Overview of Genetically Engineered Mouse Models of Papillary and Anaplastic Thyroid Cancers: Enabling Translational Biology for Patient Care Improvement.

The prognosis from thyroid cancer subtypes in humans covers a spectrum from "cured at almost 90%" to "100% lethal." Invasive and poorly differentiated forms of thyroid cancer are among the most aggressive human cancers, and there are few effective therapeutic options. Genetically engineered mice, based on mutations observed in patients, can accurately recapitulate the human disease and its progression, providing invaluable tools for the preclinical evaluation of novel therapeutic approaches. This overview details models developed to date as well as their uses for identifying novel anticancer agents. © 2015 by John Wiley & Sons, Inc.

Comparative dendritic cell biology of veterinary mammals.

Dendritic cells (DC) have a main function in innate immunity in that they sense infections and environmental antigens at the skin and mucosal surfaces and thereby critically influence decisions about immune activation or tolerance. As professional antigen-presenting cells, they are essential for induction of adaptive immune responses. Consequently, knowledge on this cell type is required to understand the immune systems of veterinary mammals, including cattle, sheep, pigs, dogs, cats, and horses. Recent ontogenic studies define bona fide DC as an independent lineage of hematopoietic cells originating from a common precursor. Distinct transcription factors control the development into the two subsets of classical DC and plasmacytoid DC. These DC subsets express a distinguishable transcriptome, which differs from that of monocyte-derived DC. Using a comparative approach based on phenotype and function, this review attempts to classify DC of veterinary mammals and to describe important knowledge gaps.

Biology of Bone Metastases in Prostate Cancer

Advanced-stage prostate cancer (PCa) patients are often diagnosed with bone metastases. Bone metastases remain incurable and therapies are palliative. PCa cells prevalently cause osteoblastic lesions, characterized by an excess of bone formation. The prevailing concept indicates that PCa cancer cell secrete an excess of paracrine factors stimulating osteoblasts directly or indirectly, thereby leading to an excess of bone formation. The exact mechanisms by which bone formation stimulates PCa cell growth are mostly elusive. In this review, the mechanisms of PCa cancer cell osteotropism, the cancer cell-induced response within the bone marrow/bone stroma, and therapeutic stromal targets will be summarized.

How Plankton Swim: An Interdisciplinary Approach for Using Mathematics & Physics to Understand the Biology of the Natural World

The authors have developed and field-tested high school-level curricular materials that guide students to use biology, mathematics, and physics to understand plankton and how these tiny organisms move in a world where their intuition does not apply. The authors chose plankton as the focus of their materials primarily because the challenges faced by plankton are novel problems to most students, forcing adoption of new perspectives and making the study of plankton exciting. Additional reasons that they chose plankton to focus on include their ecological importance, their availability to most teachers and students, the ease with which they can be collected and observed, and the current focus of some scientific researchers on their movement and behavior. These curricular materials include a series of inquiry-based, hands-on exercises designed to be accessible to students with a range of backgrounds. Many of these materials could be adapted for use by middle-school, and/or college-level students. In this article, the authors describe sample lessons, summarize what worked well, and flag obstacles they encountered while integrating mathematics and physics into the biology classroom.

Reproductive Biology of the Deep-Sea Polychaete Gorgoniapolynoe Caeciliae (Polynoidae), a Commensal Species Associated with Octocorals

Some aspects of the reproductive biology of the polychaete Gorgoniapolynoe caeciliae have been described for the first time. Gorgoniapolynoe caeciliae is a deep-sea commensal species associated with Candidella imbricala, all octocoral that populates the New England Seamount chain. Gorgoniapolynoe caeciliae is a dioccious species with an equal sex ratio and fertile segments throughout most of the adult body. The gonads of both sexes are associated with genital blood vessels emerging from the posterior surface of most intersegmental septa. In the female, oogenesis is intraovarian with oocytes being retained within the ovary until vitellogenesis is completed. The largest female examined contained over 3000 eggs with a maximum diameter of 80-90 mu m. In the male, the testes are repeated in numerous segments and consist of small clusters of spermatogonia, spermatocytes and early spermatids associated with the walls of the genital blood vessels. Early spermatids are shed into the coelom where they complete differentiation into mature ect-aquasperm with a spherical head (4 mu m), a small cap-like acrosome, and a short mid-piece with four mitochondria. Indirect evidence suggests that this species is an annual breeder that releases its gametes into seawater and produces a planktotrophic larva following fertilization. The reproductive biology of G. caeciliae is consistent with that of most other polynoids including many shallow water species suggesting that phylogenetic history strongly shapes its biology.

Ovarian cancer angiogenesis, biology and therapy

The progressive growth of epithelial ovarian cancer tumor is regulated by proangiogenic molecules and growth factors released by tumor cells and the microenvironment. Previous studies showed that the expression of interleukin-8 (IL-8) directly correlates with the progression of human ovarian carcinomas implanted into the peritoneal cavity of nude mice. We examined the expression level of IL-8 in archival specimens of primary human ovarian carcinoma from patients undergoing curative surgery by in situ mRNA hybridization technique. The expression of IL-8 was significantly higher in patients with stage III disease than in patients with stage I disease. To investigate the role of IL-8 in the progressive growth of ovarian cancer, we isolated high- and low-IL-8 producing clones from parental Hey-A8 human ovarian cancer cells, and compared their proliferative activity and tumorigenicity in nude mice. The effect of exogenous IL-8 and IL-8 neutralizing antibody on ovarian cancer cell proliferation was investigated. Finally, we studied the modulation of IL-8 expression in ovarian cancer cells by sense and antisense IL-8 expression vector transfection and its effect on proliferation and tumorigenicity. We concluded that IL-8 has a direct growth potentiating activity in human ovarian cancer cells. ^ The expression level of IL-8 directly correlates with disease progression of human ovarian cancer, but the mechanism of induction is unknown. Since hypoxia and acidic pH are common features in solid tumors, we determined whether hypoxic and acidic conditions could regulate the expression of IL-8. Culturing the human ovarian cancer cells in hypoxic or acidic medium led to a significant increase in IL-8 mRNA and protein. Hypoxic- and acidosis-mediated transient increase in IL-8 expression involved both transcriptional activation of the IL-8 gene and enhanced stability of the IL-8 mRNA. Furthermore, we showed that IL-8 transcription activation by hypoxia or acidosis required the cooperation of NF-κB and AP-1 binding sites. ^ Finally, we studied novel therapies against human ovarian cancer. First, we determined whether inhibition of the catalytic tyrosine kinase activity of the receptors for vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) inhibits the formation of malignant ascites and the progressive growth of human ovarian carcinoma cells implanted into the peritoneal cavity of nude mice. Our results suggest that blockade of the VEGF/VPF receptor may be an efficient strategy to inhibit formation of malignant ascites and growth of VEGF/VPF-dependent human ovarian carcinomas. Secondly, we determined whether local sustained production of murine interferon-β could inhibit the growth of human ovarian cancer cells in the peritoneal cavity of nude mice. Our results showed that local production of IFN-β could inhibit the in vivo growth of human ovarian cancer cells by upregulating the expression of the inducible nitric oxide synthase (NOS) in host macrophages. ^

A study of the biology of perlecan

Extracellular matrix (ECM) is a component of a variety of organisms that provides both structural support and influence upon the cells it surrounds. The importance of the ECM is becoming more apparent as matrix defects are linked to human disease. In this study, the large, extracellular matrix heparan sulfate proteoglycan, perlecan (Pln) is examined in two systems. First, the role of Pln in the interaction between a blastocyst and uterine epithelial cells is investigated. In mice, blastocyst attachment and implantation occurs at approximately d 4.5 post coitus. In addition, a delayed implantation model has been used to distinguish between the response of the blastocyst to that of hatching and of becoming attachment competent. ^ The second series of experiments described in this study focuses on the process of chondrogenesis in mice. Pln, commonly expressed with other basement membrane (BM) proteins, was found to be expressed in cartilaginous tissue without other BM proteins. This unusual expression pattern led to further study and the development of an in vitro chondrogenesis assay using the mouse embryonic fibroblast cell line, C3H/10T1/2. When cultured on Pln in vitro, these cells form aggregates and express the cartilage proteins, collagen type II and aggrecan. In examining the participation of the heparan sulfate (HS) chains in this process, the proteoglycan was enzymatically digested to remove the HS chains before the initiation of 10T1/2 cell culture. After digestion, the ability of Pln to stimulate aggregate formation was greatly diminished. Thus, the HS chains participate in the cell induction process. To determine which domain of Pln might be responsible for this activity, recombinant fragments of Pin were used in the cell culture assay. Of all recombinant protein fragments tested, only the domain including the HS chains, domain 1, was able to initiate the morphological change exhibited by the 10T1/2 cells. Similar to native Pln, when HS chains were removed from domain I, chondrogenic activity was abolished. A variant of domain I carrying both HS and chondroitin sulfate (CS) chains retained activity when only HS chains were removed. When both HS and CS chains were removed, then activity was lost. ^ The ability to rapidly stimulate differentiation of 10T1/2 cells in vitro may lead to better control of chondrogenesis in vitro and in vivo, providing better understanding and manipulation of the chondrogenic process. This greater understanding may have benefits for study of cartilage and bone diseases and subsequent treatment options. (Abstract shortened by UMI.)^

Insights into the biology of lymphatic metastasis

Recent data suggest that the generation of new lymphatic vessels (i.e. lymphangiogenesis) may be a rate-limiting step in the dissemination of tumor cells to regional lymph nodes. However, efforts to study the cellular and molecular interactions that take place between tumor cells and lymphatic endothelial cells have been limited due to a lack of lymphatic endothelial cell lines available for study. ^ I have used a microsurgical approach to establish conditionally immortalized lymphatic endothelial cell lines from the afferent mesenteric lymphatic vessels of mice. Characterization of lymphatic endothelial cells, and tumor-associated lymphatic vessels revealed high expression levels of VCAM-1, which is known to facilitate adhesion of some tumor cells to vascular endothelial cells. Further investigation revealed that murine melanoma cells selected for high expression of α4, a counter-receptor for VCAM-1, demonstrated enhanced adhesion to lymphatic endothelial cells in vitro, and increased tumorigenicity and lymphatic metastasis in vivo, despite similar lymphatic vessel numbers. ^ Next, I examined the effects of growth factors that regulate lymphangiogenesis, and report that several growth factors are capable of activating survival and proliferation pathways of lymphatic endothelial cells. The dual protein tyrosine kinase inhibitor AEE788 (EGFR and VEGFR-2) inhibited the activation of Akt and MAPK in lymphatic endothelial cells responding to multiple growth factors. Moreover, oral treatment of mice with AEE788 decreased lymphatic vessel density and production of lymphatic metastasis by human colon cancer cells growing in the cecum of nude mice. ^ In the last set of experiments, I investigated the surgical management of lymphatic metastasis using a novel model of sentinel lymphadenectomy in live mice bearing subcutaneous B16-BL6 melanoma. The data demonstrate that this procedure when combined with wide excision of the primary melanoma, significantly enhanced survival of syngeneic C57BL/6 mice. ^ Collectively, these results indicate that the production of lymphatic metastasis depends on lymphangiogenesis, tumor cell adhesion to lymphatic endothelial cells, and proliferation of tumor cells in lymph nodes. Thus, lymphatic metastasis is a multi-step, complex, and active process that depends upon multiple interactions between tumor cells and tumor associated lymphatic endothelial cells. ^

An innovative approach to impacting student academic achievement and attitudes: Pilot study of the HEADS UP Virtual Molecular Biology Lab

Objective. To evaluate the HEADS UP Virtual Molecular Biology Lab, a computer-based simulated laboratory designed to teach advanced high school biology students how to create a mouse model. ^ Design. A randomized clinical control design of forty-four students from two science magnet high schools in Mercedes, Texas was utilized to assess knowledge and skills of molecular laboratory procedures, attitudes towards science and computers as a learning tool, and usability of the program. ^ Measurements. Data was collected using five paper-and-pencil formatted questionnaires and an internal "lab notebook." ^ Results. The Virtual Lab was found to significantly increase student knowledge over time (p<0.005) and with each use (p<0.001) as well as positively increase attitudes towards computers (p<0.001) and skills (p<0.005). No significant differences were seen in science attitude scores.^ Conclusion. These results provide evidence that the HEADS UP Virtual Molecular Biology Lab is a potentially effective educational tool for high school molecular biology education.^

An integrated molecular biology and computational approach to CYP1A1 expression in human brain

The cytochromes P450 comprise a superfamily of heme-containing mono-oxygenases. These enzymes metabolize numerous xenobiotics, but also play a role in metabolism of endogenous compounds. The P450 1A1 enzyme generally metabolizes polycyclic aromatic hydrocarbons, and its expression can be induced by aryl hydrocarbon receptor (AhR) activation. CYP1A1 is an exception to the generality that the majority of CYPs demonstrate highest expression in liver; CYP1Al is present in numerous extrahepatic tissues, including brain. This P450 has been observed in two forms, wildtype (WT) and brain variant (BV), arising from alternatively spliced mRNA transcripts. The CYP1A1 BV mRNA presented an exon deletion and was detected in human brain but not liver tissue of the same individuals. ^ Quantitative PCR analyses were performed to determine CYP1A1 WT and BV transcript expression levels in normal, bipolar disorder or schizophrenic groups. In our samples, we show that CYP1A1 BV mRNA, when present, is found alongside the full-length form. Furthermore, we demonstrate a significant decrease in expression of CYP1A1 in patients with bipolar disorder or schizophrenia. The expression level was not influenced by post-mortem interval, tissue pH, age, tobacco use, or lifetime antipsychotic medication load. ^ There is no indication of increased brain CYP1A1 expression in normal smokers versus non-smokers in these samples. We observed slightly increased CYP1A1 expression only in bipolar and schizophrenic smokers versus non-smokers. This may be indicative of complex interactions between neuronal chemical environments and AhR-mediated CYP1A1 induction in brain. ^ Structural homology modeling demonstrated that P450 1A1 BV has several alterations to positions/orientations of substrate recognition site residues compared to the WT isoform. Automated substrate docking was employed to investigate the potential binding of neurological signaling molecules and neurotropic drugs, as well as to differentiate specificities of the two P450 1A1 isoforms. We consistently observed that the BV isoform produced energetically favorable substrate dockings in orientations not observed for the same substrate in the WT isoform. These results demonstrated that structural differences, namely an expanded substrate access channel and active site, confer greater capacity for unique compound docking positions suggesting a metabolic profile distinct from the wildtype form for these test compounds. ^

From protein microarray to biology: The discovery of epigenetic regulatory molecules

Chromatin, composed of repeating nucleosome units, is the genetic polymer of life. To aid in DNA compaction and organized storage, the double helix wraps around a core complex of histone proteins to form the nucleosome, and is therefore no longer freely accessible to cellular proteins for the processes of transcription, replication and DNA repair. Over the course of evolution, DNA-based applications have developed routes to access DNA bound up in chromatin, and further, have actually utilized the chromatin structure to create another level of complexity and information storage. The histone molecules that DNA surrounds have free-floating tails that extend out of the nucleosome. These tails are post-translationally modified to create docking sites for the proteins involved in transcription, replication and repair, thus providing one prominent way that specific genomic sequences are accessed and manipulated. Adding another degree of information storage, histone tail-modifications paint the genome in precise manners to influence a state of transcriptional activity or repression, to generate euchromatin, containing gene-dense regions, or heterochromatin, containing repeat sequences and low-density gene regions. The work presented here is the study of histone tail modifications, how they are written and how they are read, divided into two projects. Both begin with protein microarray experiments where we discover the protein domains that can bind modified histone tails, and how multiple tail modifications can influence this binding. Project one then looks deeper into the enzymes that lay down the tail modifications. Specifically, we studied histone-tail arginine methylation by PRMT6. We found that methylation of a specific histone residue by PRMT6, arginine 2 of H3, can antagonize the binding of protein domains to the H3 tail and therefore affect transcription of genes regulated by the H3-tail binding proteins. Project two focuses on a protein we identified to bind modified histone tails, PHF20, and was an endeavor to discover the biological role of this protein. Thus, in total, we are looking at a complete process: (1) histone tail modification by an enzyme (here, PRMT6), (2) how this and other modifications are bound by conserved protein domains, and (3) by using PHF20 as an example, the functional outcome of binding through investigating the biological role of a chromatin reader. ^

Systems Biology Approaches to Probe Gene Regulation in Bacteria

Mechanisms that allow pathogens to colonize the host are not the product of isolated genes, but instead emerge from the concerted operation of regulatory networks. Therefore, identifying components and the systemic behavior of networks is necessary to a better understanding of gene regulation and pathogenesis. To this end, I have developed systems biology approaches to study transcriptional and post-transcriptional gene regulation in bacteria, with an emphasis in the human pathogen Mycobacterium tuberculosis (Mtb). First, I developed a network response method to identify parts of the Mtb global transcriptional regulatory network utilized by the pathogen to counteract phagosomal stresses and survive within resting macrophages. As a result, the method unveiled transcriptional regulators and associated regulons utilized by Mtb to establish a successful infection of macrophages throughout the first 14 days of infection. Additionally, this network-based analysis identified the production of Fe-S proteins coupled to lipid metabolism through the alkane hydroxylase complex as a possible strategy employed by Mtb to survive in the host. Second, I developed a network inference method to infer the small non-coding RNA (sRNA) regulatory network in Mtb. The method identifies sRNA-mRNA interactions by integrating a priori knowledge of possible binding sites with structure-driven identification of binding sites. The reconstructed network was useful to predict functional roles for the multitude of sRNAs recently discovered in the pathogen, being that several sRNAs were postulated to be involved in virulence-related processes. Finally, I applied a combined experimental and computational approach to study post-transcriptional repression mediated by small non-coding RNAs in bacteria. Specifically, a probabilistic ranking methodology termed rank-conciliation was developed to infer sRNA-mRNA interactions based on multiple types of data. The method was shown to improve target prediction in Escherichia coli, and therefore is useful to prioritize candidate targets for experimental validation.