RNA-binding proteins to assess gene expression states of co-cultivated cells in response to tumor cells

BACKGROUND: Tumors and complex tissues consist of mixtures of communicating cells that differ significantly in their gene expression status. In order to understand how different cell types influence one another's gene expression, it will be necessary to monitor the mRNA profiles of each cell type independently and to dissect the mechanisms that regulate their gene expression outcomes. RESULTS: In order to approach these questions, we have used RNA-binding proteins such as ELAV/Hu, poly (A) binding protein (PABP) and cap-binding protein (eIF-4E) as reporters of gene expression. Here we demonstrate that the epitope-tagged RNA binding protein, PABP, expressed separately in tumor cells and endothelial cells can be used to discriminate their respective mRNA targets from mixtures of these cells without significant mRNA reassortment or exchange. Moreover, using this approach we identify a set of endothelial genes that respond to the presence of co-cultured breast tumor cells. CONCLUSION: RNA-binding proteins can be used as reporters to elucidate components of operational mRNA networks and operons involved in regulating cell-type specific gene expression in tissues and tumors

Overlapping Antisense Transcription in the Human Genome

Accumulating evidence indicates an important role for non-coding RNA molecules in eukaryotic cell regulation. A small number of coding and non-coding overlapping antisense transcripts (OATs) in eukaryotes have been reported, some of which regulate expression of the corresponding sense transcript. The prevalence of this phenomenon is unknown, but there may be an enrichment of such transcripts at imprinted gene loci. Taking a bioinformatics approach, we systematically searched a human mRNA database (RefSeq) for complementary regions that might facilitate pairing with other transcripts. We report 56 pairs of overlapping transcripts, in which each member of the pair is transcribed from the same locus. This allows us to make an estimate of 1000 for the minimum number of such transcript pairs in the entire human genome. This is a surprisingly large number of overlapping gene pairs and, clearly, some of the overlaps may not be functionally significant. Nonetheless, this may indicate an important general role for overlapping antisense control in gene regulation. EST databases were also investigated in order to address the prevalence of cases of imprinted genes with associated non-coding overlapping, antisense transcripts. However, EST databases were found to be completely inappropriate for this purpose

Distal chromosome 17q loss in Barrett's esophageal and gastric cardia adenocarcinomas: Implications for tumorigenesis

The molecular genetic mechanisms underlying esophageal cancer are poorly understood. However, a novel gene that may be involved in esophageal carcinogenesis was recently localized by others to distal 17q by linkage analysis of kindreds with palmoplantar keratoderma and squamous cell carcinoma of the esophagus. To help determine whether a distal 17q gene may also be involved in the pathogenesis of primary Barrett's esophageal and gastric cardia adenocarcinomas, we performed loss of heterozygosity (LOH) analysis of 21 Barrett's and 18 gastric cardia adenocarcinomas at loci spanning 17q: cen— BRCA1—SSTR2—D17S2058—D17S929—D17S722—D17S937—D17S802— tel. Over 50% of the Barrett's and cardia adenocarcinomas demonstrated loss of an allele at one or more informative distal 17q markers. One common overlapping region of loss involved loci mapped to distal 17q24–proximal 17q25, which tentatively defines a potential chromosomal region distal to BRCA1 involved in the pathogenesis or progression of both types of adenocarcinomas. LOH analysis of DNA from matched microdissected sections of Barrett's metaplasia suggested that loss of D17S2058 in this region may be an early event in the malignant transformation of Barrett's metaplasia. No statistically significant correlations between 17q LOH and tumor stage or patient survival were noted. In summary, LOH mapping of 17q in Barrett's and cardia adenocarcinomas suggests the existence of at least one putative distal 17q tumor suppressor gene involved in the pathogenesis of these tumors. Mol. Carcinog. 22:222–228, 1998. © 1998 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/35057/1/3_ftp.pd

Exploring the Transformative Effects of Service-Immersion Programs on Faculty: Current scholarship in experiential learning considers the influence of service immersion programming on students, but the impacts on faculty can be surprising – and just as profound.

Service-immersion programs are prevalent at many universities, often housed in ministry, mission, or service-learning departments. These experiences vary from domestic to international exploration, often lasting five to twelve days. Unlike study abroad programs that usually last a semester, service immersion “trips” are shorter and often include some sort of community service component. Current scholarship in experiential learning has focused on the influence of service immersion programming on undergraduate students. What is missing from this literature is the effect of immersion participation on adult accompaniers such as faculty. Since these adults share the same basic experience as the students, they may experience some of the transformative development as their students. Using Mezirow’s Transformative Learning Theory, as our guide, we explored three areas of transformative development  – the influence of the immersion experience on faculty themselves, how the experience affected the faculty members’ relationships with students, and how the experience affected classroom instruction. The results were positive as the participating faculty reported positive personal growth, a deeper understanding of their students, and an enhanced learning environment that included the classroom as well as additional opportunities such as mentoring.   &nbsp

Redefining 3D Bioprinting: Vertical Bio-Ink Deposition for Improved Biofabrication

A novel approach to Extrusion-based 3D printing named Vertical Pillar Extrusion (VPE) is proposed, wherein living cells are deposited vertically into a supportive substrate, diverging from the established horizontal deposition method. Traditional substrate-based 3D bio-printing methodologies primarily employ horizontal layering techniques. This innovative, low-cost technique entails the utilisation of a needle-equipped nozzle, minimising substrate disturbance by abstaining from horizontal movements within the medium. Bio-Ink injection occurs exclusively from the top, while extrusion is initiated upon needle retraction from the substrate. Extrusion during retraction mitigates the sheer stresses for the living component of the bio-inks. Various bio-ink nozzle types (tapered and straight) will be analysed to identify the optimal nozzle configuration that maximises the survival rate of the living components within the bio-ink. We aim to utilise this printing technique for bio-inks derived from algae and bacteria and for printing a bio-ink containing dissolved chitosan onto a substrate with an acetylating agent to fabricate 3D-printed chitin structures. The efficacy of this 3D printing technique is analysed via Computational Fluid Dynamics (CFD) simulations to predict the behaviour and interaction of the bio-ink with the substrate. Experimental validation of the results will be conducted to ensure the accuracy and reliability of the technique's practical applicability. The proposed methodology involves coding a new g-code generator, predicated on Digital Light Processing (DLP) slicer shutter images dictating the voxel locations for material deposition. We anticipate that this pioneering 3D printing technique will yield substantial enhancements in the precision of bio-ink deposition for the fabrication of 3D-printed engineered living materials

Overexpression of Aurora-A in primary cells interferes with S-phase entry by diminishing Cyclin D1 dependent activities.

Aurora-A is a bona-fide oncogene whose expression is associated with genomic instability and malignant transformation. In several types of cancer, gene amplification and/or increased protein levels of Aurora-A are a common feature. RESULTS: In this report, we describe that inhibition of cell proliferation is the main effect observed after transient overexpression of Aurora-A in primary human cells. In addition to the known cell cycle block at the G2/M transition, Aurora-A overexpressing cells fail to overcome the restriction point at the G1/S transition due to diminished RB phosphorylation caused by reduced Cyclin D1 expression. Consequently, overexpression of Cyclin D1 protein is able to override the Aurora-A mediated G1 block. The Aurora-A mediated cell cycle arrest in G2 is not influenced by Cyclin D1 and as a consequence cells accumulate in G2. Upon deactivation of p53 part of the cells evade this premitotic arrest to become aneuploid. CONCLUSION: Our studies describe that an increase of Aurora-A expression levels on its own has a tumor suppressing function, but in combination with the appropriate altered intracellular setting it might exert its oncogenic potential. The presented data indicate that deactivation of the tumor suppressor RB is one of the requirements for overriding a cell cycle checkpoint triggered by increased Aurora-A levels