Cell-based and cell-free firefly luciferase complementation assay to quantify Human Immunodeficiency Virus type 1 Rev-Rev interaction.

Rev is an essential regulatory protein of Human Immunodeficiency Virus type 1 (HIV) that is found in the nucleus of infected cells. Rev multimerizes on the Rev-response element (RRE) of HIV RNA to facilitate the export of intron-containing HIV mRNAs from the nucleus to the cytoplasm, and, as such, HIV cannot replicate in the absence of Rev. We have developed cell-intact and cell-free assays based upon a robust firefly split-luciferase complementation system, both of which quantify Rev-Rev interaction. Using the cell-based system we show that additional Crm1 did not impact the interaction, whereas excess Rev reduced it. Furthermore, when a series of mutant Revs were tested, there was a strong correlation between the results of the cell-based assay and the results of a functional Rev trans-complementation infectivity assay. Of interest, a camelid nanobody (NB) that was known to inhibit Rev function enhanced Rev-Rev interaction in the cell-based system. We observed a similar increase in Rev-Rev interaction in a cell-free system, when cell lysates expressing Rev-NLUC or CLUC-Rev were simply mixed. In the cell-free system Rev-Rev interaction occurred within minutes and was inhibited by excess Rev. The levels of interaction between the mutant Revs tested varied by mutant type. Treatment of Rev lysates with RNAse minimally reduced the degree of interaction whereas addition of HIV RRE RNA enhanced the interaction. Purified GST-Rev protein inhibited the interaction. The Z-factor (Z') for the cell-free system was ∼0.85 when tested in 96-well format, and the anti-Rev NB enhanced the interaction in the cell-free system. Thus, we have developed both cell-intact and cell-free systems that can reliably, rapidly, and reproducibly quantify Rev-Rev interaction. These assays, particularly the cell-free one, may be useful in screening and identifying compounds that inhibit Rev function on a high throughput basis.

Concussion occurrence and recognition in professional boxing and MMA matches: toward a concussion protocol in combat sports.

Objectives: Determine, through video reviews, how often concussions occur in combat sport matches, what influence they have on the outcome, and how well non-physician personnel can be trained to recognize concussions.Methods: This is a retrospective video analysis by an 8-person panel of 60 professional fights (30 boxing and 30 mixed martial arts). Through video review, physician and non-physician personnel recorded details about each probable concussion and determined if and when they would have stopped the fight compared to the official stoppage time.Results: A concussion was recorded in 47/60 fights. The mean number of concussions per minute of fight time was 0.061 (0.047 for boxers and 0.085 for MMA). When stratifying by outcome of the bout, the mean number of concussions per minute for the winner was 0.010 compared to the loser at 0.111 concussions per minute. The fighter that sustained the first concussion ultimately lost 98% of the time. The physician and non-physician raters had high agreement regarding the number of concussions that occurred to each fighter per match. The physician raters judged that 24 of the 60 fights (11 boxing [37%]; 13 MMA [43%]) should have been stopped sooner than what occurred.Conclusion: Recognizing that the concussions often occur in combat sport matches, that the losing fighter almost always is concussed first and tends to sustain more concussions during the fight, along with the demonstration that non-physician personnel can be taught to recognize concussion, may guide policy changes that improve brain health in combat sports.