Best practices for user consultation in flow cytometry shared resource laboratories.

This "Best Practices in User Consultation" article is the result of a 2022 International Society for the Advancement of Cytometry (ISAC) membership survey that collected valuable insights from the shared research laboratory (SRL) community and of a group discussion at the CYTO 2022 workshop of the same name. One key takeaway is the importance of initiating a consultation at the outset of a flow cytometry project, particularly for trainees. This approach enables the improvement and standardization of every step, from planning experiments to interpreting data. This proactive approach effectively mitigates experimental bias and avoids superfluous trial and error, thereby conserving valuable time and resources. In addition to guidelines, the optimal approaches for user consultation specify communication channels, methods, and critical information, thereby establishing a structure for productive correspondence between SRL and users. This framework functions as an exemplar for establishing robust and autonomous collaborative relationships. User consultation adds value by providing researchers with the necessary information to conduct reproducible flow cytometry experiments that adhere to scientific rigor. By following the steps, instructions, and strategies outlined in these best practices, an SRL can readily tailor them to its own setting, establishing a personalized workflow and formalizing user consultation services. This article provides a pragmatic guide for improving the caliber and efficacy of flow cytometry research and aggregates the flow cytometry SRL community's collective knowledge regarding user consultation.

Direct measurement of the structural change associated with amorphous solidification using static scattering of coherent radiation.

In this paper, we demonstrate that the weak temperature dependence of the structure factor of supercooled liquids, a defining feature of the glass transition, is a consequence of the averaging of the scattering intensity due to angular averaging. We show that the speckle at individual wavevectors, calculated from a simulated glass former, exhibits a Debye-Waller factor with a sufficiently large temperature dependence to represent a structural order parameter capable of distinguishing liquid from glass. We also extract from the speckle intensities a quantity proportional to the variance of the local restraint, i.e., a direct experimental measure of the amplitude of structural heterogeneity.

Comparison of electrostatic and mechanical cell sorting with limited starting material.

Isolation of multiple cell populations from limited starting material and with minimal influence on cell homeostasis and viability are common requirements in both basic and clinical research. Fluorescence-activated cell sorting (FACS) is the most commonly applied sorting methodology with the majority of instruments being based on high pressure and electrostatic deflection. A more recent technology is based on a mechanical valve, operating at low pressure. In the present work we compared the two technologies by parallel sorting of small amounts of peripheral blood and umbilical cord blood on a BD FACSAria™ III and Miltenyi MACSQuant® Tyto® instrument. Concurrent manually performed magnetic-based cell sorting served as reference. Sorting metrics, including purity and viability, were compared. Expression of the heat-shock protein HSPA1A immediately post sorting and the proliferation potential of sorted T-cells in vitro was assessed. In general, there was little to distinguish the two fluorescence-activated technologies with regard to sorting metrics and HSPA1A expression. Variation, however, with respect to recovery and viability, was much smaller among Tyto sorted samples. The proliferation potential of Tyto-sorted T-cells was significantly higher compared to Aria-sorted T-cells, indicating that T-cells of the Tyto instrument are less perturbed. In summary, cell types of blood origin including CD34+ cells could effectively be isolated from small input amounts with either fluorescence-activated technology with little immediate effect on viability. The mechanical valve-based sorting by the Tyto instrument; however, appeared to perturb the cells to a lesser extent as judged by their proliferation potential.

Equilibrium distribution functions: connection with microscopic dynamics.

Standard textbook derivations of the equilibrium distribution function rely on assumptions that may not satisfy all readers. Here, we present a straightforward approach to derive the equilibrium distribution function from the microscopic dynamics, and review how it can be used to obtain the expected expressions. In molecular dynamics simulations the equations of motion are often modified to simulate different ensembles or phenomena. We show that in some cases these equations will sample an equilibrium ensemble whereas in other cases they will not. For example, we find that for charged particles driven by a field, an equilibrium distribution is only possible when the system is confined. Furthermore, the approach correctly predicts that neither SLLOD shear flow dynamics nor constant temperature dynamics with a Berendsen thermostat sample any time-independent phase space distributions.

Six weeks of intensive rehearsals for the Swan Lake ballet shows ultrasound tissue characterization changes of the Achilles tendons in dancers.

The objective was to investigate, first, whether six weeks of intensive ballet dance exposure is associated with structural and clinical changes in the Achilles tendon; second, the importance of demographics, self-reported Achilles pain, and generalized joint hypermobility (GJH). Data were collected at baseline and at six weeks' follow-up, using Achilles tendon ultrasound tissue characteristics (UTC) as primary outcome (percentage distribution of echo-type I-IV: type I = intact and aligned bundles, type II = discontinuous/wavy bundles, type III = fibrillar, and type IV = amorphous cells/fluid). Secondary outcomes included clinical signs of Achilles tendinopathy, Achilles tendon pain during single-leg heel raise, self-reported symptoms (VISA-A questionnaire), and GJH. Sixty-three ballet dancers (aged 18-41) participated. From baseline to follow-up, UTC echo-type I decreased significantly (ß = -3.6, p = 0.001; 95% CI: -5.8;-1.4), whereas echo-type II increased significantly (ß = 3.2, p < 0.0001, 95% CI: 1.6;4.8). Furthermore, a significant effect of limb (left limb showed decreased echo-type I and increased echo-type III + IV) and sex (women showed decreased echo-type I and increased in type II) was found. No significant changes in the remaining secondary outcomes were found. Ballet dancers showed structural changes in UTC, corresponding to a decreased echo-type I distribution after six weeks of rehearsing for Swan Lake ballet. No changes in self-reported symptoms, clinical signs of Achilles tendinopathy, and single-leg heel raise test were seen from pre- to post-rehearsal. Thus, UTC changes in the Achilles tendon seem to appear earlier than clinical signs of tendinopathy.

Application Settings versus Fixed Photomultiplier Tube Voltages for Optimal Cytometer Stability over Time, and Effect on Reusing a Compensation Matrix.

In flow cytometry, a compensation matrix is commonly reused over time, especially in clinical laboratories, to save time and reagents. However, generating a same-day compensation matrix is considered the best practice by many experts. BD Biosciences developed Cytometer Setup and Tracking software to deliver proper instrument characterization, performance tracking, and stability. BD's "Application Settings" enable daily cytometer adjustments of photomultiplier tube (PMT) settings to correct for day-to-day variations in instrument performance. Here, we investigated if using Application Settings would improve data stability over time, including the impact on data stability when reusing a compensation matrix. We consecutively analyzed peripheral blood mononuclear cell (PBMC) aliquots from a single healthy donor together with 8-peak Rainbow beads and daily compensation controls (22 runs in total over 6.5 months). We found larger variation within both PBMC subset quantifications and median fluorescence intensity (MFI) levels when using Application Settings (i.e., daily adjusted PMTs) compared to fixed PMT voltages (both with the same Day 0 compensation matrix applied). This larger variation was partly due to errors in compensation, but was also seen for Rainbow beads MFI data (not impacted by compensation), and thus likely produced by imprecise adjustments of PMTs by Applications Settings. Notably, the larger variation observed with Application Settings was most pronounced on a few days of the experiment with very large deviations, whereas on most days Application Settings and Fixed PMTs performed similar. The present results call for caution in using Application Settings in longitudinal studies, especially if also reusing a compensation matrix. In contrast, reusing a compensation matrix over time with fixed PMT voltages yielded stable results comparable with running same-day compensation controls. © 2020 International Society for Advancement of Cytometry.

Geometrical Frustration and Planar Triangular Antiferromagnetism in Quasi-Three-Dimensional Artificial Spin Architecture.

We present a realization of highly frustrated planar triangular antiferromagnetism achieved in a quasi-three-dimensional artificial spin system consisting of monodomain Ising-type nanomagnets lithographically arranged onto a deep-etched silicon substrate. We demonstrate how the three-dimensional spin architecture results in the first direct observation of long-range ordered planar triangular antiferromagnetism, in addition to a highly disordered phase with short-range correlations, once competing interactions are perfectly tuned. Our work demonstrates how escaping two-dimensional restrictions can lead to new types of magnetically frustrated metamaterials.

Improved microRNA suppression by WPRE-linked tough decoy microRNA sponges.

Our genes are post-transcriptionally regulated by microRNAs (miRNAs) inducing translational suppression and degradation of targeted mRNAs. Strategies to inhibit miRNAs in a spatiotemporal manner in a desired cell type or tissue, or at a desired developmental stage, can be crucial for understanding miRNA function and for pushing forward miRNA suppression as a feasible rationale for genetic treatment of disease. For such purposes, RNA polymerase II (RNA Pol II)-transcribed tough decoy (TuD) miRNA inhibitors are particularly attractive. Here, we demonstrate augmented miRNA suppression capacity of TuD RNA hairpins linked to the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). This effect is position-dependent and evident only when the WPRE is positioned upstream of the TuD. In accordance, inclusion of the WPRE does not change nuclear export, translation, total levels of TuD-containing RNA transcripts, or cytoplasmic P-body localization, suggesting that previously reported WPRE functions are negligible for improved TuD function. Notably, deletion analysis of TuD-fused WPRE unveils truncated WPRE variants resulting in optimized miRNA suppression. Together, our findings add to the guidelines for production of WPRE-supported anti-miRNA TuDs.

Anomalous transport in the soft-sphere Lorentz model.

The sensitivity of anomalous transport in crowded media to the form of the inter-particle interactions is investigated through computer simulations. We extend the highly simplified Lorentz model towards realistic natural systems by modeling the interactions between the tracer and the obstacles with a smooth potential. We find that the anomalous transport at the critical point happens to be governed by the same universal exponent as for hard exclusion interactions, although the mechanism of how narrow channels are probed is rather different. The scaling behavior of simulations close to the critical point confirm this exponent. Our result indicates that the simple Lorentz model may be applicable to describing the fundamental properties of long-range transport in real crowded environments.

Dissipation in monotonic and non-monotonic relaxation to equilibrium.

Using molecular dynamics simulations, we study field free relaxation from a non-uniform initial density, monitored using both density distributions and the dissipation function. When this density gradient is applied to colour labelled particles, the density distribution decays to a sine curve of fundamental wavelength, which then decays conformally towards a uniform distribution. For conformal relaxation, the dissipation function is found to decay towards equilibrium monotonically, consistent with the predictions of the relaxation theorem. When the system is initiated with a more dramatic density gradient, applied to all particles, non-conformal relaxation is seen in both the dissipation function and the Fourier components of the density distribution. At times, the system appears to be moving away from a uniform density distribution. In both cases, the dissipation function satisfies the modified second law inequality, and the dissipation theorem is demonstrated.

Growing older in the context of needing geriatric assessment: a qualitative study.

AIM: The number of older adults will increase worldwide in the next 30 years, with many expected to develop chronic diseases and consequently require additional medical assessments and adequate care. The aim of this study was to describe how a group of older adults who need geriatric assessment experience growing older. METHODOLOGY: A qualitative study was performed. The empirical data consisted of qualitative in-depth interviews with eight older persons referred to geriatric assessment (median age 76, range 65-86). The interviews were analysed based on Giorgi's descriptive phenomenological methodology. RESULTS: The results led to an overall essence and five themes. The five themes were identified: (i) considerations to avoid weakness, (ii) compensation for the inability to perform certain activities, (iii) aides - a symbol of freedom or limitation, (iv) lifestyle considered being particularly significant and (v) considerations about the end of life as a component of growing older. The essence was defined as: illness essential for the experience of growing older and pointed to the experience of growing older that highlighted that experiencing illnesses and limitations served as reminders of their advancing age. CONCLUSION: Growing older while in need of comprehensive geriatric assessment led to participants feeling they were no longer able to do the same things, they used to do. Consequently, compensation became an essential part of adaption to the altered situation. Aides that helped to overcome limitations in every daily life had the status of either creating freedom or being limiting in things they wished to do. Considerations about lifestyle were focused on housing and change of dwelling. This also led to thoughts about the future and end of life. Additional research regarding accommodations in older age is warranted to offer the best solutions for older people.

Cell sorting enables interphase fluorescence in situ hybridization detection of low BCR-ABL1 producing stem cells in chronic myeloid leukaemia patients beyond deep molecular remission.

The exact disease state of chronic myeloid leukaemia (CML) patients in deep molecular remission is unknown, because even the most sensitive quantitative reverse transcription polymerase chain reaction (qPCR) methods cannot identify patients prone to relapse after treatment withdrawal. To elucidate this, CD34(+) stem cell and progenitor cell subpopulations were isolated by fluorescence-activated cell sorting (FACS), and their content of residual Philadelphia positive (Ph(+) ) cells was evaluated in 17 CML patients (major molecular response, n = 6; 4-log reduction in BCR-ABL1 expression (MR(4) ), n = 11) using both sensitive qPCR and interphase fluorescence in situ hybridization (iFISH). Despite evaluating fewer cells, iFISH proved superior to mRNA-based qPCR in detecting residual Ph(+) stem cells (P = 0·005), and detected Ph(+) stem- and progenitor cells in 9/10 patients at frequencies of 2-14%. Moreover, while all qPCR(+) samples also were iFISH(+) , 9/33 samples were qPCR-/iFISH(+) , including all positive samples from MR(4) patients. Our findings show that residual Ph(+) cells are low BCR-ABL1 producers, and that DNA-based methods are required to assess the content of persisting Ph(+) stem cells in these patients. This approach demonstrates a clinically applicable manner of assessing residual disease at the stem cell level in CML patients in MR(4) , and may enable early and safe identification of candidates for tyrosine kinase inhibitor withdrawal.

Practicalities of Cell Sorting.

Cell sorting is a technique commonly used in academic and biotechnology laboratories in order to separate out cells or particles of interest from heterogeneous populations. Cell sorters use the same principles as flow cytometry analyzers, but instead of cell populations passing to the waste of the instrument, they can be collected for further studies including DNA sequencing as well as other genomic, in vitro and in vivo experiments. This chapter aims to give an overview of cell sorting, the different types of cell sorters, details on how a cell sorter works, as well as protocols that are useful when embarking on a journey with cell sorting.

The instantaneous fluctuation theorem.

We give a derivation of a new instantaneous fluctuation relation for an arbitrary phase function which is odd under time reversal. The form of this new relation is not obvious, and involves observing the system along its transient phase space trajectory both before and after the point in time at which the fluctuations are being compared. We demonstrate this relation computationally for a number of phase functions in a shear flow system and show that this non-locality in time is an essential component of the instantaneous fluctuation theorem.

Investigation of sleep and circadian rhythm disorders.

Diagnosis of a sleep disorders is multimodal. An overview is presented in this review. The medical history leads to a tentative diagnosis supported by questionnaires, sleep diary and objective methods. Examination may reveal upper airway problems in a patient suspected to have obstructive sleep apnoea or rigidity in an elderly patient with shouting during sleep, suggestive of rapid eye movement sleep behaviour disorder. The choice of diagnostic sleep test is based on the tentative diagnosis. Other tests (e.g., lumbar puncture, brain scan) may be indicated. Wearables pose the advantage of documenting the patients' habitual sleep and circadian rhythm.

A piriformis-preserving posterior approach reduces dislocation rate of the hemiarthroplasty in patients with femoral neck fracture.

INTRODUCTION: The posterior approach (PA) for hemiarthroplasty (HA) in patients with femoral neck fracture (FFN) has a high risk of dislocation; however, by preserving the piriformis muscle, the dislocation rate could be lowered considerably. The aim of this study was to compare the surgical complications of the piriformis-preserving posterior approach (PPPA) and the PA in patients with FNF treated with HA. METHODS: On 1 January 2019, the PPPA was introduced at two hospitals as the new treatment standard. The sample size was calculated based on a 5 percentage point dislocation reduction and 25% censoring; a sample of 264 patients per group was determined. An approximately 2-year inclusion period with 1-year follow-up was estimated, including a historical cohort from 2 years before the PPPA introduction. Data were retrieved from health care records and X-ray images from the hospitals' administrative databases. Relative risk (RR) and 95% confidence intervals were calculated using Cox regression and adjusted for age, sex, comorbidity, smoking, surgeon experience and implant type. RESULTS: There were 527 patients included in the study, of which 72% were women and 43% were above 85 years old. There were no baseline differences between the PPPA and PA groups in sex, age, comorbidity, body mass index, smoking, alcohol, mobility, length of surgery, blood loss or implant positioning, but there were differences in 30-day mortality, surgeon experience and implant type. The dislocation rate reduced from 11.6% in the PA group to 4.7% in the PPPA group (p = 0.004), with an RR of 2.5 (1.2; 5.1). The reoperation rate reduced from 6.8% with the PA to 3.3% with the PPPA (p = 0.022), with an RR of 2.1 (0.9; 5.2), and total surgery-related complications reduced from 14.7% with the PA to 6.9% with the PPPA (p = 0.003), with an RR of 2.4 (1.3; 4.4). INTERPRETATION: Changing from PA to PPPA in patients with FNF treated with HA resulted in a more than 50% reduction in dislocation and reoperation rates. This approach was easily introduced and may enable further lowering of dislocation rates through the sparing of all short external rotators.

Facilitating In-House Mobile App Development Within Psychiatric Outpatient Services for Patients Diagnosed With Borderline Personality Disorder: Rapid Application Development Approach.

BACKGROUND: Mobile app development within mental health is often time- and resource-consuming, challenging the development of mobile apps for psychiatry. There is a continuum of software development methods ranging from linear (waterfall model) to continuous adaption (Scrum). Rapid application development (RAD) is a model that so far has not been applied to psychiatric settings and may have some advantages over other models. OBJECTIVE: This study aims to explore the utility of the RAD model in developing a mobile app for patients with borderline personality disorder (BPD) in a psychiatric outpatient setting. METHODS: The 4 phases of the RAD model: (1) requirements planning, (2) user design, (3) construction, and (4) cutover, were applied to develop a mobile app within psychiatric outpatient services for patients diagnosed with BPD. RESULTS: For the requirements planning phase, a short time frame was selected to minimize the time between product conceptualization and access within a clinical setting. Evidenced-based interactive content already developed was provided by current staff to enhance usability and trustworthiness. For the user design phase, activity with video themes and a discrete number of functions were used to improve the app functionality and graphical user interface. For the construction phase, close collaboration between clinicians, researchers, and software developers yielded a fully functional, in-house-developed app ready to be tested in clinical practice. For the cutover phase, the mobile app was tested successfully with a small number (n=5) of patients with a BPD. CONCLUSIONS: The RAD model could be meaningfully applied in a psychiatric setting to develop an app for BPD within a relatively short time period from conceptualization to implementation in the clinic. Short time frames and identifying a limited number of stakeholders with relevant skills in-house facilitated the use of this model. Despite some limitations, RAD could be a useful model in the development of apps for clinical populations to enable development and access to evidence-based technology.

SARS-CoV-2 and autoantibodies in the cerebrospinal fluid of COVID-19 patients: prospective multicentre cohort study.

Disease mechanisms underlying neurological and neuropsychiatric symptoms after coronavirus disease 2019 (COVID-19), termed neuro-COVID, are poorly understood. Investigations of the cerebrospinal fluid (CSF) for the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and antibodies, as well as autoantibodies against neuronal surface antigens, could improve our understanding in that regard. We prospectively collected CSF and blood from patients investigated by lumbar puncture for neurological or neuropsychiatric symptoms during or after COVID-19. Primary outcomes were the presence of (i) SARS-CoV-2 RNA in CSF via polymerase chain reaction (PCR), (ii) SARS-CoV-2 immunoglobulin G (IgG) anti-S receptor-binding-domain antibodies via the Euroimmun and Wantai assays and (iii) IgG autoantibodies against neuronal surface antigens using commercial cell- and tissue-based assays (Euroimmun). Secondary outcomes were (i) routine CSF investigations and (ii) correlation between SARS-CoV-2 antibody levels in CSF with serum levels, blood-brain barrier permeability and peripheral inflammation. We obtained CSF from 38 COVID-19 patients (mean age 56.5 ± 19.2 years, 53% women) who developed neurological and neuropsychiatric symptoms. CSF pleocytosis (>5 cells) was observed in 9/38 patients (23.7%), elevated CSF protein (>0.50 g/L) in 13/38 (34.2%) and elevated CSF/serum albumin ratio in 12/35 (34.3%). PCR for SARS-CoV-2 RNA in CSF was negative in all. SARS-CoV-2 CSF antibodies were detected in 15/34 (44.1%; Euroimmun assay) and 7/31 (22.6%; Wantai assay) individuals, but there were no signs of intrathecal SARS-CoV-2 IgG production. SARS-CoV-2 CSF antibodies were positively correlated with serum levels (R = 0.93, P < 0.001), blood-brain barrier permeability (R = 0.47, P = 0.006), peripheral inflammation (R = 0.51, P = 0.002) and admission to the intensive care unit [odds ratio (OR) 17.65; 95% confidence interval (CI) 1.18-264.96; P = 0.04; n = 15]. Cell-based assays detected weakly positive NMDAR, LGI1 and CASPR2 antibodies in serum of 4/34 (11.8%) patients but not in CSF. The tissue-based assay showed anti-neuronal fluorescence in CSF from one individual, staining for Purkinje cells. In summary, whereas we did not detect active SARS-CoV-2 infection in the CSF, SARS-CoV-2 antibodies were prevalent. The absence of intrathecal antibody production points towards blood-brain barrier impairment as the origin of CSF SARS-CoV-2 antibodies. In contrast, CSF autoantibodies against neuronal surface antigens were rare. There was no evidence for a clinical correlate of these antibodies. We conclude that, rather than specific autoimmune neuronal injury, non-specific effects of critical illness including an impaired blood-brain barrier are more likely to contribute to neuro-COVID.

New Framework for Computing a General Local Self-Diffusion Coefficient Using Statistical Mechanics.

Widely applicable, modified Green-Kubo expressions for the local diffusion coefficient (Dl) are obtained using linear response theory. In contrast to past definitions in use, these expressions are statistical mechanical results. Molecular simulations of systems with anisotropic diffusion and an inhomogeneous density profile confirm the validity of the results. Diffusion coefficients determined from different expressions in terms of currents and velocity correlations agree in the limit of large systems. Furthermore, they apply to arbitrarily small local regions, making them readily applicable to nanoscale and inhomogeneous systems where knowledge of Dl is important.