Fas ligand expression and tumour immune evasion; the role of prostaglandin E2 and the EP1 receptor

Background and Aim: During carcinogenesis, tumours develop multiple mechanisms to evade the immune system and suppress the anti-tumour immune response. Upregulation of Fas Ligand (FasL/CD95L) expression may represent one such mechanism. FasL is a member of the tumour necrosis factor superfamily that triggers apoptotic cell death following ligation to its receptor Fas. Numerous studies have demonstrated upregulated FasL expression in tumor cells, with FasL expression associated with numerous pro-tumorigenic effects. However, little is known about the mechanisms that regulate FasL expression in tumours. The cyclooxgenase (COX) signalling pathway may play an important role in colon carcinogenesis, via the production of prostaglandins, in particular PGE2. PGE2 signals through four different receptor subtypes, EP1 – EP4. Thus, the aim of this study was to investigate the effect of targeting the PGE2-FasL signaling pathway. Results: (i) PGE2 induces FasL expression via the EP1 receptor in colon cancer cells. (ii) Suppression of FasL expression in colon tumour cells in vivo significantly delays and reduces tumour growth. (iii) Blocking EP1 receptor signaling, or suppression of the EP1 receptor in colon tumour cells, reduces tumour growth in vivo. Suppression of tumour growth correlates in part with suppression of FasL expression. (iv) The reduction in tumour growth is associated with an improved anti-tumour immune response. Tumour infiltration by Treg cells and macrophages was reduced, and the cytotoxic activity of CTL generated from splenocytes isolated from these mice increased. Conclusion: 1) Targeting FasL expression by blocking PGE2-EP1 receptor signalling reduces tumour development in vivo. 2) The mechanism is indirect but is associated with an increased anti-tumour immune response. Thus, unraveling the mechanisms regulating FasL expression and the pro-tumorigenic effects of the EP1 receptor may aid in the search for new therapeutic targets against colon cancer.

The study of molecular variation in Atlantic salmon (Salmo salar L.) and brown trout (Salmo trutta L.)

Polymorphic microsatellite DNA loci were used here in three studies, one on Salmo salar and two on S. trutta. In the case of S. salar, the survival of native fish and non-natives from a nearby catchment, and their hybrids, were compared in a freshwater common garden experiment and subsequently in ocean ranching, with parental assignment utilising microsatellites. Overall survival of non-natives was 35% of natives. This differential survival was mainly in the oceanic phase. These results imply a genetic basis and suggest local adaptation can occur in salmonids across relatively small geographic distances which may have important implications for the management of salmon populations. In the first case study with S trutta, the species was investigated throughout its spread as an invasive in Newfoundland, eastern Canada. Genetic investigation confirmed historical records that the majority of introductions were from a Scottish hatchery and provided a clear example of the structure of two expanding waves of spread along coasts, probably by natural straying of anadromous individuals, to the north and south of the point of human introduction. This study showed a clearer example of the genetic anatomy of an invasion than in previous studies with brown trout, and may have implications for the management of invasive species in general. Finally, the genetics of anadromous S. trutta from the Waterville catchment in south western Ireland were studied. Two significantly different population groupings, from tributaries in geographically distinct locations entering the largest lake in the catchment, were identified. These results were then used to assign very large rod caught sea trout individuals (so called “specimen” sea trout) back to region of origin, in a Genetic Stock Identification exercise. This suggested that the majority of these large sea trout originated from one of the two tributary groups. These results are relevant for the understanding of sea trout population dynamics and for the future management of this and other sea trout producing catchments. This thesis has demonstrated new insights into the population structuring of salmonids both between and within catchments. While these chapters look at the existence and scale of genetic variation from different angles, it might be concluded that the overarching message from this thesis should be to highlight the importance of maintaining genetic diversity in salmonid populations as vital for their long-term productivity and resilience.

A craniometric study of population dynamics and social organisation in the European upper Palaeolithic and Mesolithic

The purpose of this study is to explore aspects of social organisation during the Upper Palaeolithic and Mesolithic periods using craniometric data. Different hypotheses were tested using geometric morphometrics, alongside traditional craniometric data. The clustering of individuals from the same site, as well as a correspondence to an isolation-by-distance model—particular in the Mesolithic samples—points to population structure within these groups. Moreover, discontinuities in cranial traits between the early Upper Palaeolithic and later periods could suggest that the Last Glacial Maximum had a disruptive effect on populations in Europe. Differences in social organisation can often result from cultural norms regarding post-marital residence. Such differences can be tested by comparing cranial data to that of geographic information. Greater variation in male cranial traits relative to females, after controlling for location, suggests that the overall pattern of residence during the Upper Palaeolithic and Mesolithic was one of matrilocality. It has been suggested that coastal occupation was density dependent and these populations show a greater degree of sedentism than their inland counterparts. Moreover, it has been proposed that coastal areas were not continuously occupied until the Late Pleistocene due to spatial restrictions that would adversely affect reproductive opportunities. This study corroborates the pattern seen in cranial traits corresponded with that of a more sedentary population. The results are consistent with the hypothesis that coastal populations are more sedentary than inland populations during these periods. This study adds new information regarding the social dynamics of prehistoric populations in Europe and sheds light on some of the conditions that may have paved the way for the transition to agriculture

Variability in frontotemporal brain structure: the importance of recruitment of African Americans in neuroscience research.

BACKGROUND: Variation in brain structure is both genetically and environmentally influenced. The question about potential differences in brain anatomy across populations of differing race and ethnicity remains a controversial issue. There are few studies specifically examining racial or ethnic differences and also few studies that test for race-related differences in context of other neuropsychiatric research, possibly due to the underrepresentation of ethnic minorities in clinical research. It is within this context that we conducted a secondary data analysis examining volumetric MRI data from healthy participants and compared the volumes of the amygdala, hippocampus, lateral ventricles, caudate nucleus, orbitofrontal cortex (OFC) and total cerebral volume between Caucasian and African-American participants. We discuss the importance of this finding in context of neuroimaging methodology, but also the need for improved recruitment of African Americans in clinical research and its broader implications for a better understanding of the neural basis of neuropsychiatric disorders. METHODOLOGY/PRINCIPAL FINDINGS: This was a case control study in the setting of an academic medical center outpatient service. Participants consisted of 44 Caucasians and 33 ethnic minorities. The following volumetric data were obtained: amygdala, hippocampus, lateral ventricles, caudate nucleus, orbitofrontal cortex (OFC) and total cerebrum. Each participant completed a 1.5 T magnetic resonance imaging (MRI). Our primary finding in analyses of brain subregions was that when compared to Caucasians, African Americans exhibited larger left OFC volumes (F (1,68) = 7.50, p = 0.008). CONCLUSIONS: The biological implications of our findings are unclear as we do not know what factors may be contributing to these observed differences. However, this study raises several questions that have important implications for the future of neuropsychiatric research.

Relative growth of the limbs and trunk in sifakas: heterochronic, ecological, and functional considerations.

Limb, trunk, and body weight measurements were obtained for growth series of Milne-Edwards's diademed sifaka, Propithecus diadema edwardsi, and the golden-crowned sifaka, Propithecus tattersalli. Similar measures were obtained also for primarily adults of two subspecies of the western sifaka: Propithecus verreauxi coquereli, Coquerel's sifaka, and Propithecus verreauxi verreauxi, Verreaux's sifaka. Ontogenetic series for the larger-bodied P. d. edwardsi and the smaller-bodied P. tattersalli were compared to evaluate whether species-level differences in body proportions result from the differential extension of common patterns of relative growth. In bivariate plots, both subspecies of P. verreauxi were included to examine whether these taxa also lie along a growth trajectory common to all sifakas. Analyses of the data indicate that postcranial proportions for sifakas are ontogenetically scaled, much as demonstrated previously with cranial dimensions for all three species (Ravosa, 1992). As such, P. d. edwardsi apparently develops larger overall size primarily by growing at a faster rate, but not for a longer duration of time, than P. tattersalli and P. verreauxi; this is similar to results based on cranial data. A consideration of Malagasy lemur ecology suggests that regional differences in forage quality and resource availability have strongly influenced the evolutionary development of body-size variation in sifakas. On one hand, the rainforest environment of P. d. edwardsi imposes greater selective pressures for larger body size than the dry-forest environment of P. tattersalli and P. v. coquereli, or the semi-arid climate of P. v. verreauxi. On the other hand, as progressively smaller-bodied adult sifakas are located in the east, west, and northwest, this apparently supports suggestions that adult body size is set by dry-season constraints on food quality and distribution (i.e., smaller taxa are located in more seasonal habitats such as the west and northeast). Moreover, the fact that body-size differentiation occurs primarily via differences in growth rate is also due apparently to differences in resource seasonality (and juvenile mortality risk in turn) between the eastern rainforest and the more temperate northeast and west. Most scaling coefficients for both arm and leg growth range from slight negative allometry to slight positive allometry. Given the low intermembral index for sifakas, which is also an adaptation for propulsive hindlimb-dominated jumping, this suggests that differences in adult limb proportions are largely set prenatally rather than being achieved via higher rates of postnatal hindlimb growth.(ABSTRACT TRUNCATED AT 400 WORDS)

Eco-evolutionary trophic dynamics: loss of top predators drives trophic evolution and ecology of prey.

Ecosystems are being altered on a global scale by the extirpation of top predators. The ecological effects of predator removal have been investigated widely; however, predator removal can also change natural selection acting on prey, resulting in contemporary evolution. Here we tested the role of predator removal on the contemporary evolution of trophic traits in prey. We utilized a historical introduction experiment where Trinidadian guppies (Poecilia reticulata) were relocated from a site with predatory fishes to a site lacking predators. To assess the trophic consequences of predator release, we linked individual morphology (cranial, jaw, and body) to foraging performance. Our results show that predator release caused an increase in guppy density and a "sharpening" of guppy trophic traits, which enhanced food consumption rates. Predator release appears to have shifted natural selection away from predator escape ability and towards resource acquisition ability. Related diet and mesocosm studies suggest that this shift enhances the impact of guppies on lower trophic levels in a fashion nuanced by the omnivorous feeding ecology of the species. We conclude that extirpation of top predators may commonly select for enhanced feeding performance in prey, with important cascading consequences for communities and ecosystems.

The Oud Across Arabic Culture

This study is a compilation and compendium of information on the oud, the most important instrument in Arabic classical music. It has grown out of my own long-time involvement in studying and playing the oud, and in particular out of my interest in the lack of sources and knowledge available to the vast majority of oud players and researchers, as well as for the readers. My own path started from an intensive study of the oud, which included exposure to several treaties; some housed in museums around the globe, and some only available in the Arabic language. The study combines archival research (including Arabic poetry and pre-Islamic Era and medieval treaties), symbolism, new archaeological discoveries, field interviews, and analysis of existing scholarship, and draws on my professional performance experience for detailed stylistic analysis of the oud's performance practice and its historical development. The study consists of participant observation, personal performance, and interviews conducted in person, via telephone, and/or via e-mail, according to the choice of the performers. The performers have been selected from networks of musicians who perform regularly at lounges, concert halls, and private events. These performers have been chosen according to their musical knowledge, technical skill, experience, and activity in Arabic music and oud performance. Chapter one deals with the purpose of this study and the methods of investigation, as well as giving a brief overview of the history of the oud. In addition, there will be an introduction to the Arabic musical system (mâqâm), which is primarily based on the mechanics and sound production of the oud. Chapter two deals with the oud in Arabic sources: the first source is Arabic poetry in the pre-Islamic Era. The second source is Arabic poetry in the medieval era, in which I found a significant number of poets who allude to the oud, providing accurate descriptions of the player, singers, and the scenes within the contexts of oud performance. The third source is the Arab scholars' intensive treatises with meticulous accounts of the instrument's apparatii, including descriptions and measurements of the parts, strings, and tuning. While chapter three deals with the classification, the development of the oud, chapter four deals with topics such as: the symbolism of the oud and its relation to cosmology, astronomy, mathematics and anatomy. In most of the pertinent Arabic writings, philosophers mention a significant correlation between the oud and the other sciences. Chapter five deals with recreating the performance practice of the oud. A case study of the oud performers focuses on their style, technique, training, and personal experiences. Topics such as improvisation and ornamentation, the oud in the Arabic musical ensemble, the social uses and functions, and gender in musical performance practices will be included in detailed analysis. Other important topics will be analyzed such as traditional vs. modern technique, and the repertoire of the oud. Specifically, in regard to technique, the study outline the style of the music, the role of the oud in Arabic ensembles, the function of the oud in music composition, and the form of the ensembles in Arabic performance and practice.

Characterization of basal pseudopod-like processes in ileal and colonic PYY cells.

The peptide tyrosine tyrosine (PYY) is produced and secreted from L cells of the gastrointestinal mucosa. To study the anatomy and function of PYY-secreting L cells, we developed a transgenic PYY-green fluorescent protein mouse model. PYY-containing cells exhibited green fluorescence under UV light and were immunoreactive to antibodies against PYY and GLP-1 (glucagon-like peptide-1, an incretin hormone also secreted by L cells). PYY-GFP cells from 15 μm thick sections were imaged using confocal laser scanning microscopy and three-dimensionally (3D) reconstructed. Results revealed unique details of the anatomical differences between ileal and colonic PYY-GFP cells. In ileal villi, the apical portion of PYY cells makes minimal contact with the lumen of the gut. Long pseudopod-like basal processes extend from these cells and form an interface between the mucosal epithelium and the lamina propria. Some basal processes are up to 50 μm in length. Multiple processes can be seen protruding from one cell and these often have a terminus resembling a synapse that appears to interact with neighboring cells. In colonic crypts, PYY-GFP cells adopt a spindle-like shape and weave in between epithelial cells, while maintaining contact with the lumen and lamina propria. In both tissues, cytoplasmic granules containing the hormones PYY and GLP-1 are confined to the base of the cell, often filling the basal process. The anatomical arrangement of these structures suggests a dual function as a dock for receptors to survey absorbed nutrients and as a launching platform for hormone secretion in a paracrine fashion.

Evolutionary characters, phenotypes and ontologies: curating data from the systematic biology literature.

BACKGROUND: The wealth of phenotypic descriptions documented in the published articles, monographs, and dissertations of phylogenetic systematics is traditionally reported in a free-text format, and it is therefore largely inaccessible for linkage to biological databases for genetics, development, and phenotypes, and difficult to manage for large-scale integrative work. The Phenoscape project aims to represent these complex and detailed descriptions with rich and formal semantics that are amenable to computation and integration with phenotype data from other fields of biology. This entails reconceptualizing the traditional free-text characters into the computable Entity-Quality (EQ) formalism using ontologies. METHODOLOGY/PRINCIPAL FINDINGS: We used ontologies and the EQ formalism to curate a collection of 47 phylogenetic studies on ostariophysan fishes (including catfishes, characins, minnows, knifefishes) and their relatives with the goal of integrating these complex phenotype descriptions with information from an existing model organism database (zebrafish, http://zfin.org). We developed a curation workflow for the collection of character, taxonomic and specimen data from these publications. A total of 4,617 phenotypic characters (10,512 states) for 3,449 taxa, primarily species, were curated into EQ formalism (for a total of 12,861 EQ statements) using anatomical and taxonomic terms from teleost-specific ontologies (Teleost Anatomy Ontology and Teleost Taxonomy Ontology) in combination with terms from a quality ontology (Phenotype and Trait Ontology). Standards and guidelines for consistently and accurately representing phenotypes were developed in response to the challenges that were evident from two annotation experiments and from feedback from curators. CONCLUSIONS/SIGNIFICANCE: The challenges we encountered and many of the curation standards and methods for improving consistency that we developed are generally applicable to any effort to represent phenotypes using ontologies. This is because an ontological representation of the detailed variations in phenotype, whether between mutant or wildtype, among individual humans, or across the diversity of species, requires a process by which a precise combination of terms from domain ontologies are selected and organized according to logical relations. The efficiencies that we have developed in this process will be useful for any attempt to annotate complex phenotypic descriptions using ontologies. We also discuss some ramifications of EQ representation for the domain of systematics.

Overview of FEED, the feeding experiments end-user database.

The Feeding Experiments End-user Database (FEED) is a research tool developed by the Mammalian Feeding Working Group at the National Evolutionary Synthesis Center that permits synthetic, evolutionary analyses of the physiology of mammalian feeding. The tasks of the Working Group are to compile physiologic data sets into a uniform digital format stored at a central source, develop a standardized terminology for describing and organizing the data, and carry out a set of novel analyses using FEED. FEED contains raw physiologic data linked to extensive metadata. It serves as an archive for a large number of existing data sets and a repository for future data sets. The metadata are stored as text and images that describe experimental protocols, research subjects, and anatomical information. The metadata incorporate controlled vocabularies to allow consistent use of the terms used to describe and organize the physiologic data. The planned analyses address long-standing questions concerning the phylogenetic distribution of phenotypes involving muscle anatomy and feeding physiology among mammals, the presence and nature of motor pattern conservation in the mammalian feeding muscles, and the extent to which suckling constrains the evolution of feeding behavior in adult mammals. We expect FEED to be a growing digital archive that will facilitate new research into understanding the evolution of feeding anatomy.

Micro-Anatomical Quantitative Imaging Towards Enabling Automated Diagnosis of Thick Tissues at the Point of Care

Histopathology is the clinical standard for tissue diagnosis. However, histopathology has several limitations including that it requires tissue processing, which can take 30 minutes or more, and requires a highly trained pathologist to diagnose the tissue. Additionally, the diagnosis is qualitative, and the lack of quantitation leads to possible observer-specific diagnosis. Taken together, it is difficult to diagnose tissue at the point of care using histopathology.

Several clinical situations could benefit from more rapid and automated histological processing, which could reduce the time and the number of steps required between obtaining a fresh tissue specimen and rendering a diagnosis. For example, there is need for rapid detection of residual cancer on the surface of tumor resection specimens during excisional surgeries, which is known as intraoperative tumor margin assessment. Additionally, rapid assessment of biopsy specimens at the point-of-care could enable clinicians to confirm that a suspicious lesion is successfully sampled, thus preventing an unnecessary repeat biopsy procedure. Rapid and low cost histological processing could also be potentially useful in settings lacking the human resources and equipment necessary to perform standard histologic assessment. Lastly, automated interpretation of tissue samples could potentially reduce inter-observer error, particularly in the diagnosis of borderline lesions.

To address these needs, high quality microscopic images of the tissue must be obtained in rapid timeframes, in order for a pathologic assessment to be useful for guiding the intervention. Optical microscopy is a powerful technique to obtain high-resolution images of tissue morphology in real-time at the point of care, without the need for tissue processing. In particular, a number of groups have combined fluorescence microscopy with vital fluorescent stains to visualize micro-anatomical features of thick (i.e. unsectioned or unprocessed) tissue. However, robust methods for segmentation and quantitative analysis of heterogeneous images are essential to enable automated diagnosis. Thus, the goal of this work was to obtain high resolution imaging of tissue morphology through employing fluorescence microscopy and vital fluorescent stains and to develop a quantitative strategy to segment and quantify tissue features in heterogeneous images, such as nuclei and the surrounding stroma, which will enable automated diagnosis of thick tissues.

To achieve these goals, three specific aims were proposed. The first aim was to develop an image processing method that can differentiate nuclei from background tissue heterogeneity and enable automated diagnosis of thick tissue at the point of care. A computational technique called sparse component analysis (SCA) was adapted to isolate features of interest, such as nuclei, from the background. SCA has been used previously in the image processing community for image compression, enhancement, and restoration, but has never been applied to separate distinct tissue types in a heterogeneous image. In combination with a high resolution fluorescence microendoscope (HRME) and a contrast agent acriflavine, the utility of this technique was demonstrated through imaging preclinical sarcoma tumor margins. Acriflavine localizes to the nuclei of cells where it reversibly associates with RNA and DNA. Additionally, acriflavine shows some affinity for collagen and muscle. SCA was adapted to isolate acriflavine positive features or APFs (which correspond to RNA and DNA) from background tissue heterogeneity. The circle transform (CT) was applied to the SCA output to quantify the size and density of overlapping APFs. The sensitivity of the SCA+CT approach to variations in APF size, density and background heterogeneity was demonstrated through simulations. Specifically, SCA+CT achieved the lowest errors for higher contrast ratios and larger APF sizes. When applied to tissue images of excised sarcoma margins, SCA+CT correctly isolated APFs and showed consistently increased density in tumor and tumor + muscle images compared to images containing muscle. Next, variables were quantified from images of resected primary sarcomas and used to optimize a multivariate model. The sensitivity and specificity for differentiating positive from negative ex vivo resected tumor margins was 82% and 75%. The utility of this approach was further tested by imaging the in vivo tumor cavities from 34 mice after resection of a sarcoma with local recurrence as a bench mark. When applied prospectively to images from the tumor cavity, the sensitivity and specificity for differentiating local recurrence was 78% and 82%. The results indicate that SCA+CT can accurately delineate APFs in heterogeneous tissue, which is essential to enable automated and rapid surveillance of tissue pathology.

Two primary challenges were identified in the work in aim 1. First, while SCA can be used to isolate features, such as APFs, from heterogeneous images, its performance is limited by the contrast between APFs and the background. Second, while it is feasible to create mosaics by scanning a sarcoma tumor bed in a mouse, which is on the order of 3-7 mm in any one dimension, it is not feasible to evaluate an entire human surgical margin. Thus, improvements to the microscopic imaging system were made to (1) improve image contrast through rejecting out-of-focus background fluorescence and to (2) increase the field of view (FOV) while maintaining the sub-cellular resolution needed for delineation of nuclei. To address these challenges, a technique called structured illumination microscopy (SIM) was employed in which the entire FOV is illuminated with a defined spatial pattern rather than scanning a focal spot, such as in confocal microscopy.

Thus, the second aim was to improve image contrast and increase the FOV through employing wide-field, non-contact structured illumination microscopy and optimize the segmentation algorithm for new imaging modality. Both image contrast and FOV were increased through the development of a wide-field fluorescence SIM system. Clear improvement in image contrast was seen in structured illumination images compared to uniform illumination images. Additionally, the FOV is over 13X larger than the fluorescence microendoscope used in aim 1. Initial segmentation results of SIM images revealed that SCA is unable to segment large numbers of APFs in the tumor images. Because the FOV of the SIM system is over 13X larger than the FOV of the fluorescence microendoscope, dense collections of APFs commonly seen in tumor images could no longer be sparsely represented, and the fundamental sparsity assumption associated with SCA was no longer met. Thus, an algorithm called maximally stable extremal regions (MSER) was investigated as an alternative approach for APF segmentation in SIM images. MSER was able to accurately segment large numbers of APFs in SIM images of tumor tissue. In addition to optimizing MSER for SIM image segmentation, an optimal frequency of the illumination pattern used in SIM was carefully selected because the image signal to noise ratio (SNR) is dependent on the grid frequency. A grid frequency of 31.7 mm-1 led to the highest SNR and lowest percent error associated with MSER segmentation.

Once MSER was optimized for SIM image segmentation and the optimal grid frequency was selected, a quantitative model was developed to diagnose mouse sarcoma tumor margins that were imaged ex vivo with SIM. Tumor margins were stained with acridine orange (AO) in aim 2 because AO was found to stain the sarcoma tissue more brightly than acriflavine. Both acriflavine and AO are intravital dyes, which have been shown to stain nuclei, skeletal muscle, and collagenous stroma. A tissue-type classification model was developed to differentiate localized regions (75x75 µm) of tumor from skeletal muscle and adipose tissue based on the MSER segmentation output. Specifically, a logistic regression model was used to classify each localized region. The logistic regression model yielded an output in terms of probability (0-100%) that tumor was located within each 75x75 µm region. The model performance was tested using a receiver operator characteristic (ROC) curve analysis that revealed 77% sensitivity and 81% specificity. For margin classification, the whole margin image was divided into localized regions and this tissue-type classification model was applied. In a subset of 6 margins (3 negative, 3 positive), it was shown that with a tumor probability threshold of 50%, 8% of all regions from negative margins exceeded this threshold, while over 17% of all regions exceeded the threshold in the positive margins. Thus, 8% of regions in negative margins were considered false positives. These false positive regions are likely due to the high density of APFs present in normal tissues, which clearly demonstrates a challenge in implementing this automatic algorithm based on AO staining alone.

Thus, the third aim was to improve the specificity of the diagnostic model through leveraging other sources of contrast. Modifications were made to the SIM system to enable fluorescence imaging at a variety of wavelengths. Specifically, the SIM system was modified to enabling imaging of red fluorescent protein (RFP) expressing sarcomas, which were used to delineate the location of tumor cells within each image. Initial analysis of AO stained panels confirmed that there was room for improvement in tumor detection, particularly in regards to false positive regions that were negative for RFP. One approach for improving the specificity of the diagnostic model was to investigate using a fluorophore that was more specific to staining tumor. Specifically, tetracycline was selected because it appeared to specifically stain freshly excised tumor tissue in a matter of minutes, and was non-toxic and stable in solution. Results indicated that tetracycline staining has promise for increasing the specificity of tumor detection in SIM images of a preclinical sarcoma model and further investigation is warranted.

In conclusion, this work presents the development of a combination of tools that is capable of automated segmentation and quantification of micro-anatomical images of thick tissue. When compared to the fluorescence microendoscope, wide-field multispectral fluorescence SIM imaging provided improved image contrast, a larger FOV with comparable resolution, and the ability to image a variety of fluorophores. MSER was an appropriate and rapid approach to segment dense collections of APFs from wide-field SIM images. Variables that reflect the morphology of the tissue, such as the density, size, and shape of nuclei and nucleoli, can be used to automatically diagnose SIM images. The clinical utility of SIM imaging and MSER segmentation to detect microscopic residual disease has been demonstrated by imaging excised preclinical sarcoma margins. Ultimately, this work demonstrates that fluorescence imaging of tissue micro-anatomy combined with a specialized algorithm for delineation and quantification of features is a means for rapid, non-destructive and automated detection of microscopic disease, which could improve cancer management in a variety of clinical scenarios.

High-Resolution CT for Small-Animal Imaging Research

Preclinical imaging has a critical role in phenotyping, in drug discovery, and in providing a basic understanding of mechanisms of disease. Translating imaging methods from humans to small animals is not an easy task. The purpose of this work is to review high-resolution computed tomography (CT) also known as micro-CT for small-animal imaging. We present the principles, the technologies, the image quality parameters, and the types of applications. We show that micro-CT can be used to provide not only morphological but also functional information such as cardiac function or vascular permeability. Another way in which micro-CT can be used in the study of both function and anatomy is by combining it with other imaging modalities, such as positron emission tomography or single-photon emission tomography. Compared to other modalities, micro-CT imaging is usually regarded as being able to provide higher throughput at lower cost and higher resolution. The limitations are usually associated with the relatively poor contrast mechanisms and the radiation damage, although the use of novel nanoparticle-based contrast agents and careful design of studies can address these limitations.

Live Imaging of Host-Parasite Interactions in a Zebrafish Infection Model Reveals Cryptococcal Determinants of Virulence and Central Nervous System Invasion.

UNLABELLED: The human fungal pathogen Cryptococcus neoformans is capable of infecting a broad range of hosts, from invertebrates like amoebas and nematodes to standard vertebrate models such as mice and rabbits. Here we have taken advantage of a zebrafish model to investigate host-pathogen interactions of Cryptococcus with the zebrafish innate immune system, which shares a highly conserved framework with that of mammals. Through live-imaging observations and genetic knockdown, we establish that macrophages are the primary immune cells responsible for responding to and containing acute cryptococcal infections. By interrogating survival and cryptococcal burden following infection with a panel of Cryptococcus mutants, we find that virulence factors initially identified as important in causing disease in mice are also necessary for pathogenesis in zebrafish larvae. Live imaging of the cranial blood vessels of infected larvae reveals that C. neoformans is able to penetrate the zebrafish brain following intravenous infection. By studying a C. neoformans FNX1 gene mutant, we find that blood-brain barrier invasion is dependent on a known cryptococcal invasion-promoting pathway previously identified in a murine model of central nervous system invasion. The zebrafish-C. neoformans platform provides a visually and genetically accessible vertebrate model system for cryptococcal pathogenesis with many of the advantages of small invertebrates. This model is well suited for higher-throughput screening of mutants, mechanistic dissection of cryptococcal pathogenesis in live animals, and use in the evaluation of therapeutic agents. IMPORTANCE: Cryptococcus neoformans is an important opportunistic pathogen that is estimated to be responsible for more than 600,000 deaths worldwide annually. Existing mammalian models of cryptococcal pathogenesis are costly, and the analysis of important pathogenic processes such as meningitis is laborious and remains a challenge to visualize. Conversely, although invertebrate models of cryptococcal infection allow high-throughput assays, they fail to replicate the anatomical complexity found in vertebrates and, specifically, cryptococcal stages of disease. Here we have utilized larval zebrafish as a platform that overcomes many of these limitations. We demonstrate that the pathogenesis of C. neoformans infection in zebrafish involves factors identical to those in mammalian and invertebrate infections. We then utilize the live-imaging capacity of zebrafish larvae to follow the progression of cryptococcal infection in real time and establish a relevant model of the critical central nervous system infection phase of disease in a nonmammalian model.

Evaluating functional network inference using simulations of complex biological systems.

MOTIVATION: Although many network inference algorithms have been presented in the bioinformatics literature, no suitable approach has been formulated for evaluating their effectiveness at recovering models of complex biological systems from limited data. To overcome this limitation, we propose an approach to evaluate network inference algorithms according to their ability to recover a complex functional network from biologically reasonable simulated data. RESULTS: We designed a simulator to generate data representing a complex biological system at multiple levels of organization: behaviour, neural anatomy, brain electrophysiology, and gene expression of songbirds. About 90% of the simulated variables are unregulated by other variables in the system and are included simply as distracters. We sampled the simulated data at intervals as one would sample from a biological system in practice, and then used the sampled data to evaluate the effectiveness of an algorithm we developed for functional network inference. We found that our algorithm is highly effective at recovering the functional network structure of the simulated system-including the irrelevance of unregulated variables-from sampled data alone. To assess the reproducibility of these results, we tested our inference algorithm on 50 separately simulated sets of data and it consistently recovered almost perfectly the complex functional network structure underlying the simulated data. To our knowledge, this is the first approach for evaluating the effectiveness of functional network inference algorithms at recovering models from limited data. Our simulation approach also enables researchers a priori to design experiments and data-collection protocols that are amenable to functional network inference.

A screw microdrive for adjustable chronic unit recording in monkeys.

A screw microdrive is described that attaches to the grid system used for recording single neurons from brains of awake behaving monkeys. Multiple screwdrives can be mounted on a grid over a single cranial opening. This method allows many electrodes to be implanted chronically in the brain and adjusted as needed to maintain isolation. rights reserved.