Rapid high-throughput processing of tissue samples for microbiological diagnosis of periprosthetic joint infections using bead-beating homogenization.

Enrichment of periprosthetic tissue samples in blood culture bottles (BCBs) for microbiological diagnosis of periprosthetic joint infections (PJI) is more reliable than the use of an enrichment broth. Nevertheless, the extremely time-consuming homogenization of the samples for BCB processing has so far limited its use, especially in high-throughput settings. We aimed to establish a highly scalable homogenization process of tissue samples for long-term incubation in BCBs. A protocol for homogenization of tissue samples using bead beating was established and validated. In a second step, the use of the homogenate for enrichment in BCBs was compared to the use of thioglycolate broth (TB) in terms of diagnostic accuracy using clinical tissue samples from 150 patients with suspected PJI. Among 150 analyzed samples, 35 samples met the microbiological criteria for PJI. Using BCB, 32 of 35 (91.4%) PJI were detected compared to 30 of 35 (85.7%) by TB. The use of BCB had a lower secondary contamination rate (2/115; 1.7% vs 4/115; 3.5%) but the trend was not significant due to low numbers of samples (P = 0.39). The time to process a batch of 12 samples using the established homogenization method was 23 ± 5 min (n = 10 batches). We established and validated a homogenization workflow that achieves the highest sensitivity in the microbiological diagnostic of PJI. The enrichment of the tissue homogenate in BCBs showed equally good results as the use of enrichment broth and allows semi-automated high-throughput processing while demonstrating lower contamination rates in our study.

The kinetochore module Okp1CENP-Q/Ame1CENP-U is a reader for N-terminal modifications on the centromeric histone Cse4CENP-A.

Kinetochores are supramolecular assemblies that link centromeres to microtubules for sister chromatid segregation in mitosis. For this, the inner kinetochore CCAN/Ctf19 complex binds to centromeric chromatin containing the histone variant CENP-A, but whether the interaction of kinetochore components to centromeric nucleosomes is regulated by posttranslational modifications is unknown. Here, we investigated how methylation of arginine 37 (R37Me) and acetylation of lysine 49 (K49Ac) on the CENP-A homolog Cse4 from Saccharomyces cerevisiae regulate molecular interactions at the inner kinetochore. Importantly, we found that the Cse4 N-terminus binds with high affinity to the Ctf19 complex subassembly Okp1/Ame1 (CENP-Q/CENP-U in higher eukaryotes), and that this interaction is inhibited by R37Me and K49Ac modification on Cse4. In vivo defects in cse4-R37A were suppressed by mutations in OKP1 and AME1, and biochemical analysis of a mutant version of Okp1 showed increased affinity for Cse4. Altogether, our results demonstrate that the Okp1/Ame1 heterodimer is a reader module for posttranslational modifications on Cse4, thereby targeting the yeast CCAN complex to centromeric chromatin.