Real‐time measurement of transport activity of the human concentrative nucleoside transporter, hCNT3, using a fluorescent reporter
Bibliographic record
Abstract
Human concentrative nucleoside transporter, hCNT3, mediates Na + /nucleoside co‐transport, with a low turnover rate (10 nucleoside molecules s −1 ). hCNT3 also supports H + ‐coupled uridine transport, as measured in X. laevis oocytes. We used a new approach to monitor H + /uridine co‐transport in mammalian cells. We fused mNectarine, a new H + ‐sensitive red fluorescent protein, to the N‐terminus of hCNT3 to generate mNect‐hCNT3. Fusion of the fluorescent H + sensor enabled measurement of pH at the intracellular surface of hCNT3. mNectarine fluorescence was monitored in human embryonic kidney 293 cells expressing mNect‐hCNT3 or, as a negative control, cytosolic mNectarine and catalytically dead mutant of the AE1 Cl − /HCO 3 − exchanger, AE1‐P652C. mNectarine fluorescence values were calibrated for pH using the nigericin/high K + clamping method. Cells were incubated at the permissive pH for H + ‐coupled nucleoside transport, pH 5.5, under Na + ‐free conditions. At this pH there was a continuous cytosolic acidification. In mNect‐hCNT3 expressing cells (but not under negative control conditions) the rate of acidification increased upon addition of 0.5 mM uridine, consistent with H + ‐coupled uridine transport. We conclude that mNect‐hCNT3 acts as a sensitive self‐referencing sensor of nucleoside transport. Research is supported by the Canadian Institutes of Health Research and the National Cancer Institute of Canada.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.000 | 0.001 |
| Science and technology studies | 0.000 | 0.001 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".