Functional analysis of the leading malaria vaccine candidate AMA-1 reveals an essential role for the cytoplasmic domain in the invasion process

A key process in the lifecycle of the malaria parasite Plasmodium falciparum is the fast invasion of human erythrocytes. Entry into the host cell requires the apical membrane antigen 1 (AMA-1), a type I transmembrane protein located in the micronemes of the merozoite. Although AMA-1 is evolving into the leading blood-stage malaria vaccine candidate, its precise role in invasion is still unclear. We investigate AMA-1 function using live video microscopy in the absence and presence of an AMA-1 inhibitory peptide. This data reveals a crucial function of AMA-1 during the primary contact period upstream of the entry process at around the time of moving junction formation. We generate a Plasmodium falciparum cell line that expresses a functional GFP-tagged AMA-1. This allows the visualization of the dynamics of AMA-1 in live parasites. We functionally validate the ectopically expressed AMA-1 by establishing a complementation assay based on strain-specific inhibition. This method provides the basis for the functional analysis of essential genes that are refractory to any genetic manipulation. Using the complementation assay, we show that the cytoplasmic domain of AMA-1 is not required for correct trafficking and surface translocation but is essential for AMA-1 function. Although this function can be mimicked by the highly conserved cytoplasmic domains of P. vivax and P. berghei, the exchange with the heterologous domain of the microneme protein EBA-175 or the rhoptry protein Rh2b leads to a loss of function. We identify several residues in the cytoplasmic tail that are essential for AMA-1 function. We validate this data using additional transgenic parasite lines expressing AMA-1 mutants with TY1 epitopes. We show that the cytoplasmic domain of AMA-1 is phosphorylated. Mutational analysis suggests an important role for the phosphorylation in the invasion process, which might translate into novel therapeutic strategies.

Understanding in-vivo abrasion fatigue or common suture materials used in arthroscopic and open shoulder surgery

In orthopaedic surgery the reattachment of tendon to bone requires suture materials that have stable and durable properties to allow healing at the tendon-bone interface. Failure rates of this type of surgery can be as high as 25%. While the tissue suture interface is a weak link, proportions of these failures are caused by in-vivo abrasion of the suture with bone and suture anchor materials. Abrasion of the suture material results from the movement of the suture through the eyelet by the surgeon during surgery, or with limb movement after surgery as the suture is not rigidly restrained within the eyelet. During movement the suture is subjected to bending and frictional forces that can lead to fatigue induced failure. This paper investigates the mechanism of bending abrasion fatigue induced failure of number two grade braided sheath only and braided sheath/multifilament core sutures. Sutures were oscillated over a stainless steel wire at low frequency under load in a dry state to simulate the bending and frictional forces between suture and eyelet. Failure mechanism was determined by video microscopy of the suture during abrasion combined with optical microscopy analysis of partially and fully abraded sutures. Braided only structures had high friction loading on the small number of fibres at the abrasion interface. This caused rapid single fibre breakages that accumulate to cause suture failure. The addition of ultra-high molecular weight polyethylene core fibres to a braided suture distributed the applied load across multiple fibres at the abrasion interface. This improved abrasion resistance by 15-20 times that of braided sheath alone.