Dogs Evaluate Threatening Facial Expressions by Their Biological Validity – Evidence from Gazing Patterns

Appropriate response to companions’ emotional signals is important for all social creatures. The emotional expressions of humans and non-human animals have analogies in their form and function, suggesting shared evolutionary roots, but very little is known about how animals other than primates view and process facial expressions. In primates, threat-related facial expressions evoke exceptional viewing patterns compared with neutral or positive stimuli. Here, we explore if domestic dogs (Canis familiaris) have such an attentional bias toward threatening social stimuli and whether observed emotional expressions affect dogs’ gaze fixation distribution among the facial features (eyes, midface and mouth). We recorded the voluntary eye gaze of 31 domestic dogs during viewing of facial photographs of humans and dogs with three emotional expressions (threatening, pleasant and neutral). We found that dogs’ gaze fixations spread systematically among facial features. The distribution of fixations was altered by the seen expression, but eyes were the most probable targets of the first fixations and gathered longer looking durations than mouth regardless of the viewed expression. The examination of the inner facial features as a whole revealed more pronounced scanning differences among expressions. This suggests that dogs do not base their perception of facial expressions on the viewing of single structures, but the interpretation of the composition formed by eyes, midface and mouth. Dogs evaluated social threat rapidly and this evaluation led to attentional bias, which was dependent on the depicted species: threatening conspecifics’ faces evoked heightened attention but threatening human faces instead an avoidance response. We propose that threatening signals carrying differential biological validity are processed via distinctive neurocognitive pathways. Both of these mechanisms may have an adaptive significance for domestic dogs. The findings provide a novel perspective on understanding the processing of emotional expressions and sensitivity to social threat in non-primates.     

Rapid and accurate processing method for amide proton exchange rate measurement in proteins

Exchange between protein backbone amide hydrogen and water gives relevant information about solvent accessibility and protein secondary structure stability. NMR spectroscopy provides a convenient tool to study these dynamic processes with saturation transfer experiments. Processing of this type of NMR spectra has traditionally required peak integration followed by exponential fitting, which can be tedious with large data sets. We propose here a computer-aided method that applies inverse Laplace transform in the exchange rate measurement. With this approach, the determination of exchange rates can be automated, and reliable results can be acquired rapidly without a need for manual processing.     

Atomic Structures of Two Novel Immunoglobulin-like Domain Pairs in the Actin Cross-linking Protein Filamin

Filamins are actin filament cross-linking proteins composed of an N-terminal actin-binding domain and 24 immunoglobulin-like domains (IgFLNs). Filamins interact with numerous proteins, including the cytoplasmic domains of plasma membrane signaling and cell adhesion receptors. Thereby filamins mechanically and functionally link the cell membrane to the cytoskeleton. Most of the interactions have been mapped to the C-terminal IgFLNs 16–24. Similarly, as with the previously known compact domain pair of IgFLNa20–21, the two-domain fragments IgFLNa16–17 and IgFLNa18–19 were more compact in small angle x-ray scattering analysis than would be expected for two independent domains. Solution state NMR structures revealed that the domain packing in IgFLNa18–19 resembles the structure of IgFLNa20–21. In both domain pairs the integrin-binding site is masked, although the details of the domain-domain interaction are partly distinct. The structure of IgFLNa16–17 revealed a new domain packing mode where the adhesion receptor binding site of domain 17 is not masked. Sequence comparison suggests that similar packing of three tandem filamin domain pairs is present throughout the animal kingdom, and we propose that this packing is involved in the regulation of filamin interactions through a mechanosensor mechanism.Actin cytoskeleton is a dynamic network that is involved in many fundamental cellular processes such as cell differentiation, morphology, endocytosis, exocytosis, cytokinesis, and cell movement. These events are regulated by proteins that interact with monomeric and filamentous actin. Filamins are actin filament-binding and cross-linking proteins. Filamin A and filamin B are both ubiquitously expressed, and their mutations in human patients cause developmental abnormalities in brain, cartilage, bones, and epithelial tissues (1). Filamin C is muscle-specific, and mutations thereof cause myofibrillar myopathy (2). Mice with targeted deletion of any of the filamin genes die either during development or soon after birth (3–6). These phenotypes are thought to reflect the roles of filamins as scaffolds of signaling pathways required for cell differentiation, regulators of cell migration, and stabilizers of cytoskeleton and cell membranes (1, 7).Filamins bind to actin filaments mainly via their N-terminal actin-binding domains and interact with other proteins via the 24 filamin type immunoglobulin-like domains (IgFLN),³ also called filamin repeats (8). Especially the C-terminal IgFLNs 16–24 contain several protein-protein interaction sites (1). Our previous structural studies have revealed that many proteins interact with filamins by forming an additional β-strand next to strand C of an individual IgFLN. The platelet von Willebrand factor receptor, glycoprotein (GP) Ibα, interacts in this way with IgFLNa17 (9). The integrin family adhesion receptor β subunits interact with IgFLNa21 and to a lesser extent with IgFLNa19 (10, 11). Furthermore, some signaling proteins use a similar interaction mode: the adaptor protein migfilin interacts with IgFLNa21 (12), and the Rho family GTPase-activating protein FilGAP interacts with IgFLNa23 (13, 14).Although structural details are known from many filamin interactions, it is not completely clear how these interactions are regulated. In some cases the regulation involves competition between multiple binding partners (10, 11). Alternative splicing (15), proteolysis of filamin (16–18), and ligand phosphorylation (11) also contribute to the regulation. Recently, it has become apparent that conformational changes in filamins may also be involved. For instance, actomyosin contraction exposes hidden cysteine residues in filamins (19). This opens the possibility that forces transmitted through actin filament may open up binding sites, and filamin may thus be involved in mechanosensor signaling.We have recently found a structural mechanism by which mechanical forces could regulate interactions at the C-terminal part of filamin. Our recent crystal structure revealed that IgFLNa20 forms a compact pair with IgFLNa21, and in this pair the N-terminal part of IgFLNa20 masks the integrin-binding site on IgFLNa21 (15). It is possible that this masking could be released by mechanical forces. Four lines of evidence led us to hypothesize that in addition to the IgFLNa20–21 pair, other similar domain pairs could exist at the C terminus of filamin: (i) the overall structure of the C-terminal part (IgFLNs 16–24) of filamin is relatively more compact than the N-terminal part of the molecule (IgFLNs 1–15) (8); (ii) the N-terminal sequences of even-numbered domains 16, 18, and 20 differ from other IgFLNs (20) (sequence alignment is shown in supplemental Fig. S1); (iii) in single-domain solution NMR structures of IgFLNc16, IgFLNb16, 18, and 20, the N-terminal part is not folded with the rest of the domain; and (iv) according to biochemical experiments, IgFLNa18 masks integrin binding to IgFLNa19 (15). We report here small angle x-ray scattering (SAXS) analysis showing that IgFLNa16–17 and 18–19 have overall dimensions very similar to those of the previously known domain pair IgFLNa20–21. The IgFLNa22–23 construct was much more elongated, which is indicative for two independently folded noninteracting domains. Further, the atomic structures solved with NMR spectroscopy show that IgFLNa18–19 forms a pair similar to IgFLNa20–21, but the details of the interaction and orientation of the domains differ. On the other hand, IgFLNa16–17 forms an entirely novel type of domain pair. Sequence comparisons predict that these three interdependent domain pairs are conserved from nematodes to vertebrates, suggesting that the arrangement has special regulatory functions.     

CCHCR1 Is Up-Regulated in Skin Cancer and Associated with EGFR Expression

Despite chronic inflammation, psoriatic lesions hardly ever progress to skin cancer. Aberrant function of the CCHCR1 gene (Coiled-Coil α-Helical Rod protein 1, HCR) within the PSORS1 locus may contribute to the onset of psoriasis. As CCHCR1 is expressed in certain cancers and regulates keratinocyte (KC) proliferation in a transgenic mouse model, we studied its relation to proliferation in cutaneous squamous cell cancer (SCC) cell lines by expression arrays and quantitative RT-PCR and in skin tumors by immunohistochemistry. CCHCR1 protein was detected in the pushing border of SCC and lining basal cell carcinoma islands. Different from psoriasis, Ki67 had a similar expression pattern as CCHCR1. The most intense CCHCR1 staining occurred in areas positive for epidermal growth factor receptor (EGFR). Expression of CCHCR1 mRNA was upregulated 30–80% in SCC lines when compared to normal KCs and correlated positively with Ki67 expression. The most aggressive and invasive tumor cell lines (RT3, FaDu) expressed CCHCR1 mRNA less than non-tumorigenic HaCaT cells. Moreover, the tumor promoters okadaic acid and menadione downregulated CCHCR1 mRNA. We conclude that both in psoriasis and the early stages of KC transformation, CCHCR1 may function as a negative regulator of proliferation, but beyond a certain point in oncogenesis cannot control this phenomenon any longer.     

Search for nucleotide diversity patterns of local adaptation in dehydrins and other cold-related candidate genes in Scots pine (Pinus sylvestris L.)

Nucleotide variation at several cold candidate genes including seven members of the dehydrin gene family was surveyed in haplotypes of Scots pine (Pinus sylvestris) sampled in populations showing divergence for cold tolerance in Europe. Patterns of nucleotide diversity, linkage disequilibrium, and frequency spectrum of alleles were compared between north and south populations to search for signs of directional selection potentially underlying adaptation to cold. Significant differentiation between populations in allelic frequency or haplotype structure was detected at dhn1, dhn3, and abaH loci. Allelic dimorphism with no evidence of haplotype clustering by geographical distribution was found at dhn9. An excess of fixed non-synonymous mutations as compared to the outgroup P. pinaster pine species was found at dhn1. Differences in nucleotide polymorphisms were found between the members of the Kn class of dehydrin upregulated during cold acclimation (average π_sil = 0.004) as compared to the SKn class (average π_sil = 0.024). The multilocus nucleotide diversity at silent sites (θ _W = 0.009) was moderate compared to other conifer species, but higher than previous estimates for Scots pine. There was an excess of rare and high frequency derived variants as revealed by significantly negative multilocus value of Tajima’s D (D = −0.72, P < 0.01) and negative mean value of Fay and Wu H statistics (H = −0.50). The level of linkage disequilibrium decayed rapidly with an average expected r ² of 0.2 at about 200 bp. Overall, there was a positive correlation between polymorphism and divergence at ten loci when outgroup sequence was available. The discovered polymorphism will be used for further evaluation of the adaptive role of genes through association mapping studies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Metal-mediated allylation of enzymatically oxidized methyl α-D-galactopyranoside

The C-6 unit of methyl α-d-galactopyranoside was selectively modified by combining enzymatic oxidation with an indium-mediated allylation reaction. The Barbier-Grignard type reaction, where a carbonyl group reacts with an allyl halide, proceeds in aqueous solution, even with water as the only solvent; thus carbohydrates can be modified without the need for drying or protection-deprotection steps. The corresponding homoallyl alcohols are produced in high yields of >90% in the reactions with allyl bromide and cinnamyl chloride. The main products were isolated and characterized by GC-MS and NMR spectroscopy.     

MQ-HNCO-TROSY for the measurement of scalar and residual dipolar couplings in larger proteins: application to a 557-residue IgFLNa16-21

We describe a novel pulse sequence, MQ-HNCO-TROSY, for the measurement of scalar and residual dipolar couplings between amide proton and nitrogen in larger proteins. The experiment utilizes the whole 2T_N polarization transfer delay for labeling of ¹⁵N chemical shift in a constant time manner, which efficiently doubles the attainable resolution in ¹⁵N dimension with respect to the conventional HNCO-TROSY experiment. In addition, the accordion principle is employed for measuring (J + D)_NHs, and the multiplet components are selected with the generalized version of the TROSY scheme introduced by Nietlispach (J Biomol NMR 31:161–166, 2005). Therefore, cross peak overlap is diminished while the time period during which the ¹⁵N spin is susceptible to fast transverse relaxation associated with the anti-TROSY transition is minimized per attainable resolution unit. The proposed MQ-HNCO-TROSY scheme was employed for measuring RDCs in high molecular weight protein IgFLNa16-21 of 557 residues, resulting in 431 experimental RDCs. Correlations between experimental and back-calculated RDCs in individual domains gave relatively low Q-factors (0.19–0.39), indicative of sufficient accuracy that can be obtained with the proposed MQ-HNCO-TROSY experiment in high molecular weight proteins.     

Genetic diversity of the 2009 pandemic influenza A(H1N1) viruses in Finland

Background

In Finland, the first infections caused by the 2009 pandemic influenza A(H1N1) virus were identified on May 10. During the next three months almost all infections were found from patients who had recently traveled abroad. In September 2009 the pandemic virus started to spread in the general population, leading to localized outbreaks and peak epidemic activity was reached during weeks 43–48.

Methods/Results

The nucleotide sequences of the hemagglutinin (HA) and neuraminidase (NA) genes from viruses collected from 138 patients were determined. The analyzed viruses represented mild and severe infections and different geographic regions and time periods. Based on HA and NA gene sequences, the Finnish pandemic viruses clustered in four groups. Finnish epidemic viruses and A/California/07/2009 vaccine virus strain varied from 2–8 and 0–5 amino acids in HA and NA molecules, respectively, giving a respective maximal evolution speed of 1.4% and 1.1%. Most amino acid changes in HA and NA molecules accumulated on the surface of the molecule and were partly located in antigenic sites. Three severe infections were detected with a mutation at HA residue 222, in two viruses with a change D222G, and in one virus D222Y. Also viruses with change D222E were identified. All Finnish pandemic viruses were sensitive to oseltamivir having the amino acid histidine at residue 275 of the neuraminidase molecule.

Conclusions

The Finnish pandemic viruses were quite closely related to A/California/07/2009 vaccine virus. Neither in the HA nor in the NA were changes identified that may lead to the selection of a virus with increased epidemic potential or exceptionally high virulence. Continued laboratory-based surveillance of the 2009 pandemic influenza A(H1N1) is important in order to rapidly identify drug resistant viruses and/or virus variants with potential ability to cause severe forms of infection and an ability to circumvent vaccine-induced immunity.     

Β-amyloid 1-42 oligomers impair function of human embryonic stem cell-derived forebrain cholinergic neurons

Cognitive impairment in Alzheimer's disease (AD) patients is associated with a decline in the levels of growth factors, impairment of axonal transport and marked degeneration of basal forebrain cholinergic neurons (BFCNs). Neurogenesis persists in the adult human brain, and the stimulation of regenerative processes in the CNS is an attractive prospect for neuroreplacement therapy in neurodegenerative diseases such as AD. Currently, it is still not clear how the pathophysiological environment in the AD brain affects stem cell biology. Previous studies investigating the effects of the β-amyloid (Aβ) peptide on neurogenesis have been inconclusive, since both neurogenic and neurotoxic effects on progenitor cell populations have been reported. In this study, we treated pluripotent human embryonic stem (hES) cells with nerve growth factor (NGF) as well as with fibrillar and oligomeric Aβ1-40 and Aβ1-42 (nM-μM concentrations) and thereafter studied the differentiation in vitro during 28-35 days. The process applied real time quantitative PCR, immunocytochemistry as well as functional studies of intracellular calcium signaling. Treatment with NGF promoted the differentiation into functionally mature BFCNs. In comparison to untreated cells, oligomeric Aβ1-40 increased the number of functional neurons, whereas oligomeric Aβ1-42 suppressed the number of functional neurons. Interestingly, oligomeric Aβ exposure did not influence the number of hES cell-derived neurons compared with untreated cells, while in contrast fibrillar Aβ1-40 and Aβ1-42 induced gliogenesis. These findings indicate that Aβ1-42 oligomers may impair the function of stem cell-derived neurons. We propose that it may be possible for future AD therapies to promote the maturation of functional stem cell-derived neurons by altering the brain microenvironment with trophic support and by targeting different aggregation forms of Aβ.