Cristina Iftode

Replication Protein A (RPA): The Eukaryotic SSB

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein that is highly conserved in eukaryotes. RPA plays essential roles in many aspects of nucleic acid metabolism, including DNA replication, nucleotide excision repair, and homologous recombination. In this review, we provide a comprehensive overview of RPA structure and function and highlight the more recent developments in these areas. The last few years have seen major advances in our understanding of the mechanism of RPA binding to DNA, including the structural characterization of the primary DNA-binding domains (DBD) and the identification of two secondary DBDs. Moreover, evidence indicates that RPA utilizes a multistep pathway to bind single-stranded DNA involving a particular molecular polarity of RPA, a mechanism that is apparently used to facilitate origin denaturation. In addition to its mechanistic roles, RPA interacts with many key factors in nucleic acid metabolism, and we discuss the critical nature of many of these interactions to DNA metabolism. RPA is a phosphorylation target for DNA-dependent protein kinase (DNA-PK) and likely the ataxia telangiectasia-mutated gene (ATM) protein kinase, and recent observations are described that suggest that RPA phosphorylation plays a significant modulatory role in the cellular response to DNA damage.

Biomimetic hydrogels for tissue engineering of the intervertebral disc

Tissue engineering is a multidisciplinary field that aims to repair or regenerate lost or damaged tissues and organs in the body. One such area with significant medical applications is the degeneration of the intervertebral disc (IVD). The objective of this work is to generate a bioadhesive polymer that, in addition to bonding with tissue, can support and cell survival post-adhesion. A thermosensitive poly(N-isopropylacrylamide) (PNIPAAm) and chondroitin sulfate (CS) scaffold with aldehyde-modified CS adhesive and extracellular matrix (ECM) loaded lipid vesicles was examined as a potential minimally invasive method for repair and regeneration of degenerated IVD tissue. Samples containing varying percentages of aldehyde-modified CS and presence or absence of ECM loaded lipid vesicles were evaluated for physiological relevant performance with porcine skin. Maximum stress and work of adhesion were calculated for each polymer formulation based on force-distance data. Currently, work is being done to investigate the biocompatibility of various polymer compositions to optimize polymer blends for maximum work, stress and biocompatibility for use in vivo.

Tissue Engineering of the Intervertebral Disc

Tissue engineering is a rapidly growing field of research that aims to repair damaged tissues within the body. Among tissue engineering approaches is the use of scaffolds to help regenerate lost tissues. Scaffolds provide structural support for specific areas within the body, namely load bearing regions, and allow for cells to be seeded within the scaffold for tissue regeneration. Scaffolds that specifically replicate the properties and/or composition of native tissues are referred to as biomimetic scaffolds.Copyright

Analysis of the Efficiency of Adenovirus Transcription

This method is designed to measure rates of transcription from adenoviral promoters as a function of the concentrations within infected cells of the promoter(s) of interest. The latter parameter is assessed by quantification of viral DNA by hybridization of membrane-bound DNA following purification of DNA from nuclear fractions of adenovirus-infected cells. Two alternative protocols, primer extension and quantitative reverse transcription polymerase chain reaction, are described for determination of the concentrations of viral mRNAs purified from the cytoplasmic fractions of the same infected cell samples. An alternative procedure to measure rates of transcription directly using run-on transcription in isolated nuclei is also presented.