Development and validation of a liquid chromatography-tandem mass spectrometry assay to quantify plasma 24(S)-hydroxycholesterol and 27-hydroxycholesterol: A new approach integrating the concept of ion ratio.

Accurate quantification of 24(S)-hydroxycholesterol and 27-hydroxycholesterol holds substantial biological significance due to their involvement in pivotal cellular processes, encompassing cholesterol homeostasis, inflammatory responses, neuronal signaling, and their potential as disease biomarkers. The plasma determination of these oxysterols is challenging considering their low concentrations and similarities in terms of empirical formulae, molecular structure, and physicochemical properties across all human endogenous plasma oxysterols. To overcome these sensitivity and specificity issues, we developed and validated a quantification method using liquid chromatography coupled to a tandem mass spectrometry instrument. Validation studies were designed inspired by Clinical and Laboratory Standards Institute (CLSI) C62-A Guidelines. The linearity ranged between 20 and 300 nM for both oxysterols with limits of quantification at 20 nM and 30 nM for 24(S)-OHC and 27-OHC, respectively. Inter-day precision coefficient variations (CV) were lower than 10% for both oxysterols. An optimal separation of 25-OHC was obtained from 24(S)-OHC and 27-OHC with a resolution (Rs) > 1.25. The determination and validation of ion ratios for 24(S)-OHC and 27-OHC enabled another quality check in identifying interferents that could impact the quantification. Our developed and validated LC-MS/MS method allows consistent and reliable quantification of human plasmatic 24(S)-OHC and 27-OHC that is warranted in fundamental and clinical research projects.

Establishment of reference intervals for the salivary cortisol circadian cycle, by electrochemiluminescence (ECLIA), in healthy adults.

OBJECTIVES: To determine salivary cortisol reference intervals in a healthy adult population, at 6 different time points during a 24-hour (h) period. METHODS: In a prospective study, salivary cortisol concentrations were measured upon waking, one-hour post-waking and at specific times of the day: at 12 h00, 16 h00, 20 h00 and midnight. Samples were analyzed by the first and second-generation electrochemiluminescence assays (ECLIA) from Roche Cobas Cortisol®. RESULTS: Salivary cortisol values were obtained from 134 healthy volunteers. Reference intervals for the first-generation assay were 6.14-33.19 nmol/L (95% prediction interval) at waking, 5.42-28.06 nmol/L one-hour post-waking, 3.62-16.23 nmol/L at 12 h00, 2.78-15.27 nmol/L at 16 h00, 2.08-14.90 nmol/L at 20 h00 and 2.09-16.92 nmol/L at midnight. Mean salivary cortisol values were 14.63 nmol/L at waking and 6.44 nmol/L at midnight. Reference intervals for the second-generation assay were 1.50-22.02 nmol/L (2.5th to 97.5th percentiles) at waking, 1.50-20.87 nmol/L one-hour post-waking, 1.50-12.51 nmol/L at 12 h00, 1.50-13.03 nmol/L at 16 h00, 1.50-9.52 nmol/L at 20 h00 and 1.50-6.28 nmol/L at midnight. Values for the second-generation assay at all 6 different time points were almost half of the first-generation assay. The second-generation assay showed a better correlation with LC-MS/MS (r = 0,97). CONCLUSION: Our study confirms that reference intervals for salivary cortisol are not comparable across first and second-generation Roche Cobas Cortisol® assays. Furthermore, the second-generation assay has a better correlation with LC-MS/MS and a better analytical performance (accuracy and precision).

Comparison of two methods for measuring 25-OH vitamin D in the follow-up of patients after bilio-pancreatic diversion bariatric surgery.

OBJECTIVE: Our objective was to compare the ECLIA from Roche versus the LC-MS/MS method for quantitation of serum 25-hydroxy-vitamin D in patients who have undergone bariatric surgery. DESIGN AND METHODS: Cross-sectional and correlational studies were performed on three different groups for the 25-OH-D levels quantitated by both methods. The control group of apparently healthy subjects was randomly selected in a clinical chemistry laboratory. Test groups were patients who had undergone bilio-pancreatic diversion (BPD) and were supplemented either with vitamin D2 or with vitamin D3. The number of samples per group was established according to the CLSI recommendation protocol (EPO9-A2-IR). RESULTS: The agreement of LC-MS/MS with the Roche method was acceptable in the apparently healthy subjects group and in the post-BPD D3-supplemented group with an average bias of -1.7% and -9.2%, respectively. However, this agreement was unacceptable in the post-BPD D2-supplemented group with an average bias of -45.3%. The LC-MS/MS enabled us to detect four patients who had excess vitamin D or poisoning with vitamin D for which it was necessary to stop the supplementation with vitamin D in the D2 -supplemented group. CONCLUSION: Despite the apparent good agreement between the Roche method and LC-MS/MS in the healthy subjects group and in the post-DBP D3-supplemented patient group, a considerable bias seems to exist, particularly in the presence of D2. The LC-MS/MS method is therefore the most accurate method to follow the vitamin D2 -supplemented bariatric population.

Multiple Transcriptional and Post-transcriptional Pathways Collaborate to Control Sense and Antisense RNAs of Tf2 Retroelements in Fission Yeast.

Retrotransposons are mobile genetic elements that colonize eukaryotic genomes by replicating through an RNA intermediate. As retrotransposons can move within the host genome, defense mechanisms have evolved to repress their potential mutagenic activities. In the fission yeast Schizosaccharomyces pombe, the mRNA of Tf2 long terminal repeat retrotransposons is targeted for degradation by the 3'-5' exonucleolytic activity of the exosome-associated protein Rrp6. Here, we show that the nuclear poly(A)-binding protein Pab2 functions with Rrp6 to negatively control Tf2 mRNA accumulation. Furthermore, we found that Pab2/Rrp6-dependent RNA elimination functions redundantly to the transcriptional silencing mediated by the CENP-B homolog, Abp1, in the suppression of antisense Tf2 RNA accumulation. Interestingly, the absence of Pab2 attenuated the derepression of Tf2 transcription and the increased frequency of Tf2 mobilization caused by the deletion of abp1 Our data also reveal that the expression of antisense Tf2 transcripts is developmentally regulated and correlates with decreased levels of Tf2 mRNA. Our findings suggest that transcriptional and post-transcriptional pathways cooperate to control sense and antisense RNAs expressed from Tf2 retroelements.

A proline-tyrosine nuclear localization signal (PY-NLS) is required for the nuclear import of fission yeast PAB2, but not of human PABPN1.

Nuclear poly(A)-binding proteins (PABPs) are evolutionarily conserved proteins that play key roles in eukaryotic gene expression. In the fission yeast Schizosaccharomyces pombe, the major nuclear PABP, Pab2, functions in the maturation of small nucleolar RNAs as well as in nuclear RNA decay. Despite knowledge about its nuclear functions, nothing is known about how Pab2 is imported into the nucleus. Here, we show that Pab2 contains a proline-tyrosine nuclear localization signal (PY-NLS) that is necessary and sufficient for its nuclear localization and function. Consistent with the role of karyopherin ß2 (Kapß2)-type receptors in the import of PY-NLS cargoes, we show that the fission yeast ortholog of human Kapß2, Kap104, binds to recombinant Pab2 and is required for Pab2 nuclear localization. The absence of arginine methylation in a basic region N-terminal to the PY-core motif of Pab2 did not affect its nuclear localization. However, in the context of a sub-optimal PY-NLS, we found that Pab2 was more efficiently targeted to the nucleus in the absence of arginine methylation, suggesting that this modification can affect the import kinetics of a PY-NLS cargo. Although a sequence resembling a PY-NLS motif can be found in the human Pab2 ortholog, PABPN1, our results indicate that neither a functional PY-NLS nor Kapß2 activity are required to promote entry of PABPN1 into the nucleus of human cells. Our findings describe the mechanism by which Pab2 is imported into the nucleus, providing the first example of a PY-NLS import system in fission yeast. In addition, this study suggests the existence of alternative or redundant nuclear import pathways for human PABPN1.