Haubner, M. | bioRxiv Science

bioRxiv2025

Overcoming Preferred Orientation in Cryo-EM With Ultrasonic Excitation During Vitrification

Preferred particle orientation remains a frequently encountered problem in cryo-electron microscopy that arises when proteins adsorb to the air-water interface in only a limited number of orientations. This issue can significantly increase the data acquisition time required to reach a desired resolution or even make it impossible to obtain a reconstruction altogether. Here, we show that preferred orientation can be overcome by continuously exciting the sample with ultrasonic waves during vitrification. Our experiments suggest that mechanical oscillations induced in the sample support continuously shake proteins loose from the air-water interface, thereby scrambling their orientations. The simple, physical nature of this mechanism should make it applicable to a wide range of proteins. Since our method can be easily implemented in existing vitrification devices, we expect it to find widespread adoption.

biophysics↗

bioRxiv2024

Genome-guided, field-deployable loop-mediated isothermal amplification (LAMP) assay for specific detection of Dickeya dadantii

Potatoes, among the most economically significant crops worldwide, are susceptible to various plant pathogens that significantly impact their propagation, production, storage, and distribution. Soft rot disease, caused primarily by Dickeya and Pectobacterium, results in substantial economic losses to the agricultural industry annually. In this study, we developed a rapid, reliable, and field-deployable loop-mediated isothermal amplification (LAMP) assay for detecting D. dadantii, a common soft rot causing bacteria. The D. dadantii-specific LAMP primers were designed targeting a highly conserved genomic region within D. dadantii, the TetR/AcrR family transcriptional regulator CDS and its flanking sequences. This assay was thoroughly validated with the members of inclusivity (nine strains of D. dadantii) and exclusivity panels (85 strains, including all Dickeya species, related taxa, and host DNA), detecting no false positives or negatives. The limit of detection (LOD) was established by performing assays with 10-fold serially diluted pure gDNA of D. dadantii and gDNA spiked with host crude extract; the assay detected the target pathogen down to 1 pg (188 copies) without being adversely affected by the host crude extract. The developed LAMP assay specifically detected the target pathogen in infected plant materials. Additional multi-operator blind and multi-instrument tests were conducted to assess the assays robustness and applicability, consistently yielding accurate results without false positives or negatives. These findings demonstrate the assays potential utility for biosecurity, routine diagnostics, and epidemiological studies.

microbiology↗