The amniotic fluid proteome changes across gestation in humans and rhesus macaques

Abstract Amniotic fluid is a complex biological medium that offers protection to the fetus and plays a key role in normal fetal nutrition, organogenesis, and potentially fetal programming. Amniotic fluid is also critically involved in longitudinally shaping the in utero milieu during pregnancy. Yet, the molecular mechanism(s) of action by which amniotic fluid regulates fetal development is ill-defined partly due to an incomplete understanding of the evolving composition of the amniotic fluid proteome. Prior research consisting of cross-sectional studies suggests that the amniotic fluid proteome changes as pregnancy advances, yet longitudinal alterations have not been confirmed because repeated sampling is prohibitive in humans. We therefore performed serial amniocenteses at early, mid, and late gestational time-points within the same pregnancies in a rhesus macaque model. Longitudinally-collected rhesus amniotic fluid samples were paired with gestational-age matched cross-sectional human samples. Utilizing LC–MS/MS isobaric labeling quantitative proteomics, we demonstrate considerable cross-species similarity between the amniotic fluid proteomes and large scale gestational-age associated changes in protein content throughout pregnancy. This is the first study to compare human and rhesus amniotic fluid proteomic profiles across gestation and establishes a reference amniotic fluid proteome. The non-human primate model holds promise as a translational platform for amniotic fluid studies

Conditional RAG-1 Mutants Block the Hairpin Formation Step of V(D)J Recombination

Hairpin formation serves an important regulatory role in V(D)J recombination because it requires synapsis of an appropriate pair of recombination sites. How hairpin formation is regulated and which regions of the RAG proteins perform this step remain unknown. We analyzed two conditional RAG-1 mutants that affect residues quite close in the primary sequence to an active site amino acid (D600), and we found that they exhibit severely impaired recombination in the presence of certain cleavage site sequences. These mutants are specifically defective for the formation of hairpins, providing the first identification of a region of the V(D)J recombinase necessary for this reaction. Substrates containing mismatched bases at the cleavage site rescued hairpin formation by both mutants, which suggests that the mutations affect the generation of a distorted or unwound DNA intermediate that has been implicated in hairpin formation. Our results also indicate that this region of RAG-1 may be important for coupling hairpin formation to synapsis