Statistical analysis of the autocorrelation function in fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is a powerful method to measure concentration, mobility, and stoichiometry in solution and in living cells, but quantitative analysis of FCS data remains challenging due to the correlated noise in the autocorrelation function (ACF) of FCS. We demonstrate here that least-squares fitting of the conventional ACF is incompatible with the χ2 goodness-of-fit test and systematically underestimates the true fit parameter uncertainty. To overcome this challenge, a simple method to fit the ACF is introduced that allows proper calculation of goodness-of-fit statistics and that provides more tightly constrained parameter estimates than the conventional least-squares fitting method, achieving the theoretical minimum uncertainty. Because this method requires significantly more data than the standard method, we further introduce an approximate method that requires fewer data. We demonstrate both these new methods using experiments and simulations of diffusion. Finally, we apply our method to FCS data of the peripheral membrane protein HRas, which has a slow-diffusing membrane-bound population and a fast-diffusing cytoplasmic population. Despite the order-of-magnitude difference of the diffusion times, conventional FCS fails to reliably resolve the two species, whereas the new method identifies the correct model and provides robust estimates of the fit parameters for both species.

Autocorrelation function of finite-length data in fluorescence correlation spectroscopy.

The experimental autocorrelation function of fluorescence correlation spectroscopy calculated from finite-length data is a biased estimator of the theoretical correlation function. This study presents a new theoretical framework that explicitly accounts for the data length to allow for unbiased analysis of experimental autocorrelation functions. To validate our theory, we applied it to experiments and simulations of diffusion and characterized the accuracy and precision of the resulting parameter estimates. Because measurements in living cells are often affected by instabilities of the fluorescence signal, autocorrelation functions are typically calculated on segmented data to improve their robustness. Our reformulated theory extends the range of usable segment times down to timescales approaching the diffusion time. This flexibility confers unique advantages for live-cell data that contain intensity variations and instabilities. We describe several applications of short segmentation to analyze data contaminated with unwanted fluctuations, drifts, or spikes in the intensity that are not suited for conventional fluorescence correlation analysis. These results demonstrate the potential of our theoretical framework to significantly expand the experimental systems accessible to fluorescence correlation spectroscopy.

Measuring Parental Presence in the Neonatal Intensive Care Unit.

Parental presence is believed to improve outcomes for infants hospitalized in the neonatal intensive care unit (NICU). As a result, NICU policies and procedures have evolved to support parental presence, and a growing number of studies examine the role of parental presence in the NICU. However, the measurement of parental presence is not standardized, complicating assessment of its impact on child and parent outcomes across studies. We reviewed 29 studies that presented 27 distinct methods of quantifying parental presence in the NICU and reported associations of presence with patient demographics, parental engagement in the NICU, and outcomes for both infants and parents. This overview provides a foundation for standardizing and improving routine measurement of parental presence in the NICU. KEY POINTS: · NICUs encourage visiting ill newborns.. · Measurement of presence is not standardized.. · A uniform method to assess presence is needed..

Dissemination of Quality Improvement Project Results After Local Presentation.

Among quality improvement (QI) projects submitted for local presentation, the authors sought to understand how often project results were eventually disseminated through national/international presentation or peer-reviewed journal publication. Projects submitted for local presentation from 2016 to 2019 were linked to resulting publications or national/international conference presentations. Submitting authors were surveyed about their intentions, experience, and satisfaction with the process of disseminating their project results. Of 83 projects, 5 were published and another 10 were presented nationally/internationally. External dissemination was more likely with fewer project cycles and cost-focused outcomes. Survey responses indicated that most project leaders wanted to see their results published but held mixed opinions about resources and encouragement available to reach this goal. Few QI projects submitted for local presentation resulted in wider dissemination of project results. Sharing results and lessons learned beyond the local institution requires long-term planning, education, and support beginning early in the QI process.

Fathers' Visitation of Very Low Birth Weight Infants in the Neonatal Intensive Care Unit during the First Week of Life.

OBJECTIVE: The perceived fragility of extremely preterm neonates may deter paternal visitation early during the neonatal intensive care unit (NICU) stay. We retrospectively analyzed the correlation between paternal visitation of very low birth weight (VLBW) infants in our NICU and sociodemographic characteristics. STUDY DESIGN: We identified inborn VLBW infants admitted to our NICU from 2017 to 2018. The rate of visit days in the first week of life was analyzed using Spearman's correlation and Poisson's regression. RESULTS: The analysis included 292 infants (median gestational age [GA]: 29 weeks), with fathers present on a median of 3 days of the first week of life. GA was not correlated with visitation (rho = -0.04). On multivariable regression, fathers visited less frequently if they did not live with the mother or if the mother lived 25 to 75 km from the hospital versus < 25 km. CONCLUSION: Fathers' visitation in our NICU was constrained by socioeconomic factors rather than VLBW infants' characteristics.

Differentiating Luminal and Membrane-Associated Nuclear Envelope Proteins.

The nuclear envelope (NE) consists of two concentric nuclear membranes separated by the lumen, an ∼40-nm-wide fluid layer. NE proteins are implicated in important cellular processes ranging from gene expression to nuclear positioning. Although recent progress has been achieved in quantifying the assembly states of NE proteins in their native environment with fluorescence fluctuation spectroscopy, these studies raised questions regarding the association of NE proteins with nuclear membranes during the assembly process. Monitoring the interaction of proteins with membranes is important because the binding event is often associated with conformational changes that are critical to cellular signaling pathways. Unfortunately, the close physical proximity of both membranes poses a severe experimental challenge in distinguishing luminal and membrane-associated NE proteins. This study seeks to address this problem by introducing new, to our knowledge, fluorescence-based assays that overcome the restrictions imposed by the NE environment. We found that luminal proteins violate the Stokes-Einstein relation, which eliminates a straightforward use of protein mobility as a marker of membrane association within the NE. However, a surprising anomaly in the temperature-dependent mobility of luminal proteins was observed, which was developed into an assay for distinguishing between soluble and membrane-bound NE proteins. We further introduced a second independent tool for distinguishing both protein populations by harnessing the previously reported undulations of the nuclear membranes. These membrane undulations introduce local volume changes that produce an additional fluorescence fluctuation signal for luminal, but not for membrane-bound, proteins. After testing both methods using simple model systems, we apply the two assays to investigate a previously proposed model of membrane association for the luminal domain of SUN2, a constituent protein of the linker of nucleoskeleton and cytoskeleton complex. Finally, we investigate the effect of C- and N-terminal tagging of the luminal ATPase torsinA on its ability to associate with nuclear membranes.

Identifying Heteroprotein Complexes in the Nuclear Envelope.

The nucleus is delineated by the nuclear envelope (NE), which is a double membrane barrier composed of the inner and outer nuclear membranes as well as a ∼40-nm wide lumen. In addition to its barrier function, the NE acts as a critical signaling node for a variety of cellular processes, which are mediated by protein complexes within this subcellular compartment. Although fluorescence fluctuation spectroscopy is a powerful tool for characterizing protein complexes in living cells, it was recently demonstrated that conventional fluorescence fluctuation spectroscopy methods are not suitable for applications in the NE because of the presence of slow nuclear membrane undulations. We previously addressed this challenge by developing time-shifted mean-segmented Q (tsMSQ) analysis and applied it to successfully characterize protein homo-oligomerization in the NE. However, many NE complexes, such as the linker of the nucleoskeleton and cytoskeleton complex, are formed by heterotypic interactions, which single-color tsMSQ is unable to characterize. Here, we describe the development of dual-color (DC) tsMSQ to analyze NE heteroprotein complexes built from proteins that carry two spectrally distinct fluorescent labels. Experiments performed on model systems demonstrate that DC tsMSQ properly identifies heteroprotein complexes and their stoichiometry in the NE by accounting for spectral cross talk and local volume fluctuations. Finally, we applied DC tsMSQ to study the assembly of the linker of the nucleoskeleton and cytoskeleton complex, a heteroprotein complex composed of Klarsicht/ANC-1/SYNE homology and Sad1/UNC-84 (SUN) proteins, in the NE of living cells. Using DC tsMSQ, we demonstrate the ability of the SUN protein SUN2 and the Klarsicht/ANC-1/SYNE homology protein nesprin-2 to form a heterocomplex in vivo. Our results are consistent with previously published in vitro studies and demonstrate the utility of the DC tsMSQ technique for characterizing NE heteroprotein complexes.

Improved Use of Human Milk, Growth, and Central Line Utilization With Standard Feeding Roadmap in an Academic NICU.

BACKGROUND: Before the initiation of a standardized feeding roadmap in our regional, level IV academic neonatal intensive care unit, utilization of central lines was high, and initiation of enteral feeds delayed in the very low-birth-weight population (<1500 g). Given our review of the literature, it appeared that the standardization of feeding advancement would likely result in improved performance in both issues. METHODS: This was a retrospective cohort comparison of very low-birth-weight patients before initiation of any feeding roadmap with a second cohort following completion of the final roadmap. Infants were examined retrospectively in 2 historical cohorts: Phase 1, infants fed before roadmap development and rollout, October 1, 2012-March 31, 2013; and Phase 2, following promulgation of the final feeding roadmap, January 1, 2017-June 30, 2017. RESULTS: During Phase 2, we observed a significant reduction in median (interquartile range) days to first feed (3 [1] vs 1 [1] [P < 0.0001]) and utilization of a second central line (35% vs 12% [P < 0.01]). Weight gain was significantly improved from before roadmap implementation to final, mean (SD) (g/d, 21 [5] vs 24 [4]; [P < .0001]). Percentage of first enteral feedings that were human milk also increased significantly from 71% to 91% (P = 0.0007). CONCLUSION: Implementation of a standardized feeding roadmap was associated with a reduction in days to first enteral feeds, an increase in the primary use of human milk for initiation of enteral feeds, and a decrease in the utilization of central lines while improving weight gain in very low-birth-weight infants.

Publication Bias Among Conference Abstracts Reporting on Pediatric Quality Improvement Projects.

This study evaluated progress to publication of pediatric quality improvement (QI) projects initially presented as national conference abstracts, according to project findings and other characteristics. QI abstracts were identified among presentations at the 2010-2015 American Academy of Pediatrics National Conference & Exhibition, and publications were tracked through June 2018. Positive findings (improvement on at least 1 quantitative project outcome), interventions, and analyses were correlated with journal publication. Of 142 abstracts, 128 (90%) reported positive findings. Forty-nine positive abstracts and 3 abstracts reporting negative results resulted in publication (38% vs 21%, respectively; P = .256). Median time to publication was 1.2 years for projects with positive findings, compared to >3 years for abstracts with negative findings (P = .029). Ninety percent of abstracts reported positive findings, and these abstracts progressed to publication more quickly. Overcoming publication bias for pediatric QI projects may enhance selection of promising interventions as new projects are designed.

Bright split red fluorescent proteins for the visualization of endogenous proteins and synapses.

Self-associating split fluorescent proteins (FPs) are split FPs whose two fragments spontaneously associate to form a functional FP. They have been widely used for labeling proteins, scaffolding protein assembly and detecting cell-cell contacts. Recently developments have expanded the palette of self-associating split FPs beyond the original split GFP1-10/11. However, these new ones have suffered from suboptimal fluorescence signal after complementation. Here, by investigating the complementation process, we have demonstrated two approaches to improve split FPs: assistance through SpyTag/SpyCatcher interaction and directed evolution. The latter has yielded two split sfCherry3 variants with substantially enhanced overall brightness, facilitating the tagging of endogenous proteins by gene editing. Based on sfCherry3, we have further developed a new red-colored trans-synaptic marker called Neuroligin-1 sfCherry3 Linker Across Synaptic Partners (NLG-1 CLASP) for multiplexed visualization of neuronal synapses in living C. elegans, demonstrating its broad applications.

Libman-Sacks endocarditis in pediatric patient with systemic lupus erythematosus.

A 16 year old female patient with systemic lupus erythematosus presented to rheumatology clinic with a new I-II/VI honking-quality mitral regurgitation murmur. The patient was initially evaluated by transthoracic echocardiogram that revealed mitral valve regurgitation and a large band of tissue under the mitral valve leaflets. Blood cultures were obtained and were negative. Transesophageal echocardiogram provided better visualization of the lesion and showed the band of tissue involving most of the chordae of the posterior mitral leaflet. A diagnosis of Libman-Sacks endocarditis was made given the aseptic nature of the lesions and the patient's underlying lupus. Aggressive management of the lupus showed reduction of the mitral regurgitation and the size of the lesion. Libman-Sacks endocarditis is best evaluated by transesophageal echocardiogram.

Optical techniques for tracking multiple myeloma engraftment, growth, and response to therapy.

Multiple myeloma (MM), the second most common hematological malignancy, initiates from a single site and spreads via circulation to multiple sites in the bone marrow (BM). Methods to track MM cells both in the BM and circulation would be useful for developing new therapeutic strategies to target MM cell spread. We describe the use of complementary optical techniques to track human MM cells expressing both bioluminescent and fluorescent reporters in a mouse xenograft model. Long-term tumor growth and response to therapy are monitored using bioluminescence imaging (BLI), while numbers of circulating tumor cells are detected by in-vivo flow cytometry. Intravital microscopy is used to detect early seeding of MM cells to the BM, as well as residual cancer cells that remain in the BM after the bulk of the tumor is eradicated following drug treatment. Thus, intravital microscopy provides a powerful, albeit invasive, means to study cellular processes in vivo at the very early stage of the disease process and at the very late stage of therapeutic intervention when the tumor burden is too small to be detected by other imaging methods.

The Drosophila protein palmitoylome: characterizing palmitoyl-thioesterases and DHHC palmitoyl-transferases.

Palmitoylation is the post-translational addition of a palmitate moiety to a cysteine residue through a covalent thioester bond. The addition and removal of this modification is controlled by both palmitoyl acyl-transferases and thioesterases. Using bioinformatic analysis, we identified 22 DHHC family palmitoyl acyl-transferase homologs in the Drosophila genome. We used in situ hybridization,RT-PCR, and published FlyAtlas microarray data to characterize the expression patterns of all 22 fly homologs. Our results indicate that all are expressed genes, but several, including CG1407, CG4676, CG5620, CG6017/dHIP14, CG6618, CG6627 and CG17257 appear to be enriched in neural tissues suggesting that they are important for neural function. Furthermore, we have found that several may be expressed in a sex-specific manner with adult male specific expression of CG4483 and CG17195. Using tagged versions of the DHHC genes, we demonstrate that fly DHHC proteins are primarily located in either the Golgi Apparatus or Endoplasmic Reticulum in S2 cells, except for CG1407, which was found on the plasma membrane. We also characterized the subcellular localization and expression of the three known thioesterases: Palmitoyl-protein Thioesterase 1 (Ppt1), Palmitoyl-protein Thioesterase 2 (Ppt2)and Acyl-protein Thioesterase 1 (APT1). Our results indicate that Ppt1 and Ppt2 are the major lysosomal thioesterases while APT1 is the likely cytoplasmic thioesterase. Finally, in vivo rescue experiments show that Ppt2 expression cannot rescue the neural inclusion phenotypes associated with loss of Ppt1, further supporting distinct functions and substrates for these two thioesterases. These results will serve as the basis for a more complete understanding of the protein palmitoylome's normal cellular functions in the fly and will lead to further insights into the molecular etiology of diseases associated with the mis-regulation of palmitoylation.