1,720,959 research outputs found
Elucidation of Genomic Structure, Biological Properties, and Functional Status of Canine MDA-7 and Its Receptors
Get PDFHuman melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) is a tumor suppressor gene with interleukin properties. Ectopic expression of MDA-7 protein causes growth suppression and induces apoptosis in a wide variety of cancer cells. In this study, we identified a canine ortholog of the human mda-7 gene, and elucidated its genomic structue and biological properties. Canine mda-7 was mapped to chromosome 7, located in a cluster of IL-10 family members. Canine MDA-7 was found to be endogenously expressed in cultured normal canine epidermal keratinocytes (NCEKs). When compared to human mda-7 mRNA, canine mda-7 mRNA had a very short 3’ untranslated region. Pre-mRNA transcribed from the canine MDA-7 locus was alternatively spliced (Exon skipping and use of alternative 5’ donor sites) to yield five splice variants in cultured NCEKs. Canine mda-7 splice variant1 (sv1) was the predominant splice variant expressed in NCEKs, while sv2 and sv5 were expressed at intermediate levels and sv3 and sv4 at the lowest level. These splice variants encode four isoforms of canine MDA-7 protein, which have similar amino acid sequences at their amino terminus. Canine MDA-7 is constitutively expressed in NCEKs and its expression was induced in PBMCs after lipopolysaccharide (LPS) stimulation. Similarly, expression of canine mda-7 mRNA was increased in NCEKs after LPS stimulation. Canine MDA-7 mRNA was not expressed in most canine tumor cells and tumor samples. However, one cancer cell line, canine mammary tumor-12 (CMT12) expressed canine MDA-7 at very high levels. When ectopically expressed from a plasmid vector, it suppressed the growth of canine and human tumor cells. Canine MDA-7 treated cancer cells accumulated in G2/M phase and underwent apoptosis. Canine MDA-7 has cytotoxic effects on cancer cells but not on normal canine fibroblasts (NCFs). In addition, canine MDA-7 protein isoforms also have growth inhibitory effects on cancer cells. Canine MDA-7 has a 28 amino acid long signal peptide sequence, and a possible cleavage site between the 28th and 29th amino acid. Canine MDA-7 was determined to be actively secreted and could bind to and signal through human MDA-7 receptors (IL-20R1/IL-20R2 and IL-22R1/IL-20R2). Secreted canine MDA-7 showed bystander antitumor activity against human tumor cells. In this study, we identified and showed that canine IL-20R1 and IL-22R1 subunits were expressed in NCEKs, canine tissues and tumor cells. A truncated mRNA sequence encoding for the extracellular domain of canine IL-20R2 subunit was also identified. The truncated canine IL-20R2 subunit does not have a transmembrane or intracellular domain and thus, it cannot translocate into the cell membrane to make functional canine MDA-7 receptor. Canine MDA-7 did not show bystander antitumor activity against canine tumor cells. This is either due to a lack of expression of functional MDA-7 receptors or due to expession of a truncated canine IL-20R2 subunit, which can block the activity of canine IL-24. In summary, we have shown that canine MDA-7 is indeed an ortholog of MDA-7. It has strikingly similarities in amino acid sequence, genomic and protein structure to human MDA-7. However, the canine MDA-7 receptor system appears to be defective, due to the absence of a full-length IL-20R2 subunit, leading to a failure of the mechanism responsible for the bystander antitumor effect. Although the canine MDA-7 has the potential to be used for cancer gene therapy to treat canine cancers, however, the lack of a bystander effect in dogs may limit its usefulness
Role of IL-27 in HSV-1-induced Herpetic Stromal Keratitis
No full textHerpes Stromal Keratitis (HSK) results from ocular infection with Herpes simplex virus-1 (HSV-1) and is one of the leading causes of infectious blindness. After primary lytic infection in the facial region, the virus establishes life-long latency in the trigeminal ganglion (TG). The periodic reactivations from latency involve the cornea with clinically evident HSK usually as the consequence of repeated recurrences. HSK lesions are primarily orchestrated by neutrophils and functional subsets of T cells, which infiltrate the cornea in response to the infection. The HSV-1 replication and the inflammatory reaction in the cornea lead to permanent damage to the cornea and cause vision impairment. Currently, a combination of antivirals and corticosteroids are used to treat HSK. However, there are numerous challenges in the current treatment paradigm including antiviral-resistant strains, glaucoma, and epithelial toxicity, when used for prolonged periods. There is a dire need for alternate therapeutic strategies to treat HSK.
Cytokines are critical for the activation and differentiation of effector immune cells during HSK. In this work, we show that interleukin (IL)-27, an immunoregulatory cytokine, is upregulated in macrophages during HSV-1 infection. IL-27 receptor knockout mice were more susceptible to HSV-1 infection and had more severe HSK lesions. IL-27 plays a critical role in the optimum induction of effector CD4+ T cell responses and limiting HSK progression. Further, IL-27 plays an antiviral role by regulating macrophage-mediated HSV-1 killing, and type-I interferon (IFN) response after HSV-1 infection. IL-27 is also critical for macrophage survival, antigen uptake, and the expression of costimulatory molecules involved in the optimum induction of effector T-cell responses. Our results indicate that IL-27 promotes endogenous antiviral and anti-inflammatory responses and represents a promising target for suppressing HSK progression.
IFNs are the first line of host defense against HSV-1 infection. Type-I IFNs are majorly produced by the corneal epithelial cells and immune cells such as macrophages, and dendritic cells (DCs) that infiltrate the cornea following HSV-1 infection. However, HSV-1 has evolved strategies to evade the host response and promote viral replication. In this work, we identified that HSV-1 infection regulates macrophage metabolism by promoting glycolysis and oxidative phosphorylation (OXPHOS). This leads to mitochondrial membrane depolarization and an increase in mitochondrial oxidative stress. Further, HSV-1 infection promotes itaconate in macrophages which disrupts the Kreb’s cycle as well as suppresses the IFN response. Upon exogenous administration of itaconate derivative, there was a significant reduction in the type-I IFN response and IL-27 in macrophages. Further, the absence of IL-27 signaling in macrophages promotes IRG1 expression and disrupts mitochondrial function. Therefore, targeting the itaconate/IL-27 pathway could be a therapeutic strategy for combating ocular HSV-1 infection.
This dissertation focuses on providing a comprehensive background on HSV-1 immunobiology and identifying potential therapeutic strategies for HSK. Since IL-27 has a dual anti-inflammatory and antiviral role in regulating HSK, we propose targeting IL-27 signaling for HSK management
Novel Approaches to the Molecular Characterization of Primary Canine Osteosarcoma
Get PDFOsteosarcoma (OSA) is an aggressive malignant bone tumor that often affects pediatric humans as well as dogs. Canine OSA shares many similarities with the human condition, including clinical presentation and molecular profiles, and therefore serves as an excellent model to study the disease. OSA is a difficult tumor to dissociate and sequence due to its bony and brittle composition. Furthermore, it is characterized by extreme genetic complexity and significant intra- and inter-tumoral heterogeneity. While great strides have been made in identifying key mutations driving carcinogenesis, the treatment and prognosis for OSA have remained largely unchanged for 40 years. New approaches to studying OSA are critical for improving patient outcomes for both humans and dogs.
Differential gene expression analysis represents one approach to analyzing important differences in the molecular profiles between normal and tumor tissue. A prerequisite to successful sequencing and differential analysis is the isolation of high-quality RNA from both normal and neoplastic tissue. In the case of OSA, normal bone corresponds to the non-neoplastic and pre-cancerous tissue representative of osteosarcoma. Bone is a dynamic tissue consisting of many different cell types embedded within a rigid matrix. Removal of the bone marrow is an important consideration for isolating RNA that is unique to OSA progenitors for an appropriate differential gene expression analysis. Therefore, a method to isolate RNA from normal canine bone was first established for subsequent transcriptomic sequencing of both unaffected tissue and tumorous lesions.
Transcriptomic bulk sequencing of primary OSA provides a global view of the changes involved in OSA tumorigenesis. Differential gene expression analysis identifies dysregulated genes in the tumors compared to the normal bone. Patient-matched OSA and normal bone were collected and sequenced to identify genes that were commonly dysregulated among the group. However, this approach requires a large sample size to accommodate the statistical analysis and fails to account for inter-individual differences. To circumvent this, we supplemented the group differential profile with a novel individual-level analysis by deriving individual fold-change differences from the group’s significant genes. The results confirm the hyper-variability in OSA and the need to study OSA at the individual level.
Single-cell sequencing (SCseq) has emerged in the past five years as a valuable tool for interrogating the molecular changes occurring in diseased tissue at the level of individual cells. However, an essential requirement for this approach includes highly viable cells (>60% live cells) dissociated from the primary tissue. OSA has proved difficult to manipulate and dissociate due to the matrix composition, resulting in poor cell viability unsuitable for SCseq. Single-nuclei isolation and sequencing have recently evolved as an alternative using 10x Genomics technology. This approach requires high-quality nuclei but overcomes the need for viable whole cells as input. With protocol adjustments, we have successfully isolated high-quality nuclei from a primary canine OSA used for single-nuclei multiome sequencing. Multiome sequencing includes transcriptomic profiling using RNA sequencing as well as epigenomic profiling using ATAC (Assay for Transposase-Accessible Chromatin) sequencing. This dual sequencing approach was used to characterize the tumor microenvironment and elucidate changes occurring at single-nuclei resolution in an individual patient.
This dissertation provides a thorough background of canine OSA, describes a novel method to isolate RNA from canine bone for proper comparative sequencing analysis, presents bulk RNA sequencing of 7 canine OSA tumors along with an individualized approach to differential gene analysis, and concludes with single-nuclei multiome sequencing of a primary OSA tumor to characterize the tumor microenvironment. Importantly, these approaches can be translated to study human OSA for comparison to canine OSA molecular pathways and for the development of more targeted and effective therapies
Fibroblast Growth Factor-21 (FGF21) in Domestic Cats
No full textFibroblast growth factors (FGFs) are a group of structurally homologous yet functionally pleiotropic proteins. Unlike canonical and intracellular FGFs, the FGF19 subfamily, which includes FGF15/19, FGF21, and FGF23, act as endocrine hormones that regulate phosphate, bile acid, and metabolic homeostasis, respectively. Research in human and rodent models demonstrates the potential of these endocrine FGFs to treat various diseases. Data from studies evaluating the metabolic effects of FGF21 pathway activation show improved lipid homeostasis and insulin sensitivity, decreased severity of non-alcoholic steatohepatitis, and potential use as a therapeutic for pancreatitis. Even though cats commonly suffer from metabolic diseases, including obesity, pancreatitis and hepatic lipidosis, there are no studies evaluating FGF21 in cats and only rare studies in other non-laboratory domestic species. This dissertation serves as the first exploration of FGF21 in domestic cats and probes how the FGF21 pathway can be utilized as a future therapy in veterinary medicine.
Chapter 1 of this dissertation reviews the endocrine FGF family, focusing on the physiology and current research. FGF19/FGF15 serves as a mechanism of bile homeostasis. FGF23, a bone-derived hormone, regulates urinary phosphate retention and active vitamin D levels. FGF21, the main focus of this dissertation, is an endocrine link between the liver and adipose tissue, regulating metabolic homeostasis. This literature review further discusses what is known about FGF21 within domestic animals and veterinary medicine.
Chapter 2 is a prospective preliminary cross-sectional study utilizing purpose-bred, male-neutered, 6-year-old, obese and overweight cats administered either a recombinant human FGF21 mimetic modified for increased thermal and confirmational stability or saline control over a 14-day treatment period. Treatment with FGF21 resulted in significant weight loss (~5.93%) compared to control and a trend toward decreased intrahepatic triglyceride content. Cats treated with FGF21 had reduced serum alkaline phosphatase. No significant changes were noted in liver elasticity, serum, liver, metabolic parameters, or gut microbiome composition. This chapter demonstrates that in obese and overweight cats, activation of the FGF21 pathway can safely induce weight loss with trends to improve liver lipid content. Manipulation of the FGF21 pathway has promising potential as a therapeutic for feline obesity. Further studies are needed to see if FGF21 pathway manipulation can be therapeutic for feline metabolic disease.
Chapter 3 explores the FGF21 pathway utilizing banked feline tissues in clinically normal and selected diseased cats. This study evaluated the distribution of transcript and protein expression of FGF21, and its co-receptors, Fibroblast Growth Factor Receptor 1c and β-klotho in key tissues. Relative to the lowest expressing tissue, the highest expression of FGF21 was in the liver, with strong and consistent immunohistochemical reactivity in formalin-fixed paraffin-embedded liver sections. Contrary to other species, FGF21 is not highly expressed in adipose tissues despite the presence of FGF21 co-receptors. This chapter proposes that the retention of the FGF21 endocrine pathway and the loss of the FGF21 autocrine/paracrine pathway in the adipose tissues may be responsible for the unique differences in the feline response to FGF21 mimetics. Additionally, the immunohistochemical expression of FGF21 in cases of spontaneous feline pancreatitis was examined, with strong FGF21 immunoreactivity within macrophages, which may represent M2 polarity.
This dissertation provides a starting point for evaluating the FGF21 pathway within domestic animals, including cats. These findings suggest that there are unique signaling pathways and therapeutic potential to target the FGF21 pathway, and further studies are needed to determine if FGF21 therapies will be a viable option in a clinical setting
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Association of DNA Copy Number and Structural Variation with Racial Disparities in Childhood Obesity
No full textObesity has become a major public health concern throughout the world. Alabama is 6th highest ranked with obesity in the United States. The development of obesity can be influenced by several factors, such as diet, environment, and genetics. Genomic variability among individuals is largely due to copy number variations (CNVs). Recent genome-wide association studies (GWAS) have successfully identified quite a few loci containing CNV related to obesity. Genomic diversity influences the mechanisms of expression in a variety of genders and ethnicities. Chapter 1 reviews the current literature on the relationships between obesity and the CNV of several loci. The subsequent chapters detail the connection between DNA copy number and structural variation (CNV) and childhood obesity in European American (EA) and African American (AA) elementary school children in Alabama. Chapter 2 evaluates the association between AMY1 copy number and obesity measurements as well as racial disparities between the two ethnic groups. Our findings suggest that overweight/obese children have a low AMY1 copy number and the effect is more prominent in AA children. Chapter 3 describes the relationship between the copy number of the 11q11 gene and obesity measurements. A significant inverse association between obesity measurements and 11q11 copy number was observed. EA children have a stronger association between low 11q11 copy number and obesity compared to AA children. The last chapter illustrates the connection between telomere length ratio, blood pressure, and childhood obesity. Our results demonstrated that AA children have high telomere to the single copy gene (T/S) ratio compared to EA children. The high T/S ratio is negatively associated with diastolic pressure
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
- …
