Elasticity of Carrier Fluid: A Key Factor Affecting Mechanical Phenotyping in Deformability Cytometry.

Recently, microfluidics deformability cytometry has emerged as a powerful tool for high-throughput mechanical phenotyping of large populations of cells. These methods characterize cells by their mechanical fingerprints by exerting hydrodynamic forces and monitoring the resulting deformation. These devices have shown great promise for label-free cytometry, yet there is a critical need to improve their accuracy and reconcile any discrepancies with other methods, such as atomic force microscopy. In this study, we employ computational fluid dynamics simulations and uncover how the elasticity of frequently used carrier fluids, such as methylcellulose dissolved in phosphate-buffered saline, is significantly influential to the resulting cellular deformation. We conducted CFD simulations conventionally used within the deformability cytometry field, which neglect fluid elasticity. Subsequently, we incorporated a more comprehensive model that simulates the viscoelastic nature of the carrier fluid. A comparison of the predicted stresses between these two approaches underscores the significance of the emerging elastic stresses in addition to the well-recognized viscous stresses along the channel. Furthermore, we utilize a two-phase flow model to predict the deformation of a promyelocyte (i.e., HL-60 cell type) within a hydrodynamic constriction channel. The obtained results highlight a substantial impact of the elasticity of carrier fluid on cellular deformation and raise questions about the accuracy of mechanical property estimates derived by neglecting elastic stresses.

Multidrug-resistant Shigella flexneri outbreak associated with a high-mortality spillover event into nonhuman primates.

Shigellosis is a gastrointestinal infection caused by species of Shigella. A large outbreak of Shigella flexneri serotype 2a occurred in Albuquerque, New Mexico (NM) between May 2021 and November 2023 that involved humans and nonhuman primates (NHP) from a local zoo. We analyzed the genomes of 202 New Mexico isolates as well as 15 closely related isolates from other states, and four from NHP. The outbreak was initially detected within men who have sex with men (MSM) but then predominantly affected people experiencing homelessness (PEH). Nearly 70% of cases were hospitalized and there was one human death. The outbreak extended into Albuquerque's BioPark Zoo, causing high morbidity and six deaths in NHPs. The NHP isolates were identical to those in the human outbreak. All isolates were multidrug-resistant, including towards fluoroquinolones, a first line treatment option which led to treatment failures in human and NHP populations. We demonstrate the transmission of this S. flexneri strain between humans and NHPs, causing fatalities in both populations. This study demonstrates the threat of antimicrobial resistant organisms to vulnerable human and primate populations and emphasizes the value of vigilant genomic surveillance within a One Health framework.

Fluorescence Lifetime Measurements and Analyses: Protocols Using Flow Cytometry and High-Throughput Microscopy.

This chapter focuses on applications and protocols that involve the measurement of the fluorescence lifetime as an informative cytometric parameter. The timing of fluorescence decay has been well-studied for cell counting, sorting, and imaging. Therefore, provided herein is an overview of the techniques used, how they enhance cytometry protocols, and the modern techniques used for lifetime analysis. The background and theory behind fluorescence decay kinetic measurements in cells is first discussed followed by the history of the development of time-resolved flow cytometry. These sections are followed by a review of applications that benefit from the quantitative nature of fluorescence lifetimes as a photophysical trait. Lastly, perspectives on the modern ways in which the fluorescence lifetime is scanned at high throughputs which include high-speed microscopy and machine learning are provided.

Exploitation of elasto-inertial fluid flow for the separation of nano-sized particles: Simulating the isolation of extracellular vesicles.

High throughput and efficient separation/isolation of nanoparticles such as exosomes remain a challenge owing to their small size. Elasto-inertial approaches have a new potential to be leveraged because of the ability to achieve fine control over the forces that act on extremely small particles. That is, the viscoelasticity of fluid that helps carry biological particles such as extracellular vesicles (EVs) and cells through microfluidic channels can be tailored to optimize how different-sized particles move within the chip. In this contribution, we demonstrate through computational fluid dynamics (CFD) simulations the ability to separate nanoparticles with a size comparable to exosomes from larger spheres with physical properties comparable to cells and larger EVs. Our current design makes use of an efficient flow-focusing geometry at the inlet of the device in which two side channels deliver the sample, while the inner channel injects the sheath flow. Such flow configuration results in an efficient focusing of all the particles near the sidewalls of the channel at the inlet. By dissolving a minute amount of polymer in the sample and sheath fluid, the elastic lift force arises and the initially focused particle adjacent to the wall will gradually migrate toward the center of the channel. This results in larger particles experiencing larger elastic forces, thereby migrating faster toward the center of the channel. By adjusting the size and location of the outlets, nanoparticles comparable to the size of exosomes (30-100 nm) will be effectively separated from other particles. Furthermore, the influence of different parameters such as channel geometry, flow rate, and fluid rheology on the separation process is evaluated by computational analysis.

Toward the Development of an On-Chip Acoustic Focusing Fluorescence Lifetime Flow Cytometer.

Conventional flow cytometry is a valuable quantitative tool. Flow cytometers reveal physical and biochemical information from cells at a high throughput, which is quite valuable for many biomedical, biological, and diagnostic research fields. Flow cytometers range in complexity and typically provide multiparametric data for the user at rates of up to 50,000 cells measured per second. Cytometry systems are configured such that fluorescence or scattered light signals are collected per-cell, and the integrated optical signal at a given wavelength range indicates a particular cellular feature such as phenotype or morphology. When the timing of the optical signal is measured, the cytometry system becomes "time-resolved." Time-resolved flow cytometry (TRFC) instruments can detect fluorescence decay kinetics, and such measurements are consequential for Förster Resonance Energy Transfer (FRET) studies, multiplexing, and metabolic mapping, to name a few. TRFC systems capture fluorescence lifetimes at rates of thousands of cells per-second, however the approach is challenged at this throughput by terminal cellular velocities. High flow rates limit the total number of photons integrated per-cell, reducing the reliability of the average lifetime as a cytometric parameter. In this contribution, we examine an innovative approach to address this signal-to-noise issue. The technology merges time-resolved hardware with microfluidics and acoustics. We present an "acoustofluidic" time-resolved flow cytometer so that cellular velocities can be adjusted on the fly with a standing acoustic wave (SAW). Our work shows that acoustic control can be combined with time-resolved features to appropriately balance the throughput with the optical signals necessary for lifetime data.

A Review of New High-Throughput Methods Designed for Fluorescence Lifetime Sensing From Cells and Tissues.

Though much of the interest in fluorescence in the past has been on measuring spectral qualities such as wavelength and intensity, there are two other highly useful intrinsic properties of fluorescence: lifetime (or decay) and anisotropy (or polarization). Each has its own set of unique advantages, limitations, and challenges in detection when it comes to use in biological studies. This review will focus on the property of fluorescence lifetime, providing a brief background on instrumentation and theory, and examine the recent advancements and applications of measuring lifetime in the fields of high-throughput fluorescence lifetime imaging microscopy (HT-FLIM) and time-resolved flow cytometry (TRFC). In addition, the crossover of these two methods and their outlooks will be discussed.

Investigating differences between tamoxifen resistant and sensitive breast cancer cells with flow cytometry.

The active metabolite of tamoxifen, 4-hydroxytamoxifen, functions as an anti-estrogen in breast cancer cells and thus inhibits proliferation. While tamoxifen continues to be successfully used to treat estrogen-dependent breast cancer, most patients receiving treatment will develop chemoresistance over time. Two commonly reported biomarkers of tamoxifen resistance are decreased expression of insulin-like growth factor 1 receptor (IGF-1R) and increased expression of epidermal growth factor receptor (EGFR). In prior work we have shown that these receptors facilitate chemoresistance and have unique regulatory functions measurable in resistant cell lines compared with nonresistant. Thus, we hypothesized that these receptors and a newly identified biomarker, integrin ß1, may be used to search for the presence of resistant breast cancer cells within a population of cells that are sensitive to tamoxifen therapy. We tested this by designing a straightforward cell-labeling approach to measure differences in the receptor expression of resistant vs. sensitive cells cytometrically. Our results show that separation is possible when observing the expression of IGF-1R as well as integrin ß1. Interestingly, we found no detectable difference in EGFR expression between tamoxifen resistant and -sensitive cells when measured with cytometry despite the fact that EGFR is upregulated in resistant cells. Our long-term goal is to utilize sorting to isolate tamoxifen resistant subpopulations of cells by receptor expression level. Isolating rare resistant cells that reside within a population of drug-sensitive cells will offer new insights into why chemoresistance occurs.

Evaluation of Caspase-3 Activity During Apoptosis with Fluorescence Lifetime-Based Cytometry Measurements and Phasor Analyses.

Caspase-3 is a well-described protease with many roles that impact the fate of a cell. During apoptosis, caspase-3 acts as an executioner caspase with important proteolytic functions that lead to the final stages of programmed cell death. Owing to this key role, caspase-3 is exploited intracellularly as a target of control of apoptosis for therapeutic outcomes. Yet the activation of caspase-3 during apoptosis is challenged by other roles and functions (e.g., paracrine signaling). This brief report presents a way to track caspase-3 levels using a flow cytometer that measures excited state fluorescence lifetimes and a signal processing approach that leads to a graphical phasor-based interpretation. An established Förster resonance energy transfer (FRET) bioprobe was used for this test; the connected donor and acceptor fluorophore is cleavable by caspase-3 during apoptosis induction. With the cell-by-cell decay kinetic data and phasor analyses we generate a caspase activation trajectory, which is used to interpret activation throughout apoptosis. When lifetime-based cytometry is combined with a FRET bioprobe and phasor analyses, enzyme activation can be simplified and quantified with phase and modulation data. We envision extrapolating this approach to high content screening, and reinforce the power of phasor approaches with cytometric data. Analyses such as these can be used to cluster cells by their phase and modulation "lifetime fingerprint" when the intracellular fluorescent probe is utilized as a sensor of enzyme activity. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.

Resilience in Children Exposed to Violence: A Meta-analysis of Protective Factors Across Ecological Contexts.

Children who experience violence in their families and communities are at increased risk for a wide range of psychological and behavioral difficulties, but some exhibit resilience, or adaptive functioning following adversity. Understanding what promotes resilience is critical for developing more effective prevention and intervention strategies. Over 100 studies have examined potential protective factors for children exposed to violence in the past 30 years, but there has been no quantitative review of this literature. In order to identify which protective factors have received the strongest empirical support, we conducted a meta-analysis of 118 studies involving 101,592 participants. We separately evaluated cross-sectional (n = 71) and longitudinal (n = 47) studies testing bivariate, additive, and buffering effects for eleven proposed protective factors. Effect sizes generally were stronger in cross-sectional than longitudinal studies, but four protective factors-self-regulation, family support, school support, and peer support-demonstrated significant additive and/or buffering effects in longitudinal studies. Results were consistent across type of violence experienced (i.e., maltreatment, intimate partner violence, community violence). The review highlights the most robust predictors of resilience, identifies limitations of this work, and offers directions for improving our understanding of the processes and programs that foster resilience in children exposed to violence.

Fluorescence lifetime shifts of NAD(P)H during apoptosis measured by time-resolved flow cytometry.

Autofluorescence from the intracellular metabolite, NAD(P)H, is a biomarker that is widely used and known to reliably screen and report metabolic activity as well as metabolic fluctuations within cells. As a ubiquitous endogenous fluorophore, NAD(P)H has a unique rate of fluorescence decay that is altered when bound to coenzymes. In this work we measure the shift in the fluorescence decay, or average fluorescence lifetime (1-3 ns), of NAD(P)H and correlate this shift to changes in metabolism that cells undergo during apoptosis. Our measurements are made with a flow cytometer designed specifically for fluorescence lifetime acquisition within the ultraviolet to violet spectrum. Our methods involved culture, treatment, and preparation of cells for cytometry and microscopy measurements. The evaluation we performed included observations and quantification of the changes in endogenous emission owing to the induction of apoptosis as well as changes in the decay kinetics of the emission measured by flow cytometry. Shifts in NAD(P)H fluorescence lifetime were observed as early as 15 min post-treatment with an apoptosis inducing agent. Results also include a phasor analysis to evaluate free to bound ratios of NAD(P)H at different time points. We defined the free to bound ratios as the ratio of 'short-to-long' (S/L) fluorescence lifetime, where S/L was found to consistently decrease with an increase in apoptosis. With a quantitative framework such as phasor analysis, the short and long lifetime components of NAD(P)H can be used to map the cycling of free and bound NAD(P)H during the early-to-late stages of apoptosis. The combination of lifetime screening and phasor analyses provides the first step in high throughput metabolic profiling of single cells and can be leveraged for screening and sorting for a range of applications in biomedicine. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Evaluating integrin activation with time-resolved flow cytometry.

Förster resonance energy transfer (FRET) continues to be a useful tool to study movement and interaction between proteins within living cells. When FRET as an optical technique is measured with flow cytometry, conformational changes of proteins can be rapidly measured cell-by-cell for the benefit of screening and profiling. We exploit FRET to study the extent of activation of α4ß1 integrin dimers expressed on the surface of leukocytes. The stalk-like transmembrane heterodimers when not active lay bent and upon activation extend outward. Integrin extension is determined by changes in the distance of closest approach between an FRET donor and acceptor, bound at the integrin head and cell membrane, respectively. Time-resolved flow cytometry analysis revealed donor emission increases up to 17%, fluorescence lifetime shifts over 1.0 ns during activation, and FRET efficiencies of 37% and 26% corresponding to the inactive and active integrin state, respectively. Last, a graphical phasor analysis, including population clustering, gating, and formation of an FRET trajectory, added precision to a comparative analysis of populations undergoing FRET, partial donor recovery, and complete donor recovery. This work establishes a quantitative cytometric approach for profiling fluorescence donor decay kinetics during integrin conformational changes on a single-cell level.

Young infants expect an unfamiliar adult to comfort a crying baby: Evidence from a standard violation-of-expectation task and a novel infant-triggered-video task.

Do infants expect individuals to act prosocially toward others in need, at least in some contexts? Very few such expectations have been uncovered to date. In three experiments, we examined whether infants would expect an adult alone in a scene with a crying baby to attempt to comfort the baby. In the first two experiments, 12- and 4-month-olds were tested using the standard violation-of-expectation method. Infants saw videotaped events in which a woman was performing a household chore when a baby nearby began to cry; the woman either comforted (comfort event) or ignored (ignore event) the baby. Infants looked significantly longer at the ignore than at the comfort event, and this effect was eliminated if the baby laughed instead of cried. In the third experiment, 8-month-olds were tested using a novel forced-choice violation-of-expectation method, the infant-triggered-video method. Infants faced two computer monitors and were first shown that touching the monitors triggered events: One monitor presented the comfort event and the other monitor presented the ignore event. Infants then chose which event they wanted to watch again by touching the corresponding monitor. Infants significantly chose the ignore over the comfort event, and this effect was eliminated if the baby laughed. Thus, across ages and methods, infants provided converging evidence that they expected the adult to comfort the crying baby. These results indicate that expectations about individuals' actions toward others in need are already present in the first year of life, and, as such, they constrain theoretical accounts of early prosociality and morality.

Overview of Fluorescence Lifetime Measurements in Flow Cytometry.

The focus of this chapter is time-resolved flow cytometry, which is broadly defined as the ability to measure the timing of fluorescence decay from excited fluorophores that pass through cytometers or high-throughput cell counting and cell sorting instruments. We focus on this subject for two main reasons: first, to discuss the nuances of hardware and software modifications needed for these measurements because currently, there are no widespread time-resolved cytometers nor a one-size-fits-all approach; and second, to summarize the application space for fluorescence lifetime-based cell counting/sorting owing to the recent increase in the number of investigators interested in this approach. Overall, this chapter is structured into three sections: (1) theory of fluorescence decay kinetics, (2) modern time-resolved flow cytometry systems, and (3) cell counting and sorting applications. These commentaries are followed by conclusions and discussion about new directions and opportunities for fluorescence lifetime measurements in flow cytometry.

Shifts in the fluorescence lifetime of EGFP during bacterial phagocytosis measured by phase-sensitive flow cytometry.

Phase-sensitive flow cytometry (PSFC) is a technique in which fluorescence excited state decay times are measured as fluorescently labeled cells rapidly transit a finely focused, frequency-modulated laser beam. With PSFC the fluorescence lifetime is taken as a cytometric parameter to differentiate intracellular events that are challenging to distinguish with standard flow cytometry. For example PSFC can report changes in protein conformation, expression, interactions, and movement, as well as differences in intracellular microenvironments. This contribution focuses on the latter case by taking PSFC measurements of macrophage cells when inoculated with enhanced green fluorescent protein (EGFP)-expressing E. coli. During progressive internalization of EGFP-E. coli, fluorescence lifetimes were acquired and compared to control groups. It was hypothesized that fluorescence lifetimes would correlate well with phagocytosis because phagosomes become acidified and the average fluorescence lifetime of EGFP is known to be affected by pH. We confirmed that average EGFP lifetimes consistently decreased (3 to 2 ns) with inoculation time. The broad significance of this work is the demonstration of how high-throughput fluorescence lifetime measurements correlate well to changes that are not easily tracked by intensity-only cytometry, which is affected by heterogeneous protein expression, cell-to-cell differences in phagosome formation, and number of bacterium engulfed.

Predicting aggression in late adolescent romantic relationships: A short-term longitudinal study.

This study sought to prospectively predict aggression in the romantic relationships of 1180 college students from the United States (807 females; 373 males) over the course of two months with a set of intrapersonal risk and protective factors, including personality characteristics that rarely have been examined in this population. After accounting for prior dating aggression, perpetration of verbal aggression was predicted uniquely by aggressive attitudes, emotion regulation, and for females, narcissism. Perpetration of physical aggression was predicted by aggressive attitudes, but only at low levels of emotion regulation, and the interaction of callous-unemotional traits, emotion regulation, and gender: males with low levels of callous-unemotional traits perpetrated less physical aggression when they reported greater emotion regulation. These findings are among the first to show that personality traits and emotion regulation prospectively predict partner aggression in late adolescence and suggest mechanisms for continuity in interpersonal aggression from early adolescence to adulthood.