Forensic issues in pain: review of current practice.

Forensic activity in pain practice is reviewed with reference to the differing roles of the pain clinician and the independent expert. Ethical guidelines and recommendations for assessment, documentation, record review, and court testimony are discussed. Specific issues include the assessment of disability and impairment, malingering, and application of the Daubert standard in forensic pain practice. Examples of case law are reviewed for civil liability and CRPS, malpractice with opioid prescribing, and practice issues in a correctional setting.

Telomerase RNA template mutations reveal sequence-specific requirements for the activation and repression of telomerase action at telomeres.

Telomeric DNA is maintained within a length range characteristic of an organism or cell type. Significant deviations outside this range are associated with altered telomere function. The yeast telomere-binding protein Rap1p negatively regulates telomere length. Telomere elongation is responsive to both the number of Rap1p molecules bound to a telomere and the Rap1p-centered DNA-protein complex at the extreme telomeric end. Previously, we showed that a specific trinucleotide substitution in the Saccharomyces cerevisiae telomerase gene (TLC1) RNA template abolished the enzymatic activity of telomerase, causing the same cell senescence and telomere shortening phenotypes as a complete tlc1 deletion. Here we analyze effects of six single- and double-base changes within these same three positions. All six mutant telomerases had in vitro enzymatic activity levels similar to the wild-type levels. The base changes predicted from the mutations all disrupted Rap1p binding in vitro to the corresponding duplex DNAs. However, they caused two classes of effects on telomere homeostasis: (i) rapid, RAD52-independent telomere lengthening and poor length regulation, whose severity correlated with the decrease in in vitro Rap1p binding affinity (this is consistent with loss of negative regulation of telomerase action at these telomeres; and (ii) telomere shortening that, depending on the template mutation, either established a new short telomere set length with normal cell growth or was progressive and led to cellular senescence. Hence, disrupting Rap1p binding at the telomeric terminus is not sufficient to deregulate telomere elongation. This provides further evidence that both positive and negative cis-acting regulators of telomerase act at telomeres.

Telomerase: Dr Jekyll or Mr Hyde?

The past year has seen the ectopic expression of human telomerase and the consequent increased replicative lifespan of cells, whereas mice lacking telomerase have lived and reproduced for six generations. Core telomerase activity from various organisms was reconstituted in vitro, yet how its action is regulated remains largely unknown. Telomerase activation preceded oncogenic transformation in some human cell types, yet was lacking in other transformed cells. These advances highlight the potentials of telomerase-based therapeutics and warn of their pitfalls.

Sequence-mediated regulation of adenovirus gene expression by repression of mRNA accumulation.

Gene expression in complex transcription units can be regulated at virtually every step in the production of mature cytoplasmic mRNA, including transcription initiation, elongation, termination, pre-mRNA processing, nucleus-to-cytoplasm mRNA transport, and alterations in mRNA stability. We have been characterizing alternative poly(A) site usage in the adenovirus major late transcription unit (MLTU) as a model for regulation at the level of pre-mRNA 3'-end processing. The MLTU contains five polyadenylation sites (L1 through L5). The promoter proximal site (L1) functions as the dominant poly(A) site during the early stage of adenovirus infection and in plasmid transfections when multiple poly(A) sites are present at the 3' end of a reporter plasmid. In contrast, stable mRNA processed at all five poly(A) sites is found during the late stage of adenovirus infection, after viral DNA replication has begun. Despite its dominance during early infection, L1 is a comparatively poor substrate for 3'-end RNA processing both in vivo and in vitro. In this study we have investigated the basis for the early L1 dominance. We have found that mRNA containing an unprocessed L1 poly(A) site is compromised in its ability to enter the steady-state pool of stable mRNA. This inhibition, which affects either the nuclear stability or nucleus-to-cytoplasm transport of the pre-mRNA, requires a cis-acting sequence located upstream of the L1 poly(A) site.

Varied poly(A) site efficiency in the adenovirus major late transcription unit.

Regulation of adenovirus major late transcription unit (MLTU) mRNA biosynthesis involves poly(A) site selection between five available sites, L1 through L5. The 5' proximal site completely dominates during early infection, whereas all five sites are used during late infection with L3 being favored slightly over the others. Previous studies have shown this early to late poly(A) switch will occur in the absence of MLTU-specific splicing patterns and hinges in large part on the character of the first poly(A) site. We have used in vitro assays to characterize basic features of the L1 and L3 pre-mRNAs which may help define how processing at poly(A) sites is controlled. We have found that L1 is 5-10-fold less efficient than L3 as a substrate for RNA cleavage. A primary difference between the L1 and L3 sites lies in the kinetics of their use, with cleavage at L3 occurring at twice the rate of cleavage at L1. In addition, L1 is 20-fold less effective than L3 in competing for processing factors. To investigate the sequence elements that contribute to poly(A) site efficiency, we have used competition assays in which the competitor RNAs lack upstream or downstream elements.