Standardization of 8-color flow cytometry across different flow cytometer instruments: A feasibility study in clinical laboratories in Switzerland.

The EuroFlow Consortium developed a fully standardized flow cytometric approach from instrument settings, through antibody panel, reagents and sample preparation protocols, to data acquisition and analysis. The Swiss Cytometry Society (SCS) promoted a study to evaluate the feasibility of using such standardized measurements of 8-color data across two different flow cytometry platforms - Becton Dickinson (BD) FACSCanto II and Beckman Coulter (BC) Navios, aiming at increasing reproducibility and inter-laboratory comparability of immunophenotypic data in clinical laboratories in Switzerland. The study was performed in two phases, i.e. a learning phase (round 1) and an analytical phase (rounds 2 and 3) consisting of a total of three rounds. Overall, 10 laboratories using BD FACSCanto II (n=6) or BC Navios (n=4) flow cytometers participated. Each laboratory measured peripheral blood samples from healthy donors stained with a uniform antibody panel of reagents - EuroFlow Lymphoid Screening Tube (LST) - applying the EuroFlow standardized protocols for instrument setup and sample preparation (www.EuroFlow.org). All data files were analyzed centrally and median fluorescence intensity (MedFI) values for individual markers on defined lymphocyte subsets were recorded; variability from reference MedFI values was assessed using performance scores. Data troubleshooting and discussion of the results with the participants followed after each round at SCS meetings. The results of the learning phase demonstrated that standardized instrument setup and data acquisition are feasible in routine clinical laboratories without previous experience with EuroFlow. During the analytical phase, highly comparable data were obtained at the different laboratories using either BD FACSCanto II or BC Navios. The coefficient of variation of MedFI for 7 of 11 markers performed repeatedly below 30%. In the last study round, 89% of participants scored over 90% MedFI values within the acceptance criteria (P-score), in line with the results of the EuroFlow quality assessment rounds performed by the EuroFlow expert laboratories(Kalina et al., 2015). Central analysis of data allowed identification of deviations from the standardized procedures and technical issues (e.g. failure to perform correct instrument setup and improper compensation). In summary, here we show that inter-laboratory cross-platform standardization of 8-color flow cytometric measurements in clinical laboratories is feasible and allows for fully comparable MedFI results across BD FACSCanto II and BC Navios instruments. However, adherence to standardized protocols is crucial. Thus, training of the laboratory personnel in the EuroFlow standardized procedures is highly recommended to prevent errors in instrument setup and sample preparation.

Exploring the Relationship between Sexual Compulsivity and Attentional Bias to Sex-Related Words in a Cohort of Sexually Active Individuals.

BACKGROUND/AIMS: If sexual compulsivity and other addictive behaviours share common aetiology, contemporary proposals about the role of attentional processes in understanding addictive behaviours are relevant. METHODS: To examine attentional biases for sex-related words among sexually active individuals and the relationship between sexual compulsivity and sexual behavioural engagement with attentional bias, 55 sexually active individuals completed a modified Stroop task and the sexual compulsivity scale. RESULTS: Findings showed attentional bias towards sex-related stimuli among sexually active participants. In addition, among those with low levels of sexual compulsivity, levels of attentional bias were the same across all levels of sexual experience. Among those with higher levels of sexual compulsivity, greater attentional bias was linked with lower levels of sexual experience. CONCLUSION: Attentional preference for concern-related stimuli varies as a function of the interaction between how long a person has been active sexually and how compulsive their sexual behaviour is.

A new phosphate-starvation response in fission yeast requires the endocytic function of myosin I.

Endocytosis is essential for uptake of many substances into the cell, but how it links to nutritional signalling is poorly understood. Here, we show a new role for endocytosis in regulating the response to low phosphate in Schizosaccharomyces pombe. Loss of function of myosin I (Myo1), Sla2/End4 or Arp2, proteins involved in the early steps of endocytosis, led to increased proliferation in low-phosphate medium compared to controls. We show that once cells are deprived of phosphate they undergo a quiescence response that is dependent on the endocytic function of Myo1. Transcriptomic analysis revealed a wide perturbation of gene expression with induction of stress-regulated genes upon phosphate starvation in wild-type but not Δmyo1 cells. Thus, endocytosis plays a pivotal role in mediating the cellular response to nutrients, bridging the external environment and internal molecular functions of the cell.

Fission yeast Iec1-ino80-mediated nucleosome eviction regulates nucleotide and phosphate metabolism.

Ino80 is an ATP-dependent nucleosome-remodeling enzyme involved in transcription, replication, and the DNA damage response. Here, we characterize the fission yeast Ino80 and find that it is essential for cell viability. We show that the Ino80 complex from fission yeast mediates ATP-dependent nucleosome remodeling in vitro. The purification of the Ino80-associated complex identified a highly conserved complex and the presence of a novel zinc finger protein with similarities to the mammalian transcriptional regulator Yin Yang 1 (YY1) and other members of the GLI-Krüppel family of proteins. Deletion of this Iec1 protein or the Ino80 complex subunit arp8, ies6, or ies2 causes defects in DNA damage repair, the response to replication stress, and nucleotide metabolism. We show that Iec1 is important for the correct expression of genes involved in nucleotide metabolism, including the ribonucleotide reductase subunit cdc22 and phosphate- and adenine-responsive genes. We find that Ino80 is recruited to a large number of promoter regions on phosphate starvation, including those of phosphate- and adenine-responsive genes that depend on Iec1 for correct expression. Iec1 is required for the binding of Ino80 to target genes and subsequent histone loss at the promoter and throughout the body of these genes on phosphate starvation. This suggests that the Iec1-Ino80 complex promotes transcription through nucleosome eviction.

The regulation of ATP-dependent nucleosome remodelling factors.

The plasticity of chromatin is governed by multi-subunit protein complexes that enzymatically regulate chromosomal structure and activity. Such complexes include ATP-dependent chromatin remodelling factors that are involved in many fundamental processes such as transcription, DNA repair, replication and chromosome structure maintenance. Because ATP-dependent chromatin remodelling factors play important roles, it is not surprising to find that their functions are regulated in a plethora of ways, including post-translational modifications of their subunits and subunit composition changes. The activity of these enzymes is modulated by many factors, including linker histones, histone variants, histone chaperones, non-histone chromatin constituents such as HMG-proteins and secondary messengers, such as inositolpolyphosphates. Additionally, specific histone modifications and interaction with site-specific transcriptional regulators direct the targeting of these activities. Understanding the network of mechanisms that control ATP-dependent chromatin remodelling will constitute an important challenge towards our understanding of chromatin dynamics.