Optimizing Lectin Staining Methodology to Assess Glycocalyx Composition of Legionella-Infected Cells
Bibliographic record
Abstract
Introduction: Legionella is a gram-negative bacterium that replicates intracellularly within macrophages. Legionella utilizes effector proteins to hijack ER-Golgi vesicle trafficking to sustain proliferation in its intracellular niche. Legionella has a considerable influence on O-glycosylation but not N-glycosylation events in the Golgi of infected cells. This research aims to optimize the use of fluorescent lectins, which are proteins that bind carbohydrates, to effectively label host-cell glycocalyx during Legionella infection. Methods: Epifluorescence imaging or flow cytometry were used to optimize the lectin staining methodology. We noted that the most effective conditions for lectin-labeling were when live HeLa cells were incubated with lectins diluted in Hank’s balanced salt solution (HBSS) with 3% Bovine serum albumin (BSA) for 10-30 minutes at 4 °C. Results: Incubating suspended cells with lectins necessitated smaller lectin concentrations, whereas lectin labeling of adherent cells required considerably larger concentrations. Wheat germ agglutinin (WGA) lectin mean fluorescence intensity (MFI) was concentration-dependent, but Concanavalin A (ConA) and Maclura pomifera (MPA) MFIs did not alter substantially with increasing lectin concentrations. Discussion: The optimal lectin concentration required was lectin-specific and based on whether the lectin fluorescence was assessed using flow cytometry or epifluorescence. Furthermore, the use of phosphate-buffered saline (PBS) for lectin dilution, cell permeabilization for intracellular labelling, and incubation of lectins in fixed cells reduced productive labelling of lectins on cell surfaces because it inhibited the lectin's ability to effectively bind the associated carbohydrate structure. Conclusion: Further research using diverse lectins on U937 macrophages is necessary to reach a definitive conclusion on the effect of Legionella on the overall host-cell glycocalyx composition during infection of these relevant immune cells.
Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.
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) | 0.000 | 0.000 |
| Bibliometrics | 0.001 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
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".