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Cellular senescence has been viewed as an irreversible cell cycle arrest that acts to prevent cancer. Recent studies discovered widespread shortening of 3′ untranslated regions (3′ UTRs) by alternative cleavage and polyadenylation (APA) in cancer cells. However, the role of APA in the process of cellular senescence remains elusive. We thus applied our published PA-seq method to investigate APA regulation in different passages of mouse embryonic fibroblasts (MEFs) and aortic vascular smooth muscle cells (VSMCs) from rats of different ages. We found that genes in senescent cells tended to use distal poly(A) sites (pAs). An independent RNA-seq analysis gave rise to the same conclusion. Interestingly, the level of expression of genes preferred to use distal pAs in senescent MFEs and VSMCs tended to decrease. More importantly, genes that preferred to use distal pAs in senescent MFEs and VSMCs were enriched in common senescence-related pathways such as ubiquitin-mediated proteolysis and cell cycle. Further, the longer 3′ UTRs of the genes that tended to use distal pAs introduced more conserved binding sites of senescence-related microRNAs (miRNAs) and RNA binding proteins (RBPs). Noteworthy, the expression level of core factors involved in cleavage and the polyadenylation tended to decrease, while those factors showed opposite trend in cancer cells. In summary, we showed, for the first time, that APA is a hidden layer of post-transcriptional gene expression regulation involved in cellular senescence.

The functional matrix theory was first introduced in the orthodontic literature by Melvin Moss half century ago. Since its original introduction, several attempts have been made to test the validity of this theory with mixed results. In this paper, we present evidence generated using transgenic mice to test the role of neuronal function in craniofacial development. Our data confirmed previous observations that midface retrusion accompanied neuronal dysfunction in the HexB −/− mouse model of Sandhoff disease. Importantly, restoration of neuronal function after targeted expression of a therapeutic gene selectively in the neurons of HexB −/− mice resulted in normalization of midface development in this mouse model. Histological analysis of the cranial base revealed that abnormal development of the synchondroses underlies the attendant midface retrusion in this model: Neuronal dysfunction led to the absence of hypertrophic chondrocytes in the synchondroses, whereas restoration of neuronal function resulted in normalization of the cranial base development. Taken together, our studies suggest that neuronal function is critical for normal midface development, underscoring the importance of the functional matrix theory as originally proposed by Melvin Moss.

Similarly to humans, healthy, wild-type mice develop osteoarthritis, including of the temporomandibular joint (TMJ), as a result of aging. Pro-inflammatory cytokines, such as IL-1β, IL-6, and TNFα, are known to contribute to the development of osteoarthritis, whereas TGFβ has been associated with articular regeneration. We hypothesized that a balance between IL-1β and TGFβ underlies the development of TMJ osteoarthritis, whereby IL-1β signaling down-regulates TGFβ expression as part of disease pathology. Our studies in wild-type mice, as well as the Col1-IL1βXATmouse model of osteoarthritis, demonstrated an inverse correlation between IL-1β and TGFβ expression in the TMJ. IL-1β etiologically correlated with joint pathology, whereas TGFβ expression associated with IL-1β down-regulation and improvement of articular pathology. Better understanding of the underlying inflammatory processes during disease will potentially enable us to harness inflammation for orofacial tissue regeneration.

The trigeminal sensory system was evaluated for the retrograde transfer of gene therapy vectors into the CNS. The feline immunodeficiency viral vector, FIV(HEXB), encoding for the human HEXB gene, was injected intra-articularly in the temporomandibular joint of 12 week-old HexB −/− mice displaying clinical and histopathological signs of Sandhoff disease. This treatment regiment reduced GM 2 storage and ameliorated neuroinflammation in the brain of HexB −/− mice, as well as attenuated behavioral deficits. In conclusion, retrograde transfer along trigeminal sensory nerves may prove to be a valuable route of gene therapy administration for the treatment of lysosomal storage disorders and other neurodegenerative diseases.

Objective Pain from arthritis has been associated with peripheral sensitization of primary sensory afferents and the development of inflammation at the dorsal horns. This study was undertaken to determine whether the role of spinal interleukin-1β (IL-1β) in central processing of pain is important in the development of arthritis. Methods Col1-IL-1βXAT mice and GFAP-IL-1βXAT mice were injected with the feline immunodeficiency virus (FIV) (Cre) vector in the right and left temporomandibular joints (TMJs), or in the cisterna magna, respectively, to induce IL-1β expression in the dorsal horns of the spinal horn. To inhibit intrathecal IL-1 receptor type I (IL-1RI) signaling, FIV(IL-1Ra) vector was injected into the cisterna magna of Col1-IL-1βXAT mice. The effects of IL-1RI receptor inhibition in GFAP-IL-1βXAT mice were studied in the GFAP-IL-1βXAT–IL-1RI−/− compound mouse model. Neuroinflammatory, sensory, and behavioral changes were evaluated in conjunction with arthritic changes in the TMJ, assessed by histopathologic and immunohistochemical analyses. Results Induction of an osteoarthritis-like condition in the TMJ in the Col1-IL-1βXAT mouse model resulted in up-regulation of murine IL-1β at the dorsal horns. Moreover, intrathecal inhibition of IL-1RI in Col1-IL-1βXAT mice with arthritis led to amelioration of joint pathology and attenuation of the attendant joint pain. Overexpression of spinal IL-1β in the recently developed GFAP-IL-1βXAT somatic mosaic model of neuroinflammation led to development of arthritis-like pathology accompanied by increased pain-like behavior. Conclusion Our results indicate that joint pathology and pain are dependent on spinal IL-1β, and suggest the presence of a bidirectional central nervous system–peripheral joints crosstalk that may contribute to the development, expansion, and exacerbation of arthritis.

The incidence of skin cancer is on the rise, with over 1 million new cases yearly. Although it is known that squamous cell cancers (SCC) are caused by UV light, the mechanism(s) involved remains poorly understood. In vitro studies with epithelial cells or reports examining malignant skin lesions suggest that loss of E-cadherin–mediated cell-cell contacts may contribute to SCCs. Other studies show a pivotal role for cyclooxygenase-dependent prostaglandin E2 (PGE2) synthesis in this process. Using chronically UV-irradiated SKH-1 mice, we show a sequential loss of E-cadherin–mediated cell-cell contacts as lesions progress from dysplasia to SCCs. This E-cadherin down-regulation was also evident after acute UV exposure in vivo. In both chronic and acute UV injury, E-cadherin levels declined at a time when epidermal PGE2 synthesis was enhanced. Inhibition of PGE2 synthesis by indomethacin in vitro, targeted deletion of EP2 in primary mouse keratinocyte (PMK) cultures or deletion of the EP2 receptor in vivo abrogated this UV-induced E-cadherin down-regulation. In contrast, addition of PGE2 or the EP2 receptor agonist butaprost to PMK produced a dose- and time-dependent decrease in E-cadherin. We also show that UV irradiation, via the PGE2-EP2 signaling pathway, may initiate tumorigenesis in keratinocytes by down-regulating E-cadherin–mediated cell-cell contacts through its mobilization away from the cell membrane, internalization into the cytoplasm, and shuttling through the lysosome and proteasome degradation pathways. Further understanding of how UV-PGE2-EP2 down-regulates E-cadherin may lead to novel chemopreventative strategies for the treatment of skin and other epithelial cancers. [Cancer Res 2007;67(16):7654–64]

International Bioenergy (Shanghai) Exhibition and Asian Bioenergy Conference 2015, IBSCE 2015

The systemic effects of gene therapy have been previously described in a variety of peripheral organs following intravenous administration or intraperitoneal inoculation of viral vectors, as well as in the brain following intracranial administration. However, limited information is available on the ability of viral vectors to cross the blood–brain barrier and infect cells located within the central nervous system (CNS). We employed a VSV-G pseudotyped FIV(lacZ) vector capable of transducing dividing, growth-arrested, as well as post-mitotic cells with the reporter gene lacZ . Adult mice were injected intraperitoneally with FIV(lacZ), and the expression of β-galactosidase was studied 5 weeks following treatment in the brain, liver, spleen and kidney by X-gal histochemistry and immunocytochemistry. Interestingly, relatively low doses of FIV(lacZ) administered intraperitoneally lead to β-galactosidase detection in the brain and cerebellum. The identity of these cells was confirmed by double immunofluorescence, and included CD31-, CD3- and CD11b-positive cells. Fluorescent microspheres co-injected with FIV(lacZ) virus were identified within mononuclear cells in the brain parenchyma, suggesting infiltration of peripheral immune cells in the CNS. Cerebellar Purkinje neurons were also transduced in all adult-injected mice. Our observations indicate that relatively low doses of FIV(lacZ) administered intraperitoneally resulted in the transduction of immune cells in the brain, as well as a specific subset of cerebellar neurons.

Genetic studies often require the employment of an inducible expression system, whereby the expression of a particular gene can be regulated by the exogenous administration of an inert ligand. Cre/loxP-based systems have been previously described as the basis for inducible expression systems by exerting site-specific DNA recombination. In our effort to enhance the properties of the RU486-responsive CrePr1 construct, we have developed the dual GLVP/CrePr system, in which RU486 confers activity control at both the transcriptional and the posttranslational level of CrePr1. This was achieved by placing CrePr1 transcriptional regulation under the control of the RU486-sensitive chimeric regulator GLVP. Stable cell lines harboring the dual GLVP/CrePr as well as the single CrePr1 system were developed. Our results indicate that the dually regulated system is highly inducible by RU486 while maintaining minimal basal activity ("leakage"), characteristics that can be employed in the development of transgenic mice in which genetic pathways can be turned on or turned off after exogenous administration of RU486 at physiologically inert doses.

Objective: This study aimed to identify the role of IL-18BPa in the regulation of immune responses associated with the pathogenesis of Rheumatoid Arthritis. Methods: 65 Rheumatoid Arthritis (RA) patients, 22 Osteoarthritis (OA) patients, and 40 sex and age matched healthy donors were enrolled in this study. Synovial fluids mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC) were prepared by using Ficoll-Hypaque separation procedure. Super Array analysis was used to measure the expression profile of immune-related genes in RA synovial tissues (RA-ST) and in normal PBMC treated with recombinant human IL-18 binding protein a (IL-18BPa). The mRNA levels of T-helper 1 (TH1) and T-helper 2 (TH2) cytokines were measured by real-time PCR, and the protein levels of IFN-γ, IL-4 and IL-18BPa autoantibodies were detected by ELISA. Results: High expression of IL-18BPa protein and messenger RNA (mRNA) are found in RA-SF and RA-ST. SuperArray analysis of immune related gene expression profile in normal PBMC treated with IL-18BPa indicated decreases in the gene expression of IFN-γ. IL-18BPa downregulated the mRNA expression of IFN-γ and IL-12, as well as, upregulated the mRNA expression of IL-4 and IL-10 in RA and normal subjects. IFN-γ and IL-12 upregulated the mRNA and protein expression of IL-18BPa in RA subjects. Autoantibodies against IL-18BPa were detected in plasma of RA patients (41.7%), in healthy subjects (4.0%), and none of OA patients, and also detected in SF of RA patients (37.9%) and OA patients (9%). Conclusion: This study emphasizes the anti-inflammatory properties of IL-18BPa on cytokines milieu and the IL-18BPa auto-antibodies may play a role in aberrant regulation of IL-18BPa in RA patients.

The branched-chain amino acids (BCAAs) leucine, isoleucine, and valine are beneficial to catabolic patients by improving hepatic protein synthesis and nitrogen economy, yet their transport from the intestinal lumen is not well-defined. The leucine transport system in human and rabbit small intestine was characterized using a brush border membrane vesicle (BBMV) model. Sodium and pH dependence and transport activity along the longitudinal axis of the small bowel were determined. Transport kinetics and inhibition profiles were defined. Although previous studies in other tissues show leucine transport to be mostly a Na+-independent process, our studies show that leucine transport is a predominantly Na+-dependent process occurring mainly via a single saturable pH-independent transporter resembling system B0 in the intestine. This system B0 transporter demonstrates stereoisomeric specificity. There is also a minor Na+-independent transport component (

Background.Changes in amino acid transport after massive enterectomy occur in a nutrient-dependent fashion and may affect long-term outcome. Epidermal growth factor (EGF) can enhance nutrient transport and a defective epidermal growth factor receptor (EGF-R) has been noted to attenuate adaptation. Most animal studies, however, have examined only a single site of resection. This does not mimic the clinical situation where disease dictates the site of resection leading to proximal, middle, or distal enterectomies. We hypothesize that the site of massive enterectomy will alter nutrient transport and EGF-R levels in the residual gut. Materials and methods.New Zealand White rabbits were randomized to control, midgut division, or 70% resection (proximal, midgut, or distal). After 1 week, sodium-dependent transport of glucose, glutamine, alanine, and leucine into brush border membrane vesicles was quantitated. EGF-R protein was determined by Western blot analysis. Results.At baseline, amino acid transport was greater in ileum than jejunum. Surgery alone elevated glutamine and leucine jejunal transport by 130 and 97%, respectively, over controls (P< 0.05). Midgut resection increased jejunal glutamine transport 61% over control (P< 0.05). In contrast, distal resection increased jejunal alanine transport by 150% over controls with no change in glutamine (P< 0.05). After midgut resection, EGF-R was significantly greater (124%) in ileum then in jejunum in whole mucosa homogenates. Proximal resection significantly lowered ileal EGF-R compared to that seen in midgut resected residual ileum. Conclusions.Site of massive resection is important in determining postoperative changes in nutrient transport and EGF-R.

Glucocorticoids mediate skeletal muscle proteolysis during critical illness to provide substrates for hepatic acute-phase protein synthesis and gluconeogenesis. The effects of hypercortisolemia on splanchnic substrate uptake are not well defined. This study characterizes intestinal nutrient transport in response to acute elevations of plasma glucocorticoid levels. New Zealand White rabbits were randomized to receive either dexamethasone (2 mg/kg intramuscularly) or vehicle and were killed 8, 16, or 24 hours after steroid treatment. Brush-border membrane vesicles were prepared from pooled small intestinal mucosa and the uptake of tritiated substrates was quantified. Serum insulin-like growth factor 1 (IGF-1) levels, mucosal DNA content, and mucosal morphology were determined. Glucocorticoids increased glucose and leucine uptake at 8 hours (80% and 24%, respectively) and 24 hours (147% and 50%, respectively). Glutanmine, alanine, and arginine transport increased by 42%, 96%, and 236%, respectively, at 24 hours. Sodium-independent transport (diffusion) of all substrates was increased by 240% by dexamethasone treatment at 24 hours. Mucosal DNA content increased by 32%, whereas microvillus heights decreased by 27% at 24 hours. No effects were noted on IGF-1 levels or gross villus heights. Glucocorticoids acutely accelerate intestinal nutrient transport in a time-related and substrate-specific fashion. Although the mechanism of glucocorticoid action remains unclear, both genomic and plasma membrane effects are implicated.

Morphological and physiological adaptation in residual small intestine occurs after massive enterectomy and is influenced significantly by different growth factors and hormones. The mechanism of adaptation occurs through hypertrophy and hyperplasia as well as nutrient transporter changes. These transporters are classified into different classes dependent on its biological properties. The adaptation process evolves over time and different nutrient absorption profiles occur at different postoperative stages. There is an initial decrease in amino acid transport after resection followed by a return to approximately normal levels. Glucose also follows a similar pattern of changes but returns to normal later than amino acids. The time course of these changes are different for different animals with rat adaptation being much faster than rabbit. Growth hormone (GH) induces increased amino acid transport during this adaptation period, however, appears not to affect small intestine hypertrophy or hyperplasia. The increase in transport occurs via an increase in transport numbers rather than affinity. Epidermal growth factor (EGF) also increases amino acid transport in postoperative animals. Its advantage is it is orally stable when given with a protease inhibitor. EGF also reverses the down-regulating effects of the somatostatin analogue Octreotide (SMS) post resection. EGF in combination with GH has additive effects. However, the effects of the growth factors are site specific. GH and EGF combination therapy significantly increased alanine and arginine transport in distal small bowel after 70 % enterectomy but not in the proximal small bowel. The same combination increases leucine and glutamine transport in the proximal small intestine only. Understanding the specific changes that occur with these therapies may improve quality of life for patients and also reduce that need for total parenteral nutrition.

Studies in animals with short bowel syndrome (SBS) suggest that up-regulation of nutrient transporter activity occurs as an adaptive response to the loss of absorptive area. It is unclear, however, whether nutrient transport is altered at the cell membrane in SBS. The purpose of this study is to clarify amino acid and glucose transport in small intestinal luminal mucosa after 70% small bowel resection in rabbits.New Zealand white rabbits underwent 70% jejunoileal resection (n = 27) or a sham operation (n = 19). Brush border membrane vesicles were prepared from small intestinal mucosa at 1 week, 1 month, and 3 months by magnesium aggregation-differential centrifugation. Transport of L-glutamine, L-alanine, L-leucine, L-arginine, and D-glucose was assayed by a rapid mixing-filtration technique.We observed no difference in uptake of all amino acids and glucose at 1 week. The uptake of amino acids and glucose was decreased by 20% to 80% in animals with SBS at 1 month. By 3 months all uptake values except that of glucose returned to normal. Kinetic studies of the system B transporter for glutamine indicate that the decrease in uptake at 1 month was caused by a reduction in the Vmax (1575 +/- 146 versus 2366 +/- 235, p0.05) consistent with a decrease in the number of functional carriers on the brush border membrane.In addition to the anatomic loss of absorptive area after massive bowel resection, alterations in enterocyte transport function may be responsible for malabsorption in patients with SBS.

This study evaluated whether GM2 ganglioside storage is necessary for neurodegeneration and neuroinflammation by performing β-hexosaminidase rescue experiments in neurons of HexB−/− mice. We developed a novel mouse model, whereby the expression of the human HEXB gene was targeted to neurons of HexB−/− mice by the Thy1 promoter. Despite β-hexosaminidase restoration in neurons was sufficient in rescuing HexB−/− mice from GM2 neuronal storage and neurodegeneration, brain inflammation persisted, including the presence of large numbers of reactive microglia/macrophages due to persisting GM2 presence in this cell type. In conclusion, our results suggest that neuroinflammation is not sufficient to elicit neurodegeneration as long as neuronal function is restored.

The chicken (Gallus gallus) is an important model organism in genetics, developmental biology, immunology, evolutionary research, and agricultural science. The completeness of the draft chicken genome sequence provided new possibilities to study genomic changes during evolution by comparing the chicken genome to that of other species. The development of long oligonucleotide microarrays based on the genome sequence made it possible to survey genome-wide gene expression in chicken. This thesis describes two gene expression surveys across a range of healthy chicken tissues in both adult and embryonic stages. Specifically, we focus on the mechanisms of regulation of gene transcription and their evolution in the vertebrate genome. Chapter 1 provides a brief history of the chicken as a model organism in biological and genomics research. In particular a brief overview is presented about expression profiling experiments, followed by an introduction to gene transcription regulation in general. Finally, the aim and outline of this thesis is presented. An important aim of this thesis is to generate surveys of genome-wide gene expression data in chicken using microarrays. In chapter 2, we introduce microarray data normalization including background correction, within-array normalization and between-array normalization. Based on these results an analysis approach is recommended for the analysis of two-color microarray data as performed in the experiments described in this thesis. We also briefly explain the relevant methodology for the identification of differentially expressed genes and how to translate resulting gene lists into biological knowledge. Finally, specific issues related to updating microarray probe annotation in farm animals, is discussed. For the analysis of the microarray data in this thesis re-annotation of the probes on the chicken 20K oligoarray was done using the oligoRAP, analysis pipeline. The vast amount of data generated from a single transcriptomics study makes it impossible to extract meaningful biological knowledge by manually going through individual genes from a list with hundreds and thousands of differentially expressed genes. In chapter 3, we present a practical approach using a collection of R/Bioconductor packages to extract biological knowledge from a microarray experiment in farm animals. Furthermore, a locally adaptive statistical procedure (LAP) analysis approach is used to identify differentially expressed chromosomal regions in a microarray experiment. Chapter 4 presents a genome-wide gene expression survey across eight different tissues (brain, bursa of Fabricius, kidney, liver, lung, small intestine, spleen, and thymus from 10-week old chickens) in adult birds using a chicken 20K microarray. To a certain extent, most genes show some tissue-specific pattern of expression. Housekeeping and tissue-specific genes are identified based on gene expression patterns across the eight different tissues. The results show that housekeeping genes are more compact, i.e. are smaller, with shorter, coding sequence length, intron length, and smaller length of the intergenic regions. This observed compactness of housekeeping genes may be a result of selection on economy of transcription during evolution. Furthermore, a comparative analysis of gene expression among mouse, chicken, and frog showed that the expression patterns of orthologous genes are conserved during evolution between mammals, birds, and amphibians. The chicken embryo has been a very popular model for developmental biology. To study the overall gene expression pattern in whole chicken embryos at different developmental stages and/or embryonic tissues, a genome-wide gene expression survey across different developmental and embryonic stages was performed (chapter 5). The study included four different developmental stages (HH stage 3, 10, 15, 22) and eight different embryonic tissues (brain, bursa of Fabricius, heart, kidney, liver, lung, small intestine, and spleen from HH stage 36). We were able to identify several embryonic stage- and tissue-specific genes in our analysis. Genomic features of genes widely expressed under these 12 conditions suggest that widely expressed genes are more compact than tissue-specific genes, confirming the findings described in chapter 4. The analysis of the differentially expressed genes during the different developmental stages of whole embryo indicates a gradual change in gene expression during embryo development. A comparison of the gene expression profiles between the same organs, of adults and embryos reveals both striking similarities as well as differences. The overall goal of this thesis was to improve our understanding of the mechanisms of transcriptional regulation in the chicken. In chapter 6, a transcriptome map for all chicken chromosomes is presented based on the expression data described in chapter 4. The results reveal the presence of two distinct types of chromosomal regions characterized by clusters of highly or lowly expressed genes respectively. Furthermore, these regions show a high correlation with a number of genome characteristics, like gene density, gene length, intron length, and GC content. A comparative analysis between the chicken and human transcriptome maps suggests that the regions with clusters of highly expressed genes are relatively conserved between the two genomes. Our results revealed the presence of a higher order organization of the chicken genome that affects gene expression, confirming similar observations in other species. Finally, in chapter 7 I summarize the main findings and discuss some of the limitations of the analyses described in this thesis. I also discuss the different merits and shortcomings of studying gene expression using either microarrays or next-generation sequencing technology and propose directions for future research. The rapid developments in new-generation sequencing technology will facilitate better coverage and depth of the chicken genome. This will provide a better genome assembly and an improved genome annotation. The sequence-based approaches for studying gene expression will reduce noise levels compared to hybridization-based approaches. Overall, next-generation sequencing is already providing greatly enhance tools to further improve our understanding of the chicken transcriptome and its regulation.