Increasing number of studies over the last decade have aimed to compare, contrast, and model results generated from in vivo or ex vivo iRBC samples acquired from human P. falciparum infections. Such studies are also beginning to recognize the potential impact on var gene 4-(Benzyloxy)phenol expression and switching depending on Mepiroxol whether individuals are na?ve and experiencing a more severe case of malaria or have previous exposure to the parasite and some degree of adaptive immunity. We provide evidence for the first time that SICA parasites do not produce SICA proteins, consistent with the lack of detectable full-length SICAvar transcripts in SICA parasite RNA. Most telling, we have used sensitive LC-MS/MS proteomic methods to confirm that SICA protein is not present in SICA trophozoite-infected RBCs. It is known that when cloned SICA parasites are reinoculated into an intact rhesus monkey SICA expression at the surface of the iRBCs can be reinstated. Future directions include investigating what SICA proteins become expressed under these ‘reactivation circumstances’ or after passage through mosquitoes and defining the mechanistic processes and controls. Another major finding presented here is that the SICA antigenic switch from the Pk1 phenotype to the Pk11+ phenotype is ‘complete’, in that none of the SICA proteins detected in the parent clone are detected in the progeny clone. The complete change in protein expression is quite impressive. This switch can be defined as a dramatic shift in expression and not the result of the progression and selection of iRBCs that have drifted in their expression characteristics. Among the SICAvar gene IDs detected by LC-MS/MS, curiously, PkH_040630 represents an isolated 2-exon sequence with a PEXEL motif and start and stop codons. The PkH_040630 sequence is immediately upstream from PkH_040620 on the antisense strand. This small sequence lacks the majority 
of a typical SICA protein including the C-terminal cytoplasmic domain that would be recognized by the pan-SICAcyto antiserum used for the immunoprecipitation reactions, but it may have been pulled down in a complex. Another possibility is that PkH_040630 may have been alternatively spliced to SICAvar exons downstream of PkH_040620. The published sequence downstream, however, is truncated by a gap, leaving this an open question. We have presented a compendium of expressed SICAvar genes and proteins for the first time to define the classic Pk1 and Pk11+ cloned populations. These data reveal that these parasites cannot be strictly defined by two SICA proteins representing the high molecular weight protein doublets originally observed by SDS-PAGE. Nevertheless, they continue to express specific predominant SICA proteins, and in particular the originally defined 205 kDa SICA protein in the Pk11+ parasites, even after many passages over time through intact rhesus monkeys; thus, overall, these clones can be viewed as stable with regards to their SICA expression. It remains unknown whether each of the proteins detected by LC-MS/MS is actually expressed on the surface of the respective infected RBCs. Each of the SICA proteins was identified by unique peptides, thus we are confident they were expressed, but there is presently no evidence of the transport of every protein to the surface of the infected host cells. Such investigations will require the development of specific noncrossreacting antibody reagents, which are a challenge to produce for members of the SICA family. Attempts at expressing specific recombinant SICA proteins to develop specific antibodies and study the expression at a cellular and population level have been complicated by 1) the high degree of conserved sequences across the family and 2) the facts that SICA recombinant proteins tend to be insoluble and may not represent the natural conformation of the antigens.