Background:The human proton-coupled folate transporter (PCFT) may exist as homo-oligomers. Results: PCFT monomers form oligomers, and wild-type and inactive mutant PCFT monomers show dominant-positive activity when co-expressed. Conclusion: Wild-type PCFT may rescue the mutant phenotype via formation of hetero-oligomers. Significance: Better understanding of PCFT oligomerization may identify therapeutic applications for hereditary folate maladsorption and delivery of PCFT-targeted chemotherapy drugs for cancer.

The human proton-coupled folate transporter (hPCFT) is expressed in solid tumours and is active at pHs characterizing the tumour microenvironment. Recent attention focused on exploiting hPCFT for targeting solid tumours with novel cytotoxic anti-folates. hPCFT has 12 transmembrane domains (TMDs) and forms homo-oligomers with functional significance. The hPCFT primary sequence includes GXXXG motifs in TMD2 (G93XXXG97) and TMD4 (G155XXXG159). To investigate roles of these motifs in hPCFT function, stability and surface expression, we mutated glycine to leucine to generate single or multiple substitution mutants. Only the G93L and G159L mutants preserved substantial [3H]methotrexate (Mtx) transport when expressed in hPCFT-null (R1-11) HeLa cells. Transport activity of the glycine-to-leucine mutants correlated with surface hPCFT by surface biotinylation and confocal microscopy with ECFP*-tagged hPCFTs, suggesting a role for GXXXG in hPCFT stability and intracellular trafficking. When co-expressed in R1-11 cells, haemagglutinin-tagged glycine-to-leucine mutants and His10-tagged wild-type (WT) hPCFT co-associated on nickel affinity columns, suggesting that the GXXXG motifs are not directly involved in hPCFT oligomerization. This was substantiated by in situ FRET experiments with co-expressed ECFP*- and YFP-tagged hPCFT. Molecular modelling of dimeric hPCFT structures showed juxtaposed TMDs 2, 3, 4 and 6 as potential structural interfaces between monomers. hPCFT cysteine insertion mutants in TMD3 (Q136C and L137C) and TMD6 (W213C, L214C, L224C, A227C, F228C, F230C and G231C) were expressed in R1-11 cells and cross-linked with 1,6-hexanediyl bismethanethiosulfonate, confirming TMD juxtapositions. Altogether, our results imply that TMDs 3 and 6 provide critical interfaces for formation of hPCFT oligomers, which might be facilitated by the GXXXG motifs in TMD2 and TMD4.

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Background The Resuscitation Rotation is a novel second-year emergency medicine rotation focusing on the highest acuity patients, including out-of-hospital cardiac arrest (OHCA). The resuscitation resident (RR) functions as an extra physician during resuscitation and post return of spontaneous circulation (ROSC). The objective of this study is to examine if the presence of a RR decreases the pre-induction interval of targeted temperature management (TTM) for patients following OHCA. Methods A retrospective study was conducted at a tertiary care level 1 trauma center with an annual ED census of 127,323 visits in 2019. We retrospectively reviewed consecutive OHCA patients from September 1, 2014, to July 20, 2020, who underwent TTM. Patients were identified as cases with or without a RR. Clinical characteristics were summarized by the status of RR involvement and compared by using t-test and χ 2 test for continuous and categorical variables, respectively. All tests with p < 0.05 were considered to indicate statistical significance. Results Our study population identified 198 adult OHCA patients that underwent TTM from 2014-2020. There were exclusions for missing TTM start time and for missing patient characteristics leaving 176 for final analysis, of which 55 (33.3%) had RR involvement. The mean time (hours) to TTM initiation (ie, the pre-induction phase) for patients involving the RR versus those without was not statistically significant (3.11 vs 3.34, p=0.39). Linear regression analysis indicates that the adjusted effect of RR involvement was not associated with the mean hours of pre-induction (p=0.47). Conclusion There is no statistically significant association of a RR on the duration of the pre-induction phase. Limitations include that both arms had prolonged pre-induction phases. This may represent a non-optimized TTM protocol. Future work will aim to use the RR to improve our pre-induction phase.