Biology subjects

Dela, F.

Publications and source records attributed to Dela, F..

4 recordsLinked to original sources

bioRxiv2023

Development and validation of a pharmacogenomics reporting workflow based on the Illumina Global Screening Array chip

BackgroundMicroarrays are a well-established and widely adopted technology capable of interrogating hundreds of thousands of loci across the human genome. Combined with imputation to cover common variants not included in the chip design, they offer a cost-effective solution for large-scale genetic studies. Beyond research applications, this technology can be applied for testing pharmacogenomics, nutrigenetics, and complex disease risk prediction. However, establishing clinical reporting workflows requires a thorough evaluation of the assays performance, which is achieved through validation studies. In this study, we performed pre-clinical validation of a genetic testing workflow based on the Illumina Global Screening Array for 25 pharmacogenomic-related genes. MethodsTo evaluate the accuracy of our workflow, we conducted multiple pre-clinical validation studies. Here, we present the results of accuracy and precision assessments, involving a total of 73 cell lines. These assessments encompass reference materials from the Genome-In-A-Bottle (GIAB), the Genetic Testing Reference Material Coordination Program (GeT-RM) projects, as well as additional samples from the 1000 Genomes project (1KGP). We conducted an accuracy assessment of genotype calls for target loci in each indication against established truth sets. ResultsIn our per-sample analysis, we observed a mean analytical sensitivity of 99.39% and specificity 99.98%. We further assessed the accuracy of star-allele calls by relying on established diplotypes in the GeT-RM catalogue or calls made based on 1KGP genotyping. On average, we detected a diplotype concordance rate of 96.47% across 14 pharmacogenomic-related genes with star allele-calls. Lastly, we evaluated the reproducibility of our findings across replicates and observed 99.48% diplotype and 100 % phenotype inter-run concordance. ConclusionOur comprehensive validation study demonstrates the robustness and reliability of the developed workflow, supporting its readiness for further development for applied testing.

genetics↗

bioRxiv2022

Peripheral blood mononuclear cells exhibit increased mitochondrial respiration after adjuvant chemo- and radiotherapy for early breast cancer

BackgroundAdjuvant chemo- and radiotherapy cause cellular damage not only to cancerous but also to healthy dividing cells. Antineoplastic treatments have been shown to cause mitochondrial respiratory dysfunction in non-tumorous tissues, but the effects on circulating human peripheral blood mononuclear cells (PBMCs) remain unknown. AimWe aimed to identify changes in mitochondrial respiration of PBMCs after adjuvant chemo- and radiotherapy in postmenopausal early breast cancer (EBC) patients and relate these to metabolic parameters of the patients. MethodsTwenty-three postmenopausal women diagnosed with EBC were examined before and shortly after chemotherapy treatment often administered in combination with radiotherapy (n=18). Respiration (O2 flux per million PBMCs) was assessed by high-resolution respirometry of intact and permeabilized PBMCs. Clinical metabolic characteristics were furthermore assessed. ResultsRespiration of intact and permeabilized PBMCs from EBC patients was significantly increased after adjuvant chemo- and radiotherapy (p=6x10-5 and p=1x10-7, respectively). The oxygen flux attributed to specific mitochondrial complexes and respiratory states increased by 17-43% compared to before therapy commencement. Leukocyte counts (p=1x10-4), hemoglobin levels (p=0.0003), and HDL cholesterol (p=0.003) decreased while triglyceride (p=0.01) and LDL levels (p=0.02) increased after treatment suggesting a worsened metabolic state. None of the metabolic parameters correlated significantly with PBMC respiration. ConclusionThis study shows that mitochondrial respiration in circulating PBMCs is significantly increased after adjuvant chemo- and radiotherapy in postmenopausal EBC patients. The increase might be explained by a shift in PBMC subpopulation proportions towards cells relying on oxidative phosphorylation rather than glycolysis or a generally increased mitochondrial content in PBMCs. Both parameters might be influenced by therapy-induced changes to the bone marrow or vascular microenvironment wherein PBMCs differentiate and reside.

cell biology↗

bioRxiv2022

Preponderant Myosin Super-Relaxed State In Skeletal Muscle From Endurance Athletes

It has recently been established that myosin, the molecular motor protein, is able to exist in two conformations in relaxed skeletal muscle. These conformations are known as super-relaxed (SRX) and disordered-relaxed (DRX) states and are finely balanced to optimize skeletal muscle metabo-lism. Indeed, SRX myosins are thought to have a 10-fold reduction in ATP turnover compared to DRX myosins. Here, we investigated whether chronic physical activity in humans would be associated with changes in the proportions of SRX and DRX skeletal myosins. For that, we isolated mus-cle fibres from various athletic and sedentary populations and ran a loaded Mant-ATP chase proto-col. We observed that, in endurance-trained athletes, the amounts of myosin molecules in the SRX state was significantly greater than in age-matched sedentary individuals or than in strength athletes. To further assess whether this change would have an impact on the potency of a SRX-inducing pharmacological compound, Mavacamten, we performed similar analyses as above with and without the drug in muscle fibres from endurance athletes. Surprisingly, we found that 0.3 M of Mava-camten had only marginal effects. Altogether, our results indicate that chronic endurance training-status influences resting skeletal myosin conformations, and Mavacamten potency. Our findings also emphasize that environmental stimuli such as exercise can re-wire the molecular metabolism of human skeletal muscle through myosin. SummaryLewis et al., investigate how training-status influences myosin conformations involved in the resting metabolism of skeletal muscle. They find that, in endurance-trained athletes, skeletal myosin preferentially adopts an energy-saving conformation known as super-relaxed state, lowering the metabolic rate and affecting the potency of a super-relaxed state-inducing drug, Mavacamten.

physiology↗

bioRxiv2022

Choice of medium affects PBMC quantification, cell size, and downstream respiratory analysis

High-resolution respirometry (HRR) can assess PBMC bioenergetics, but no standardized medium for PBMC preparation and HRR analysis exist. Here, we study the effect of four different media (MiR05, PBS, RPMI, Plasmax) on quantification, size, and HRR analysis (Oxygraph-O2k) of intact PBMCs. Remarkably, PBMC quantification was 21% higher in MiR05 than PBS and Plasmax, and 28% higher than in RPMI, causing O2 flux underestimation during HRR due to inherent adjustments. Moreover, smaller cell size of PBMCs and aggregation was observed in MiR05. We suggest optimization of HRR with a standardized, plasma-like medium for future HRR analysis of intact PBMCs.

cell biology↗