MicroRNA co-expression networks exhibit increased complexity in pancreatic ductal compared to Vater’s papilla adenocarcinoma

iRNA expression abnormalities in adenocarcinoma arising from pancreatic ductal system (PDAC) and Vater’s papilla (PVAC) could be associated with distinctive pathologic features and clinical cancer behaviours. Our previous miRNA expression profiling data on PDAC (n=9) and PVAC (n=4) were revaluated to define differences/ similarities in miRNA expression patterns. Afterwards, in order to uncover target genes and core signalling pathways regulated by specific miRNAs in these two tumour entities, miRNA interaction networks were wired for each tumour entity, and experimentally validated target genes underwent pathways enrichment analysis. One hundred and one miRNAs were altered, mainly over-expressed, in PDAC samples. Twenty-six miRNAs were deregulated in PVAC samples, where more miRNAs were down-expressed in tumours compared to normal tissues. Four miRNAs were significantly altered in both subgroups of patients, while 27 miRNAs were differentially expressed between PDAC and PVAC. Although miRNA interaction networks were more complex and dense in PDAC than in PVAC, pathways enrichment analysis uncovered a functional overlapping between PDAC and PVAC. However, shared signalling events were influenced by different miRNA and/or genes in the two tumour entities. Overall, specific miRNA expression patterns were involved in the regulation of a limited core signalling pathways in the biology landscape of PDAC and PVAC

Integration of bovine herpesvirus 4 genome into cultured persistently infected host cell genome

Persistent infection of macrophages with bovine herpesvirus 4 (BoHV-4) has been proposed to play a secondary causal role, along with bacterial infection, in bovine post-partum metritis. Mechanisms of maintenance of BoHV-4 persistent infection are not understood. We previously generated in vitro models of BoHV-4 persistent infection in human rhadomyosarcoma and bovine macrophage cell lines by drug selection of cells infected with BoHV-4 carrying a drug-resistance marker, and demonstrated circular episomal BoHV-4 genomes. In the present study, we used fluorescent in situ hybridization (FISH) to demonstrate BoHV-4 genomes also integrated into the genomes of these persistently infected cells

Virally and physically transgenized equine adipose-derived stromal cells as a cargo for paracrine secreted factors

<p>Abstract</p> <p>Background</p> <p>Adipose-Derived Stromal Cells have been shown to have multiple lineage differentiation properties and to be suitable for tissues regeneration in many degenerative processes. Their use has been proposed for the therapy of joint diseases and tendon injuries in the horse. In the present report the genetic manipulation of Equine Adipose-Derived Stromal Cells has been investigated.</p> <p>Results</p> <p>Equine Adipose-Derived Stromal Cells were successfully virally transduced as well as transiently and stably transfected with appropriate parameters, without detrimental effect on their differentiation properties. Moreover, green fluorescent protein alone, fused to <it>neo </it>gene, or co-expressed as bi-cistronic reporter constructs, driven by viral and house-keeping gene promoters, were tested. The better expressed cassette was employed to stably transfect Adipose-Derived Stromal Cells for cell therapy purposes. Stably transfected Equine Adipose-Derived Stromal Cells with a heterologous secreted viral antigen were able to immunize horses upon injection into the lateral wall of the neck.</p> <p>Conclusion</p> <p>This study provides the methods to successfully transgenize Adipose-Derived Stromal Cells both by lentiviral vector and by transfection using optimized constructs with suitable promoters and reporter genes. In conclusion these findings provide a working platform for the delivery of potentially therapeutic proteins to the site of cells injection via transgenized Equine Adipose-Derived Stromal Cells.</p

In Vivo Imaging of Transiently Transgenized Mice with a Bovine Interleukin 8 (CXCL8) Promoter/Luciferase Reporter Construct

One of the most remarkable properties of interleukin 8 (CXCL8/IL-8), a chemokine with known additional functions also in angiogenesis and tissue remodeling, is the variation of its expression levels. In healthy tissues, IL-8 is barely detectable, but it is rapidly induced by several folds in response to proinflammatory cytokines, bacterial or viral products, and cellular stress. Although mouse cells do not bear a clear homologous IL-8 gene, the murine transcriptional apparatus may well be capable of activating or repressing a heterologous IL-8 gene promoter driving a reporter gene. In order to induce a transient transgenic expression, mice were systemically injected with a bovine IL-8 promoter–luciferase construct. Subsequently mice were monitored for luciferase expression in the lung by in vivo bioluminescent image analysis over an extended period of time (up to 60 days). We demonstrate that the bovine IL-8 promoter–luciferase construct is transiently and robustly activated 3–5 hours after LPS and TNF-α instillation into the lung, peaking at 35 days after construct delivery. Bovine IL-8 promoter–luciferase activation correlates with white blood cell and neutrophil infiltration into the lung. This study demonstrates that a small experimental rodent model can be utilized for non-invasively monitoring, through a reporter gene system, the activation of an IL-8 promoter region derived from a larger size animal (bovine). This proof of principle study has the potential to be utilized also for studying primate IL-8 promoter regions

Bovine endometrial stromal cells display osteogenic properties

The endometrium is central to mammalian fertility. The endometrial stromal cells are very dynamic, growing and differentiating throughout the estrous cycle and pregnancy. In humans, stromal cells appear to have progenitor or stem cell capabilities and the cells can even differentiate into bone. It is not clear whether bovine endometrial stromal cells exhibit a similar phenotypic plasticity. So, the present study tested the hypothesis that bovine endometrial stromal cells could be differentiated along an osteogenic lineage. Pure populations of bovine stromal cells were isolated from the endometrium. The endometrial stromal cell phenotype was confirmed by morphology, prostaglandin secretion, and susceptibility to viral infection. However, cultivation of the cells in standard endometrial cell culture medium lead to a mesenchymal phenotype similar to that of bovine bone marrow cells. Furthermore, the endometrial stromal cells developed signs of osteogenesis, such as alizarin positive nodules. When the stromal cells were cultured in a specific osteogenic medium the cells rapidly developed the characteristics of mineralized bone. In conclusion, the present study has identified that stromal cells from the bovine endometrium show a capability for phenotype plasticity similar to mesenchymal progenitor cells. These observations pave the way for further investigation of the mechanisms of stroma cell differentiation in the bovine reproductive tract

Generation of molecular tools for Bluetongue virus (BTV) diagnosis and immunization

ABSTRACT Blutongue virus (BTV), the prototype member of the genus Orbivirus in the family Reoviridae, is an insect-borne virus that infects domestic and wild ruminants, causing a non contagious infectious disease spread by biting midges of some species of Culicoides genus, known as Bluetongue (BT). Historically, BT occurs most commonly and seriously in sheep and in some species of wild ruminants, occasionally in goats and rarely in cattle. In 2006, BTV serotype 8 (BTV-8) emerged in Norhern Europe followed by a still ongoing epidemic; the BTV-8 occurrence is remarkable for several reasons and, particularly, for its virulence not only in sheep between domestic ruminants. Currently, BTV, and particularly BTV-8, is responsible of greet farming industries losses; for this reason it is critical to have effective molecular tools to define standardized, specific and sensitive serodiagnostic assay and successful and economic prophilatic immunization plans. BTV is a double-stranded RNA (dsRNA) virus and its genome consists of 10 dsRNA segments that encode 4 nonstructural proteins (NS1-NS3 and NS3A) and 7 structural proteins (VP1-VP7). Among the 7 structural proteins, VP2, the outermost viral protein, is the cellular binding protein, elicitis virus-neutralizating antibody, and is responsible for hemagglutinin activity and serotype specifity. Although VP2 has been expressed successfully throught many systems and particularly with insect cell-baculovirus expression system, its paracrin expression as a soluble form in mammalian cells represents a difficult task. In this work we have investigared a mammalian expression platform for BTV VP2 production as a soluble form, generating several expression vector plasmids with BTV-8 VP2 sequence, whole or fragmented, by fusion peptides strategy. Starting from the assumption that VP2 is strongly cell associated, the full length of VP2 ORF was sub-cloned in frame with the immunoglobulin kappa light chain (IgK) signal peptide specifying secretion of heterologous proteins, generating pIgkVP2. In order to attempting the expression of BTV-8 VP2 with pIgkVP2 vector in mammalian cells and due to the lack of a suitable monoclonal antibody against BTV-8 VP2, a tag soluble epitope peptide belonging to the Bovine herpesvirus 1 (BoHV-1) glycoprotein D (gD) ectodomain was generated, obtaing pIgkVP2gD106. After electroporation of HEK 293T cells, we found that VP2 was well expressed from the tranfected cells, but not secreted into the medium, showing that introduction of a heterologous signal peptide in the amino-terminus of VP2 is not sufficient to specify its secretion. In previous studies was demonstrated that BTV VP2 associates with Vimentin cytoskeleton protein, and deletions of amino acids between residues 65 and 114 into VP2 sequence have shown to disrupt VP2-vimentin association. This aspect could be the cause of the missing VP2 secretion. Thus, new plasmid vectors was generated with different fragments of VP2 in frame upstream with Igk signal peptide, and downstream with gD106 tag; although all VP2 fragments are found in cell fraction, none is secreted into the medium. Based on the fact that BVDV gE2 ectodomain was very well expressed and secreted by pIgK-E2, gE2 ectodomain was exploited as a leader sequence to get VP2 and derived fragments secreted into the medium of transfected cells. Therefore, VP2 and derived fragments were sandwiched between IgkE2 and gD106 tag. We found that two constructs, pIgkE2VP2720-1425gD106 and pIgkE2VP22070-2883gD106, successfully expressed and secreted the VP2 fragments. Finally, BoHV-4 U ΔORF50 strain cloned as a bacterial artificial chromosome (BAC) was engineered to express VP22070-2883. Thus, we inserted the IgkE2VP22070-2883gD106 expression cassette into IE2 gene of a mutant BoHV-4 U ΔORF50 strain, in which IE2 locus is duplicated and one of them is inactivated by the insertion of 2004 bp KanaGalK DNA sequence stuffer double selecting cassette. So we obtained a recombinant BoHV-4 strain virus-vector able to yeald hight quantity of soluble VP22070-2883gD106 fragment from infected cells, which could be employed for immunodiagnostic assay development or vaccine purposes

Bovine herpesvirus 4 glycoprotein B is indispensable for lytic replication and irreplaceable by VSVg

Abstract Background Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus, belonging to Rhadinovirus genus, with no clear association with disease. However, there is increasing evidence of its secondary pathogenic role in cases of post-partum metritis in cattle. BoHV-4 Open Reading Frame 8 (ORF8) codifies for glycoprotein B (gB) that shows a heterodimeric structure, composed of two subunits and covalently linked by disulfide bonds and responsible for host cell adhesion through binding to heparan sulfates associated with cellular proteoglycans. Here we describe the generation of several tagged soluble forms of gB ectodomain, in order to test their ability to neutralize BoHV-4 infection. Results The results show, however, that none of these soluble forms are able to block viral infectivity. To better understand the role of gB during BoHV-4 lytic replication, a recombinant BoHV-4 was generated by homologous recombination from a BoHV-4 cloned as a Bacterial artificial chromosome (BAC) (pBAC-BoHV-4-A), in which most of the BoHV-4 gB ORF was substituted by the insertion of a DNA stuffer selectable cassette. The resulting recombinant BoHV-4 genome (pBAC-BoHV-4-AΔgB-KanaGalK) was completely unable to reconstitute infectious replicating viral particles (Infectious Replicating Viral Particles, IRVPs) and to replicate when transfected in permissive cell lines in comparison to its revertant clone (pBAC-BoHV-4-ΔgB-Rev) or pBAC-BoHV-4-A parental clone. Conclusion This demonstrates that the BoHV-4 replicating cycle is dependent on gB. Moreover, when gB was deleted from a recombinant BoHV-4 genome delivering an heterologous glycoprotein, Vesicular Stomatitis Virus Glycoprotein (VSVg), VSVg was unable to complement gB. This study provides direct evidence that gB is necessary for BoHV-4 lytic replication.</p

旋风进料落膜式直流转移弧型等离子体冶炼炉(plasmacan)的一种优化试验途径

在对装置作实验研究时,如果该装置可调节的参数很多,则如何通过最少次数的试验,以实现设计意图,发现装置的最佳工况就成了突出的问题。本文从分析原料粒子在 Plasmacan 中的两个特征时间尺度入手(它们是:①粒子的实际停留时间;②粒子为完成设计中预期的反应而必须停留的最短时间),基于在两个不同层次上能量平衡的考虑(从全体粒子这个层次以及从单个粒子这个较细的层次),对设计及实验中可调节参数的影响作了分析与研究;并在此基础上提出了一个推荐的试验流程。按此流程来安排实验研究,可望通过较少次数的实验为 Pla smacan 找出最佳工况.该建议的实质是:对可以用大致合理的理论模型找出定性(或定量)解答的影响因素,就尽量靠理论来研究;只对理论在目前实在难回答的那些因素,才安排必要次数的实验研究。用这样一种理论与实验研究相结合,彼此杨长避短的策略来大大减少实验研究的次数

The chemokine IL8 is up-regulated in bovine endometrial stromal cells by the BoHV-4 IE2 gene product, ORF50/Rta: A step ahead toward a mechanism for BoHV-4 induced endometritis.

Abstract Postpartum infections of the endometrium and metritis are common causes of delayed conception and infertility in cattle. These infections are characterized by inflammation of the endometrium and secretion of the chemokine interleukin 8 (IL8), which attracts granulocytes to the endometrium. Bovine herpesvirus 4 (BoHV-4) is tropic for the endometrium, and the only virus consistently associated with postpartum metritis. The BoHV-4 IE2 gene is the first viral gene transcribed by host cells after infection and the IE2 gene product, ORF50/Rta, transactivates host cell genes. The present study tested the hypothesis that ORF50/Rta transactivates the IL8 gene promoter during BoHV-4 infection of bovine endometrial stromal cells (BESCs). Infection of primary BESCs with BoHV-4 stimulated IL8 gene promoter activity and IL8 protein secretion. However, IL8 production was dependent on the transcription of viral genes as psoralen/UV crosslinking of the viral DNA abrogated the response to BoHV-4 infection. Furthermore, IL8 promoter serial deletion analysis revealed a specific region responsive to ORF50/Rta. These observations may represent an endometrial defense mechanism against viral infection or a virulence mechanism by which viral replication stimulates chemokine secretion to attract more susceptible host cells to the endometrium

Efficient heterologous antigen gene delivery and expression by a replication-attenuated BoHV-4-based vaccine vector

Bovine Herpesvirus 4 (BoHV-4) is a gammaherpesvirus belonging to the Rhadinovirus genus and due to its biological characteristics has been proposed as a vaccine vector for veterinary vaccines. Because viral vector-associated risk is a major concern for viral vector applications, attenuation is a desirable feature. Therefore, efforts are directed toward the development of highly attenuated viral vectors. BoHV-4 naturally exhibits limited pathogenicity and a further attenuation, in terms of replication, was obtained by disrupting the late gene encoding the 1.7-kb polyadenylated RNA (L1.7). An L1.7 deleted mutant BoHV-4 (BoHV-4-A-KanaGalKΔL1.7), as well as its revertant (BoHV-4-A-Rev), was generated by homologous recombination from the genome of a BoHV-4 isolate (BoHV-4-A) cloned as a bacterial artificial chromosome (BAC). BoHV-4-A-KanaGalKΔL1.7 showed attenuation in terms of competence to reconstitute infectious virus, viral replication, and plaque size when compared to BoHV-4-A, BoHV-4-A-Rev, and BoHV-4-A-KanaGalKΔTK, a recombinant control virus where the KanaGalK selectable marker was inserted into the thymidine kinase open reading frame. The capability of BoHV-4-A-KanaGalKΔL1.7 to deliver and express a heterologous antigen was investigated by replacing the KanaGalK cassette with a vesicular stomatitis virus glycoprotein (VSVg) expression cassette to generate BoHV-4-A-EF1αVSVgΔL1.7. BoHV-4-A-EF1αVSVgΔL1.7 infected cells robustly expressed VSVg, thus confirming that the replication deficiency resulting from L1.7 disruption did not prevent heterologous gene delivery and expression. Although further work is needed to identify the specific function of the BoHV-4 L1.7 gene, the L1.7 gene may represent an ideal targeting locus for the integration of a heterologous antigen expression cassette, resulting in attenuation of the viral vector