Immune response of healthy horses to DNA constructs formulated with a cationic lipid transfection reagent

Background Deoxyribonucleic acid (DNA) vaccines are used for experimental immunotherapy of equine melanoma. The injection of complexed linear DNA encoding interleukin (IL)-12/IL-18 induced partial tumour remission in a clinical study including 27 grey horses. To date, the detailed mechanism of the anti-tumour effect of this treatment is unknown. Results In the present study, the clinical and cellular responses of 24 healthy horses were monitored over 72 h after simultaneous intradermal and intramuscular application of equine IL-12/IL-18 DNA (complexed with a transfection reagent) or comparative substances (transfection reagent only, nonsense DNA, nonsense DNA depleted of CG). Although the strongest effect was observed in horses treated with expressing DNA, horses in all groups treated with DNA showed systemic responses. In these horses treated with DNA, rectal temperatures were elevated after treatment and serum amyloid A increased. Total leukocyte and neutrophil counts increased, while lymphocyte numbers decreased. The secretion of tumour necrosis factor alpha (TNFα) and interferon gamma (IFNγ) from peripheral mononuclear blood cells ex vivo increased after treatments with DNA, while IL-10 secretion decreased. Horses treated with DNA had significantly higher myeloid cell numbers and chemokine (C-X-C motif) ligand (CXCL)-10 expression in skin samples at the intradermal injection sites compared to horses treated with transfection reagent only, suggesting an inflammatory response to DNA treatment. In horses treated with expressing DNA, however, local CXCL-10 expression was highest and immunohistochemistry revealed more intradermal IL-12-positive cells when compared to the other treatment groups. In contrast to non-grey horses, grey horses showed fewer effects of DNA treatments on blood lymphocyte counts, TNFα secretion and myeloid cell infiltration in the dermis. Conclusion Treatment with complexed linear DNA constructs induced an inflammatory response independent of the coding sequence and of CG motif content. Expressing IL-12/IL-18 DNA locally induces expression of the downstream mediator CXCL-10. The grey horses included appeared to display an attenuated immune response to DNA treatment, although grey horses bearing melanoma responded to this treatment with moderate tumour remission in a preceding study. Whether the different immunological reactivity compared to other horses may contributes to the melanoma susceptibility of grey horses remains to be elucidated

A qualitative investigation of breast cancer survivors’ experiences with breastfeeding

This is an exploratory, qualitative investigation of breast cancer survivors’ experiences with breastfeeding. Previous studies have focused on the physiology of lactation after surgery and treatment, but have not explored factors influencing breastfeeding decisions and behavior. We used purposeful sampling to identify 11 breast cancer survivors who had a child after their diagnosis and treatment. Participants were recruited from among those in the Women’s Healthy Eating and Living (WHEL) study and a Young Survival Coalition (YSC) affiliate. We conducted semi-structured, open-ended telephone interviews lasting 45–75 min. We used social cognitive theory (SCT) to structure questions regarding influences on breastfeeding behavior. We transcribed interviews and used cross-case, inductive analysis to identify themes. Ten of 11 participants initiated breastfeeding. The following main themes emerged: 1) Cautiously hopeful, 2) Exhausting to rely on one breast, 3) Motivated despite challenges, 4) Support and lack of support, and 5) Encouraging to others. Study participants were highly motivated to breastfeed but faced considerable challenges. Participants described problems that are not unique to women with breast cancer, but experienced these to a much greater degree because they relied mostly or entirely on one lactating breast. This study revealed a need for improved access to information and support and greater sensitivity to the obstacles faced by breast cancer survivors. Results of this qualitative analysis indicate that interventions to support the efforts of breast cancer survivors who are interested in breastfeeding are warranted. Additional research would aid in the development of such interventions

A study of chronic pelvic pain after radiotherapy in survivors of locally advanced cervical cancer

Introduction: Chronic pelvic pain (persisting pain in hips, groins or lower back) is poorly described in studies of cervical cancer survivors (CCSs). The aims of this study were to describe chronic pelvic pain and associated variables in CCSs surveyed >5 years post-radiotherapy, and to compare the presence of pain in hips and lower back in CCSs with findings in the general female population. Methods: Ninety-one CCSs treated with radiotherapy between 1994 and 1999 were in 2005 included in a cross-sectional questionnaire-based study. They were asked about demographic variables, clinical symptoms, mental distress, and quality of life (QOL). Normative data (NORM) were collected from a population-study of Norwegian females. Results: Pain in lower back and hips was significantly more prevalent (p < .001) in CCSs compared to NORMs. 35/92 (38%) of the CCSs had chronic pelvic pain. These women had significantly lower QOL, higher levels of anxiety and depression and more bladder and intestinal problems than those without chronic pelvic pain. In a multivariable regression model, use of analgesics and intestinal and bladder problems were significantly associated with chronic pelvic pain in the CCSs. Conclusions: CCSs have a higher prevalence of pain in lower back and hips than women in the general population, which might be due to late effects of radiation. 35/92 (38%) of the CCSs suffer from chronic pelvic pain, shown to be associated with high overall mental and somatic morbidity. Implications for Cancer Survivors: Evaluation and management of pelvic pain is important in follow-up of CCSs treated with radiotherapy

Control of Tissue Growth and Cell Transformation by the Salvador/Warts/Hippo Pathway

The Salvador-Warts-Hippo (SWH) pathway is an important regulator of tissue growth that is frequently subverted in human cancer. The key oncoprotein of the SWH pathway is the transcriptional co-activator, Yes-associated protein (YAP). YAP promotes tissue growth and transformation of cultured cells by interacting with transcriptional regulatory proteins via its WW domains, or, in the case of the TEAD1-4 transcription factors, an N-terminal binding domain. YAP possesses a putative transactivation domain in its C-terminus that is necessary to stimulate transcription factors in vitro, but its requirement for YAP function has not been investigated in detail. Interestingly, whilst the WW domains and TEAD-binding domain are highly conserved in the Drosophila melanogaster YAP orthologue, Yorkie, the majority of the C-terminal region of YAP is not present in Yorkie. To investigate this apparent conundrum, we assessed the functional roles of the YAP and Yorkie C-termini. We found that these regions were not required for Yorkie's ability to drive tissue growth in vivo, or YAP's ability to promote anchorage-independent growth or resistance to contact inhibition. However, the YAP transactivation domain was required for YAP's ability to induce cell migration and invasion. Moreover, a role for the YAP transactivation domain in cell transformation was uncovered when the YAP WW domains were mutated together with the transactivation domain. This shows that YAP can promote cell transformation in a flexible manner, presumably by contacting transcriptional regulatory proteins either via its WW domains or its transactivation domain

FGF-2, TGF&#946;-1, PDGF-A and respective receptors expression in pleomorphic adenoma myoepithelial cells: an in vivo and in vitro study

Myoepithelial cells have an important role in salivary gland tumor development, contributing to a low grade of aggressiveness of these tumors. Normal myoepithelial cells are known by their suppressor function presenting increased expression of extracellular matrix genes and protease inhibitors. The importance of stromal cells and growth factors during tumor initiation and progression has been highlighted by recent literature. Many tumors result from the alteration of paracrine growth factors pathways. Growth factors mediate a wide variety of biological processes such as development, tissue repair and tumorigenesis, and also contribute to cellular proliferation and transformation in neoplastic cells. OBJECTIVES: This study evaluated the expression of fibroblast growth factor-2 (FGF-2), transforming growth factor &#946;-1 (TGF&#946;-1), platelet-derived growth factor-A (PDGF-A) and their respective receptors (FGFR-1, FGFR-2, TGF&#946;R-II and PDGFR-&#945;) in myoepithelial cells from pleomorphic adenomas (PA) by in vivo and in vitro experiments. MATERIAL AND METHODS: Serial sections were obtained from paraffin-embedded PA samples obtained from the school's files. Myoepithelial cells were obtained from explants of PA tumors provided by surgery from different donors. Immunohistochemistry, cell culture and immunofluorescence assays were used to evaluate growth factor expression. RESULTS: The present findings demonstrated that myoepithelial cells from PA were mainly positive to FGF-2 and FGFR-1 by immunohistochemistry and immunofluorescence. PDGF-A and PDGFR-&#945; had moderate expression by immunohistochemistry and presented punctated deposits throughout cytoplasm of myoepithelial cells. FGFR-2, TGF&#946;-1 and TGF&#946;R-II were negative in all samples. CONCLUSIONS: These data suggested that FGF-2 compared to the other studied growth factors has an important role in PA benign myoepithelial cells, probably contributing to proliferation of these cells through the FGFR-1

A Passerine Bird's Evolution Corroborates the Geologic History of the Island of New Guinea

New Guinea is a biologically diverse island, with a unique geologic history and topography that has likely played a role in the evolution of species. Few island-wide studies, however, have examined the phylogeographic history of lowland species. The objective of this study was to examine patterns of phylogeographic variation of a common and widespread New Guinean bird species (Colluricincla megarhyncha). Specifically, we test the mechanisms hypothesized to cause geographic and genetic variation (e.g., vicariance, isolation by distance and founder-effect with dispersal). To accomplish this, we surveyed three regions of the mitochondrial genome and a nuclear intron and assessed differences among 23 of the 30 described subspecies from throughout their range. We found support for eight highly divergent lineages within C. megarhyncha. Genetic lineages were found within continuous lowland habitat or on smaller islands, but all individuals within clades were not necessarily structured by predicted biogeographic barriers. There was some evidence of isolation by distance and potential founder-effects. Mitochondrial DNA sequence divergence among lineages was at a level often observed among different species or even genera of birds (5–11%), suggesting lineages within regions have been isolated for long periods of time. When topographical barriers were associated with divergence patterns, the estimated divergence date for the clade coincided with the estimated time of barrier formation. We also found that dispersal distance and range size are positively correlated across lineages. Evidence from this research suggests that different phylogeographic mechanisms concurrently structure lineages of C. megarhyncha and are not mutually exclusive. These lineages are a result of evolutionary forces acting at different temporal and spatial scales concordant with New Guinea's geological history

Xylella fastidiosa gene expression analysis by DNA microarrays

Xylella fastidiosa genome sequencing has generated valuable data by identifying genes acting either on metabolic pathways or in associated pathogenicity and virulence. Based on available information on these genes, new strategies for studying their expression patterns, such as microarray technology, were employed. A total of 2,600 primer pairs were synthesized and then used to generate fragments using the PCR technique. The arrays were hybridized against cDNAs labeled during reverse transcription reactions and which were obtained from bacteria grown under two different conditions (liquid XDM2 and liquid BCYE). All data were statistically analyzed to verify which genes were differentially expressed. In addition to exploring conditions for X. fastidiosa genome-wide transcriptome analysis, the present work observed the differential expression of several classes of genes (energy, protein, amino acid and nucleotide metabolism, transport, degradation of substances, toxins and hypothetical proteins, among others). The understanding of expressed genes in these two different media will be useful in comprehending the metabolic characteristics of X. fastidiosa, and in evaluating how important certain genes are for the functioning and survival of these bacteria in plants

Gene Expression Profiling of Preovulatory Follicle in the Buffalo Cow: Effects of Increased IGF-I Concentration on Periovulatory Events

The preovulatory follicle in response to gonadotropin surge undergoes dramatic biochemical, and morphological changes orchestrated by expression changes in hundreds of genes. Employing well characterized bovine preovulatory follicle model, granulosa cells (GCs) and follicle wall were collected from the preovulatory follicle before, 1, 10 and 22 h post peak LH surge. Microarray analysis performed on GCs revealed that 450 and 111 genes were differentially expressed at 1 and 22 h post peak LH surge, respectively. For validation, qPCR and immunocytochemistry analyses were carried out for some of the differentially expressed genes. Expression analysis of many of these genes showed distinct expression patterns in GCs and the follicle wall. To study molecular functions and genetic networks, microarray data was analyzed using Ingenuity Pathway Analysis which revealed majority of the differentially expressed genes to cluster within processes like steroidogenesis, cell survival and cell differentiation. In the ovarian follicle, IGF-I is established to be an important regulator of the above mentioned molecular functions. Thus, further experiments were conducted to verify the effects of increased intrafollicular IGF-I levels on the expression of genes associated with the above mentioned processes. For this purpose, buffalo cows were administered with exogenous bGH to transiently increase circulating and intrafollicular concentrations of IGF-I. The results indicated that increased intrafollicular concentrations of IGF-I caused changes in expression of genes associated with steroidogenesis (StAR, SRF) and apoptosis (BCL-2, FKHR, PAWR). These results taken together suggest that onset of gonadotropin surge triggers activation of various biological pathways and that the effects of growth factors and peptides on gonadotropin actions could be examined during preovulatory follicle development

Comparative 3D QSAR study on β1-, β2-, and β3-adrenoceptor agonists

A quantitative structure–activity relationship study of tryptamine-based derivatives of β1-, β2-, and β3-adrenoceptor agonists was conducted using comparative molecular field analysis (CoMFA). Correlation coefficients (cross-validated r2) of 0.578, 0.595, and 0.558 were obtained for the three subtypes, respectively, in three different CoMFA models. All three CoMFA models have different steric and electrostatic contributions, implying different requirements inside the binding cavity. The CoMFA coefficient contour plots of the three models and comparisons among these plots provide clues regarding the main chemical features responsible for the biological activity variations and also result in predictions which correlate very well with the observed biological activity. Based on the analysis, a summary regeospecific description of the requirements for improving β-adrenoceptor subtype selectivity is given

Evaluation of endogenous references for gene expression profiling in different tissues of the oriental fruit fly Bactrocera dorsalis (Diptera: Tephritidae)

<p>Abstract</p> <p>Background</p> <p>Quantitative real-time reverse transcriptase PCR (RT-qPCR) has been widely used for quantification of mRNA as a way to determine key genes involved in different biological processes. For accurate gene quantification analysis, normalization of RT-qPCR data is absolutely essential. To date, normalization is most frequently achieved by the use of internal controls, often referred to as reference genes. However, several studies have shown that the reference genes used for the quantification of mRNA expression can be affected by the experimental set-up or cell type resulting in variation of the expression level of these key genes. Therefore, the evaluation of reference genes is critical for gene expression profiling, which is often neglected in gene expression studies of insects. For this purpose, ten candidate reference genes were investigated in three different tissues (midgut, Malpighian tubules, and fat body) of the oriental fruit fly, <it>Bactrocera dorsalis </it>(Hendel).</p> <p>Results</p> <p>Two different programs, <it>geNorm </it>and <it>Normfinder</it>, were used to analyze the data. According to <it>geNorm</it>, α-TUB + ACT5 are the most appropriate reference genes for gene expression profiling across the three different tissues in the female flies, while ACT3 + α-TUB are considered as the best for males. Furthermore, we evaluated the stability of the candidate reference genes to determine the sexual differences in the same tissue. In the midgut and Malpighian tubules, ACT2 + α-TUB are the best choice for both males and females. However, α-TUB + ACT1 are the best pair for fat body. Meanwhile, the results calculated by <it>Normfinder </it>are quite the same as the results with <it>geNorm</it>; α-TUB is always one of the most stable genes in each sample validated by the two programs.</p> <p>Conclusions</p> <p>In this study, we validated the suitable reference genes for gene expression profiling in different tissues of <it>B. dorsalis. </it>Moreover, appropriate reference genes were selected out for gene expression profiling of the same tissues taking the sexual differences into consideration. This work not only formed a solid basis for future gene expression study in <it>B. dorsalis</it>, but also will serve as a resource to screen reference genes for gene expression studies in any other insects.</p