Improving individual identification of wolves (Canis lupus) using the fundamental frequency and amplitude of their howls: a new survey method

Many bioacoustic studies have been able to identify individual mammals from variations in the fundamental frequency (F0) of their vocalizations. Other characteristics of vocalization which encode individuality, such as amplitude, are less frequently used because of problems with background noise and recording fidelity over distance. In this thesis, I investigate whether the inclusion of amplitude variables improves the accuracy of individual howl identification in captive Eastern grey wolves (Canis lupus lycaon). I also explore whether the use of a bespoke code to extract the howl features, combined with histogram-derived principal component analysis (PCA) values, can improve current individual wolf howl identification accuracies. From a total of 89 solo howls from six captive individuals, where distances between wolf and observer were short, I achieved 95.5% (+9.0% improvement) individual identification accuracy of captive wolves using discriminant function analysis (DFA) to classify simple scalar variables of F0 and normalized amplitudes. Moreover, this accuracy was increased to 100% when using histogram-derived PCA values of F0 and amplitudes of the first harmonic

Benchmarking network propagation methods for disease gene identification

In-silico identification of potential target genes for disease is an essential aspect of drug target discovery. Recent studies suggest that successful targets can be found through by leveraging genetic, genomic and protein interaction information. Here, we systematically tested the ability of 12 varied algorithms, based on network propagation, to identify genes that have been targeted by any drug, on gene-disease data from 22 common non-cancerous diseases in OpenTargets. We considered two biological networks, six performance metrics and compared two types of input gene-disease association scores. The impact of the design factors in performance was quantified through additive explanatory models. Standard cross-validation led to over-optimistic performance estimates due to the presence of protein complexes. In order to obtain realistic estimates, we introduced two novel protein complex-aware cross-validation schemes. When seeding biological networks with known drug targets, machine learning and diffusion-based methods found around 2-4 true targets within the top 20 suggestions. Seeding the networks with genes associated to disease by genetics decreased performance below 1 true hit on average. The use of a larger network, although noisier, improved overall performance. We conclude that diffusion-based prioritisers and machine learning applied to diffusion-based features are suited for drug discovery in practice and improve over simpler neighbour-voting methods. We also demonstrate the large impact of choosing an adequate validation strategy and the definition of seed disease genesPeer ReviewedPostprint (published version

Sedimentary context and palaeoecology of Gigantoproductus shell beds in the Mississippian Eyam Limestone Formation, Derbyshire carbonate platform, central England

A sedimentological study was conducted at two localities exposing the Mississippian Eyam Limestone Formation of the Derbyshire carbonate platform, UK. Ricklow Quarry comprises seven facies with diverse skeletal assemblages, representing deposition on the inner to middle ramp within open marine waters. Once-a-Week Quarry comprises four facies, dominated by crinoidal debris representing deposition on the inner ramp. Both localities expose Gigantoproductus shell beds. Palaeoecological analysis of a single shell bed from each locality enabled investigation of the rapid colonization and success of this taxon on the platform. At Ricklow Quarry, on the eastern side of a localized mud mound, both life (>72% of thin and thick-shelled brachiopods in life position) and neighbourhood assemblages are present. A low-moderate diversity community (<1.37 and <0.8 Shannon diversity index) rapidly established over relict Brigantian mud mounds. Shell beds are preluded by intervals of decreased energy that allowed larvae to settle. Once established, the dominance of thick-shelled individuals enabled baffling, potentially providing localized shelter for larvae and nearby individuals. At Once-a-Week Quarry, where no mud mound is present, only thick-shelled Gigantoproductus species and a low diversity community (<1.07 Shannon diversity index) exclusively comprising neighbourhood assemblages (37% in life position) is present. The presence of inactive mud mounds at Ricklow Quarry appears to have been the key to the success of Gigantoproductus species enabling the onset of stable communities in the shelter provided by the relict mound. Once the first palaeocommunities were established, larvae dispersed and colonized higher energy settings, such as at Once-a-Week Quarry

Directing Differentiation of Pluripotent Stem Cells Toward Retinal Pigment Epithelium Lineage

Development of efficient and reproducible conditions for directed differentiation of pluripotent stem cells into specific cell types is important not only to understand early human development but also to enable more practical applications, such as in vitro disease modeling, drug discovery, and cell therapies. The differentiation of stem cells to retinal pigment epithelium (RPE) in particular holds promise as a source of cells for therapeutic replacement in age-related macular degeneration. Here we show development of an efficient method for deriving homogeneous RPE populations in a period of 45 days using an adherent, monolayer system and defined xeno-free media and matrices. The method utilizes sequential inhibition and activation of the Activin and bone morphogenetic protein signaling pathways and can be applied to both human embryonic stem cells and induced pluripotent stem cells as the starting population. In addition, we use whole genome transcript analysis to characterize cells at different stages of differentiation that provides further understanding of the developmental dynamics and fate specification of RPE. We show that with the described method, RPE develop through stages consistent with their formation during embryonic development. This characterization- together with the absence of steps involving embryoid bodies, three-dimensional culture, or manual dissections, which are common features of other protocols-makes this process very attractive for use in research as well as for clinical applications. SIGNIFICANCE: This report describes a novel method of directed differentiation to generate retinal pigment epithelium (RPE) cells from pluripotent stem cells. The employed method is based on adherent monolayer culture using xeno-free conditions and manipulation of the Activin and bone morphogenetic protein signaling pathway using small molecules and recombinant proteins. Whole genome microarray analysis was performed to characterize the differentiation process and understand the developmental path of RPE generation in vitro. This method can be applied for generation of RPE for research as well as for clinical applications

Acoustic localisation of wildlife with low-cost equipment: Lower sensitivity, but no loss of precision

Abstract Context Synchronised acoustic recorders can be used as a non-invasive tool to detect and localise sounds of interest, including vocal wildlife and anthropogenic sounds. Due to the high cost of commercial synchronised recorders, acoustic localisation has typically been restricted to small or well funded surveys. Recently, low-cost acoustic recorders have been developed, but until now their efficacy has not been compared with higher specification recorders. Aims The present study aimed to compare the efficacy of a newly developed low-cost recorder, the Conservation at Range through Audio Classification and Localisation (CARACAL), with an established, high-end recorder, the Wildlife Acoustics Song Meter (SM). Methods Four recorders of each type were deployed in a paired set-up across five nights in Wisconsin, USA. The recordings allowed for manual identification of domestic dog (Canis familiaris), grey wolf (Canis lupus), coyote (Canis latrans) and barred owl (Strix varia) calls, and then the ability of each recorder type to detect and localise the vocalising animals was compared. Key results The CARACALs were less sensitive, detecting only 47.5% of wolf, 55% of coyote, 65% of barred owl and 82.5% of dog vocalisations detected by the paired SMs. However, when the same vocalisations were detected on both recorders, localisation was comparable, with no significant difference in the precision or maximum detection ranges. Conclusions Low-cost recording equipment can be used effectively for acoustic localisation of both wild and domestic animals. However, the lower sensitivity of the CARACALs means that a denser network of these recorders would be needed to achieve the same efficacy as the SMs. Deploying a greater number of cheaper recorders increases the labour time in the field and the quantity of data to process and store. Thus, there is a trade-off between cost and time to be considered. Implications The ability to use low-cost recorders for acoustic localisation provides new avenues for tracking, managing and researching a wide range of wildlife species. Presently, CARACALs are more suited to monitoring species that have small home ranges and high amplitude vocalisations, and for when a large time investment for in situ equipment checks and data processing is feasible.Christine Stevens Wildlife Award from the Animal Welfare Institut

H3F3A (Histone 3.3) G34W Immunohistochemistry: A Reliable Marker Defining Benign and Malignant Giant Cell Tumor of Bone

Giant cell tumor of bone (GCTB) is a locally aggressive subarticular tumor. Having recently reported that H3.3 G34W mutations are characteristic of this tumor type, we have now investigated the sensitivity and specificity of the anti-histone H3.3 G34W rabbit monoclonal antibody in a wide variety of tumors including histologic mimics of GCTB to assess its value as a diagnostic marker. We also determined the incidence of H3.3 G34 mutations in primary malignant bone tumors as assessed by genotype and H3.3 G34W immunostaining. A total of 3163 tumors were tested. Totally, 213/235 GCTB (90.6%) showed nuclear H3.3 p.G34W immunoreactivity. This was not the case for the rare variants, p.G34L, M, and V, which occurred most commonly in the small bones of the hands, patella, and the axial skeleton. If these sites were excluded from the analysis, H3.3 G34W expression was found in 97.8% of GCTB. Malignant bone tumors initially classified as osteosarcomas were the only other lesions (n=11) that showed G34W expression. Notably an additional 2 previously reported osteosarcomas with a p.G34R mutation were not immunoreactive for the antibody. A total of 11/13 of these malignant H3.3-mutant tumors exhibited an osteoclast-rich component: when imaging was available all but one presented at a subarticular site. We propose that subarticular primary malignant bone sarcoma with H3.3 mutations represent true malignant GCTB, even in the absence of a benign GCTB component

High-pitch sounds small for domestic dogs: abstract crossmodal correspondences between auditory pitch and visual size

Humans possess intuitive associations linking certain non-redundant features of stimuli - e.g. high-pitched sounds with small object size (or similarly, low-pitched sounds with large object size). This phenomenon, known as crossmodal correspondence, has been identified in humans across multiple different senses. There is some evidence that non-human animals also form crossmodal correspondences, but the known examples are mostly limited to the associations between the pitch of vocalizations and the size of callers. To investigate whether domestic dogs, like humans, show abstract pitch-size association, we first trained dogs to approach and touch an object after hearing a sound emanating from it. Subsequently, we repeated the task but presented dogs with two objects differing in size, only one of which was playing a sound. The sound was either high or low pitched, thereby creating trials that were either congruent (high pitch from small object; low pitch from large objects) or incongruent (the reverse). We found that dogs reacted faster on congruent versus incongruent trials. Moreover, their accuracy was at chance on incongruent trials, but significantly above chance for congruent trials. Our results suggest that non-human animals show abstract pitch sound correspondences, indicating these correspondences may not be uniquely human but rather a sensory processing feature shared by other species

Measuring acoustic complexity in continuously varying signals: how complex is a wolf howl?

Communicative complexity is a key behavioural and ecological indicator in the study of animal cognition. Much attention has been given to measures such as repertoire size and syntactic structure in both bird and mammal vocalizations, as large repertoires and complex call combinations may give an indication of the cognitive abilities both of the sender and receiver. However, many animals communicate using a continuous vocal signal that does not easily lend itself to be described by concepts such as ‘repertoire’. For example, dolphin whistles and wolf howls both have complex patterns of frequency modulation, so that no two howls or whistles are quite the same. Is there a sense in which some of these vocalizations may be more ‘complex’ than others? Can we arrive at a quantitative metric for complexity in a continuously varying signal? Such a metric would allow us to extend familiar analyses of communicative complexity to those species where vocal behaviour is not restricted to sequences of stereotyped syllables. We present four measures of complexity in continuous signals (Wiener Entropy, Autocorrelation, Inflection Point Count, and Parsons Entropy), and examine their relevance using example data from members of the genus Canis. We show that each metric can lead to different conclusions regarding which howls could be considered complex or not. Ultimately, complexity is poorly defined and researchers must compare metrics to ensure that they reflect the properties for which the hypothesis is being tested.AK is supported by a Herchel Smith postdoctoral fellowship at the University of Cambridge. Part of this work was carried out while AK was a Postdoctoral Fellow at the National Institute for Mathematical and Biological Synthesis, an Institute sponsored by the National Science Foundation through NSF Award #DBI-1300426, with additional support from The University of Tennessee, Knoxville

Measuring acoustic complexity in continuously varying signals: how complex is a wolf howl?

Communicative complexity is a key behavioural and ecological indicator in the study of animal cognition. Much attention has been given to measures such as repertoire size and syntactic structure in both bird and mammal vocalizations, as large repertoires and complex call combinations may give an indication of the cognitive abilities both of the sender and receiver. However, many animals communicate using a continuous vocal signal that does not easily lend itself to be described by concepts such as ‘repertoire’. For example, dolphin whistles and wolf howls both have complex patterns of frequency modulation, so that no two howls or whistles are quite the same. Is there a sense in which some of these vocalizations may be more ‘complex’ than others? Can we arrive at a quantitative metric for complexity in a continuously varying signal? Such a metric would allow us to extend familiar analyses of communicative complexity to those species where vocal behaviour is not restricted to sequences of stereotyped syllables. We present four measures of complexity in continuous signals (Wiener Entropy, Autocorrelation, Inflection Point Count, and Parsons Entropy), and examine their relevance using example data from members of the genus Canis. We show that each metric can lead to different conclusions regarding which howls could be considered complex or not. Ultimately, complexity is poorly defined and researchers must compare metrics to ensure that they reflect the properties for which the hypothesis is being tested.AK is supported by a Herchel Smith postdoctoral fellowship at the University of Cambridge. Part of this work was carried out while AK was a Postdoctoral Fellow at the National Institute for Mathematical and Biological Synthesis, an Institute sponsored by the National Science Foundation through NSF Award #DBI-1300426, with additional support from The University of Tennessee, Knoxville