cell-derived TLR5 was identified by mass spectrometry. Furthermore, mutation of serine 805 to alanine abrogated responses of transfected HEK 293T cells to flagellin. Second, TLR5 interacted with PKD in coimmunoprecipitation experiments, and this association was rapidly enhanced by flagellin treatment. Third, pharmacologic inhibition of PKC or PKD with G?6976 resulted in reduced expression and secretion of IL-8 and prevented the flagellin-induced activation of p38 MAPK, but treatment with the PKC inhibitor G?6983 had no significant effects on these phenotypes. Finally, involvement of PKD in the p38-mediated IL-8 response to flagellin was confirmed by small hairpin RNA-mediated gene silencing. Together, these results suggest that phosphorylation of TLR5 by PKD may be one of the proximal elements in the cellular response to flagellin, and that this event contributes to p38 MAPK activation and production of inflammatory cytokines in epithelial cells.     

Germline variants in oculocutaneous albinism genes and predisposition to familial cutaneous melanoma

Approximately 1%–2% of cutaneous melanoma (CM) is classified as strongly familial. We sought to investigate unexplained CM predisposition in families negative for the known susceptibility genes using next‐generation sequencing of affected individuals. Segregation of germline variants of interest within families was assessed by Sanger sequencing. Several heterozygous variants in oculocutaneous albinism (OCA) genes: TYR, OCA2, TYRP1 and SLC45A2, were present in our CM cohort. OCA is a group of autosomal recessive genetic disorders, resulting in pigmentation defects of the eyes, hair and skin. Missense variants classified as pathogenic for OCA were present in multiple families and some fully segregated with CM. The functionally compromised TYR p.T373K variant was present in three unrelated families. In OCA2, known pathogenic variants: p.V443I and p.N489D, were present in three families and one family, respectively. We identified a likely pathogenic SLC45A2 frameshift variant that fully segregated with CM in a family of four cases. Another four‐case family harboured cosegregating variants (p.A24T and p.R153C) of uncertain functional significance in TYRP1. We conclude that rare, heterozygous variants in OCA genes confer moderate risk for CM.     

Interrelatedness of 5S RNA sequences investigated by correspondence analysis

Summary Correspondence analysis (a form of multivariate statistics) applied to 74 5S ribosomal RNA sequences indicates that the sequences are interrelated in a systematic, nonrandom fashion. Aligned sequences are represented as vectors in a 5N-dimensional space, where N is the number of base positions in the 5S RNA molecule. Mutually orthogonal directions (called factor axes) along which intersequence variance is greatest are defined in this hyperspace. Projection of the sequences onto planes defined by these factorial directions reveals clustering of species that is suggestive of phylogenetic relationships. For each factorial direction, correspondence analysis points to regions of importance, i.e., those base positions at which the systematic changes occur that define that particular direction. In effect, the technique provides a rapid determination of group-specific signatures. In several instances, similarities between sequences are indicated that have only recently been inferred from visual base-to-base comparisons. These results suggest that correspondence analysis may provide a valuable starting point from which to uncover the patterns of change underlying the evolution of a macromolecule, such as 5S RNA.     

Cloning, chromosomal organization and expression analysis of Neurl, the mouse homolog of Drosophila melanogaster neuralized gene

The Drosophila neuralized (neur) gene belongs to the neurogenic group of genes involved in regulating cell-cell interactions required for neural precursor development. neur mutant phenotypes include strong overcommitment to neural fates at the expense of epidermal fates. The human neuralized homolog (NEURL) has been recently determined and found to map to chromosome 10q25.1 within the region frequently deleted in malignant astrocytomas. Because of its potential importance in developmental processes, we analyzed the structure of the mouse homolog, Neurl, and its expression pattern in embryonic tissues. Neurl activity is detected from early developmental stages in several tissues and organs including neural tissues, limbs, the skeletal system, sense organs and internal organs undergoing epithelial-mesenchymal interactions. Neurl encodes a polypeptide associated with the plasma membrane but also detected in the cytoplasm. Similarly to the Drosophila gene, mammalian neuralized may code for an important regulatory factor.     

Conflict and post-conflict behavior in a small group of chimpanzees

Chimpanzee research plays a central role in the discussions of conflict negotiation. Reconciliation, or the attraction and affiliation of former opponents following conflict, has been proposed as a central element of conflict negotiation in chimpanzees and various other taxa. In an attempt to expand the database of chimpanzee conflict resolution, conflict and post-conflict behavior were recorded for a small group of socially housed chimpanzees at the Chimpanzee and Human Communication Institute, at Central Washington University. Data were collected over six 6-week periods between 1997 and 2000, for a total of 840 hours of observation, resulting in a substantial post-conflict (PC) and matched control (MC) data set. The data demonstrate this group’s tendencies to maintain visual contact and closer proximity after conflicts. Dyadic corrected conciliatory tendencies ranged between 0 – 37.5% and averaged 17.25% across all dyads. Individual corrected conciliatory tendencies ranged between 5.8 and 32%. The results of this study combined with recent publications on captive and free-ranging chimpanzee post-conflict behavior suggest that variation in post-conflict behavior may be important to our understanding of chimpanzee conflict negotiation, and may also have implications for the design and management of captive chimpanzee enclosures and social groups, respectively.     

‘Straightening What’s Crooked’? Recognition as Moral Disruption in Indonesia’s Confucianist Revival

ABSTRACT

In 2006, the Indonesian state re-recognised Confucianism as an official religion, but this did not have the straightforwardly positive consequences that either Confucianist revivalists or some theorists of recognition might have predicted. Revivalists were often – but not always – gripped by feelings of outrage and moral torment, whilst the pace of the revival itself was very uneven. These varied outcomes reflect the complex politics pervading the lives of Indonesian Confucianists (and Chinese Indonesians more generally) as post-Suharto reforms force them to grapple with their diverse histories of accommodation and resistance to the New Order’s discriminatory policies. To fully understand such material, first-person moral perspectives must be incorporated into critical anthropological studies of recognition, as a complement to approaches focused on power and domination. Doing so reveals an important general truth about recognition – its capacity to be morally disruptive – and broadens our understanding of why recognition can hurt those it ostensibly stands to benefit.