A fluorogenic complementation tool kit for interrogating lipid droplet-organelle interaction.

Contact sites between lipid droplets and other organelles are essential for cellular lipid and energy homeostasis upon metabolic demands. Detection of these contact sites at the nanometer scale over time in living cells is challenging. We developed a tool kit for detecting contact sites based on fluorogen-activated bimolecular complementation at CONtact sites, FABCON, using a reversible, low-affinity split fluorescent protein, splitFAST. FABCON labels contact sites with minimal perturbation to organelle interaction. Via FABCON, we quantitatively demonstrated that endoplasmic reticulum (ER)- and mitochondria (mito)-lipid droplet contact sites are dynamic foci in distinct metabolic conditions, such as during lipid droplet biogenesis and consumption. An automated analysis pipeline further classified individual contact sites into distinct subgroups based on size, likely reflecting differential regulation and function. Moreover, FABCON is generalizable to visualize a repertoire of organelle contact sites including ER-mito. Altogether, FABCON reveals insights into the dynamic regulation of lipid droplet-organelle contact sites and generates new hypotheses for further mechanistical interrogation during metabolic regulation.

A fluorogenic complementation tool kit for interrogating lipid droplet-organelle interaction.

Contact sites between lipid droplets and other organelles are essential for cellular lipid and energy homeostasis. Detection of these contact sites at nanometer scale over time in living cells is challenging. Here, we developed a tool kit for detecting contact sites based on Fluorogen-Activated Bimolecular complementation at CONtact sites, FABCON, using a reversible, low affinity split fluorescent protein, splitFAST. FABCON labels contact sites with minimal perturbation to organelle interaction. Via FABCON, we quantitatively demonstrated that endoplasmic reticulum (ER)- and mitochondria (mito)-lipid droplet contact sites are dynamic foci in distinct metabolic conditions, such as during lipid droplet biogenesis and consumption. An automated analysis pipeline further classified individual contact sites into distinct subgroups based on size, likely reflecting differential regulation and function. Moreover, FABCON is generalizable to visualize a repertoire of organelle contact sites including ER-mito. Altogether, FABCON reveals insights into the dynamic regulation of lipid droplet-organelle contact sites and generates new hypotheses for further mechanistical interrogation during metabolic switch.

DDX3X acts as a live-or-die checkpoint in stressed cells by regulating NLRP3 inflammasome.

The cellular stress response has a vital role in regulating homeostasis by modulating cell survival and death. Stress granules are cytoplasmic compartments that enable cells to survive various stressors. Defects in the assembly and disassembly of stress granules are linked to neurodegenerative diseases, aberrant antiviral responses and cancer1-5. Inflammasomes are multi-protein heteromeric complexes that sense molecular patterns that are associated with damage or intracellular pathogens, and assemble into cytosolic compartments known as ASC specks to facilitate the activation of caspase-1. Activation of inflammasomes induces the secretion of interleukin (IL)-1ß and IL-18 and drives cell fate towards pyroptosis-a form of programmed inflammatory cell death that has major roles in health and disease6-12. Although both stress granules and inflammasomes can be triggered by the sensing of cellular stress, they drive contrasting cell-fate decisions. The crosstalk between stress granules and inflammasomes and how this informs cell fate has not been well-studied. Here we show that the induction of stress granules specifically inhibits NLRP3 inflammasome activation, ASC speck formation and pyroptosis. The stress granule protein DDX3X interacts with NLRP3 to drive inflammasome activation. Assembly of stress granules leads to the sequestration of DDX3X, and thereby the inhibition of NLRP3 inflammasome activation. Stress granules and the NLRP3 inflammasome compete for DDX3X molecules to coordinate the activation of innate responses and subsequent cell-fate decisions under stress conditions. Induction of stress granules or loss of DDX3X in the myeloid compartment leads to a decrease in the production of inflammasome-dependent cytokines in vivo. Our findings suggest that macrophages use the availability of DDX3X to interpret stress signals and choose between pro-survival stress granules and pyroptotic ASC specks. Together, our data demonstrate the role of DDX3X in driving NLRP3 inflammasome and stress granule assembly, and suggest a rheostat-like mechanistic paradigm for regulating live-or-die cell-fate decisions under stress conditions.

Performance evaluation of extended depth of field microscopy in the presence of spherical aberration and noise.

Effectiveness of extended depth of field microscopy (EDFM) implementation with wavefront encoding methods is reduced by depth-induced spherical aberration (SA) due to reliance of this approach on a defined point spread function (PSF). Evaluation of the engineered PSF's robustness to SA, when a specific phase mask design is used, is presented in terms of the final restored image quality. Synthetic intermediate images were generated using selected generalized cubic and cubic phase mask designs. Experimental intermediate images were acquired using the same phase mask designs projected from a liquid crystal spatial light modulator. Intermediate images were restored using the penalized space-invariant expectation maximization and the regularized linear least squares algorithms. In the presence of depth-induced SA, systems characterized by radially symmetric PSFs, coupled with model-based computational methods, achieve microscope imaging performance with fewer deviations in structural fidelity (e.g., artifacts) in simulation and experiment and 50% more accurate positioning of 1-µm beads at 10-µm depth in simulation than those with radially asymmetric PSFs. Despite a drop in the signal-to-noise ratio after processing, EDFM is shown to achieve the conventional resolution limit when a model-based reconstruction algorithm with appropriate regularization is used. These trends are also found in images of fixed fluorescently labeled brine shrimp, not adjacent to the coverslip, and fluorescently labeled mitochondria in live cells.

Reducing effects of aberration in 3D fluorescence imaging using wavefront coding with a radially symmetric phase mask.

In this work, a wavefront encoded (WFE) imaging system built using a squared cubic phase mask, designed to reduce the sensitivity of the imaging system to spherical aberration, is investigated. The proposed system allows the use of a space-invariant image restoration algorithm, which uses a single PSF, to restore intensity distribution in images suffering aberration, such as sample-induced aberration in thick tissue. This provides a computational advantage over depth-variant image restoration algorithms developed previously to address this aberration. Simulated PSFs of the proposed system are shown to change up to 25% compared to the 0 µm depth PSF (quantified by the structural similarity index) over a 100 µm depth range, while the conventional system PSFs change up to 84%. Results from experimental test-sample images show that restoration error is reduced by 29% when the proposed WFE system is used instead of the conventional system over a 30 µm depth range.

Spatial light modulator phase mask implementation of wavefront encoded 3D computational-optical microscopy.

Spatial light modulator (SLM) implementation of wavefront encoding enables various types of engineered point-spread functions (PSFs), including the generalized-cubic and squared-cubic phase mask wavefront encoded (WFE) PSFs, shown to reduce the impact of sample-induced spherical aberration in fluorescence microscopy. This investigation validates dynamic experimental parameter variation of these WFE-PSFs. We find that particular design parameter bounds exist, within which the divergence of computed and experimental WFE-PSFs is of the same order of magnitude as that of computed and experimental conventional PSFs, such that model-based approaches for solving the inverse imaging problem can be applied to a wide range of SLM-WFE systems. Interferometric measurements were obtained to evaluate the SLM implementation of the numeric mask. Agreement between experiment and theory in terms of a wrapped phase, 0-2π, validates the phase mask implementation and allows characterization of the SLM response. These measurements substantiate experimental practice of computational-optical microscope imaging with an SLM-engineered PSF.

Pathways to Assisted Living: The Influence of Race and Class.

This article examines how race and class influence decisions to move to assisted living facilities. Qualitative methods were used to study moving decisions of residents in 10 assisted living facilities varying in size and location, as well as race and socioeconomic status of residents. Data were derived from in-depth interviews with 60 residents, 43 family members and friends, and 12 administrators. Grounded theory analysis identified three types of residents based on their decision-making control: proactive, compliant, and passive/resistant. Only proactive residents (less than a quarter of residents) had primary control. Findings show that control of decision making for elders who are moving to assisted living is influenced by class, though not directly by race. The impact of class primarily related to assisted-living placement options and strategies available to forestall moves. Factors influencing the decision-making process were similar for Black and White elders of comparable socioeconomic status.

Quantitative phase microscopy through differential interference imaging.

An extension of Nomarski differential interference contrast microscopy enables isotropic linear phase imaging through the combination of phase shifting, two directions of shear, and Fourier space integration using a modified spiral phase transform. We apply this method to simulated and experimentally acquired images of partially absorptive test objects. A direct comparison of the computationally determined phase to the true object phase demonstrates the capabilities of the method. Simulation results predict and confirm results obtained from experimentally acquired images.

Quantitative structured-illumination phase microscopy.

We introduce a quantitative phase imaging method for homogeneous objects with a bright field transmission microscope by using an amplitude mask and a digital processing algorithm. A known amplitude pattern is imaged on the sample plane containing a thick phase object by placing an amplitude mask in the field diaphragm of the microscope. The phase object distorts the amplitude pattern according to its optical path length (OPL) profile, and the distorted pattern is recorded in a CCD detector. A digital processing algorithm then estimates the object's quantitative OPL profile based on a closed form analytical solution, which is derived using a ray optics model for objects with small OPL gradients.

Gerontology education and research in Kenya: establishing a US-African partnership in aging.

This article reprises four presentations on Gerontology Education in Kenya, a seminar at the 2004 Annual Meeting of the Association of Gerontology in Higher Education. It describes the process by which the Gerontology Institute of Georgia State University established a 3-year gerontology education and research partnership with Kenyatta University in Nairobi, Kenya, and the field experiences of two scholars who have conducted aging research in Kenya. We provide four key elements of cultural competence and recommendations for American gerontologists wishing to establish international linkages.

Evaluating the effects of an educational symposium on knowledge, impact, and self-management of older african americans living with osteoarthritis.

The purpose of the study1 was to determine the effects of a 1-day educational symposium on knowledge, impact, and self-management of older African Americans living with osteoarthritis (OA). A 1-group pre- and posttest design was employed. The educational symposium was the intervention variable. Speakers were experts in the field of arthritis care. Participants were recruited from a community senior center. Participants completed the Short Form of the Arthritis Impact Management Scales (AIMS2-SF), the Summary of Arthritis Management Methods (SAMMS), and a 6-question Knowledge Test about OA before the symposium. The knowledge test was administered again at the completion of the program. Posttesting of the AIMS2-SF and SAMMS occurred 3 months later to assess changes in arthritis impact and self-management strategies. Knowledge scores increased significantly immediately following the symposium. The AIMS2-SF subscales of Affect, Symptom, and Physical Function decreased significantly 3 months after the symposium indicating that arthritis had less impact on participants after the conference. Participants also reported using more self-management methods 3 months after the event. The results of this pilot study indicate that this symposium was effective in increasing participants' knowledge of OA, improving self-management, and decreasing the impact of OA on daily function. The small sample size and preexperimental design limit generalizability.

Managing decline in assisted living: the key to aging in place.

OBJECTIVE: This study investigates the process of aging in place in assisted living facilities (ALFs) and seeks to gain an in-depth understanding of the factors influencing this phenomenon in a variety of ALF contexts. METHODS: Qualitative methods were used to study five ALFs for 1 year. Data collection methods included participant observation; informal and in-depth interviewing of providers, residents, and residents' families; and review of resident and facility records. Analysis was conducted using the grounded theory approach. RESULTS: The ability of residents to remain in assisted living was principally a function of the "fit" between the capacity of both residents and facilities to manage decline. Multiple community, facility, and resident factors influenced the capacity to manage decline, and resident-facility fit was both an outcome and an influence on the decline management process. Resident and facility risk also was an intervening factor and a consequence of decline management. DISCUSSION: Findings point out the complexity of aging in place in ALFs and the need for a coordinated effort by facilities, residents, and families in the management of resident decline. Findings further highlight the necessity of residents being well informed about both their own needs and the capacity of a facility to meet them.