Objective: The purpose of this study was to examine the effect of a fatigue protocol on Vestibular/Ocular Motor Screening (VOMS) performance.
Design: Within subjects, repeated measures, crossover.
...Methods: Fifteen healthy, physically-active participants (22.20 ± 1.424 years) completed 2 sessions under 2 conditions. A pretest VOMS, condition protocol, and a posttest VOMSwere performed. The control condition consisted of rest, while the experimental consisted of a fatigue protocol.
Results: The primary outcome measures were VOMS performance scores and Near Point of Convergence (NPC) measurements. Statistically significant interaction effects for NPC, F(1,14) = 9.38, p = .008, and total VOMS score, F(1,14) = 10.96, p = .005 were observed. For NPC, posttest (9.12 ± 4.99 cm) was significantly different, t(14) = −2.60, p = .021, than pretest scores (7.12 ± 3.19 cm). For total VOMS score, posttest experimental scores (4.93 ± 5.12) were significantly different, t(14) = −3.06, p = .009, than pretest severity scores (1.73 ± 3.67).
Conclusions: Significant increases were found in total VOMS and NPC scores following exertional fatigue. Exertional fatigue affects symptoms associated with vestibular, and/or ocular motor system assessments. Clinicians should use the VOMS with caution immediately following activity and allow time for recovery from acute fatigue.
Due to sensitivity limitations, global proteome measurements generally require large amounts of biological starting material, which masks heterogeneity within the samples and differential protein ...expression among constituent cell types. Methods for spatially resolved proteomics are being developed to resolve protein expression for distinct cell types among highly heterogeneous tissues, but have primarily been applied to mammalian systems. Here we evaluate the performance of cell-type-specific proteome analysis of tomato fruit pericarp tissues by a platform integrating laser-capture microdissection (LCM) and a recently developed automated sample preparation system (nanoPOTS, nanodroplet processing in one pot for trace samples). Tomato fruits were cryosectioned prior to LCM and tissues were dissected and captured directly into nanoPOTS chips for processing. Following processing, samples were analyzed by nanoLC-MS/MS. Approximately 1900 unique peptides and 422 proteins were identified on average from ∼0.04 mm2 tissues comprising ∼8–15 parenchyma cells. Spatially resolved proteome analyses were performed using cells of outer epidermis, collenchyma, and parenchyma. Using ≤200 cells, a total of 1,870 protein groups were identified and the various tissues were easily resolved. The results provide spatial and tissue-specific insights into key enzymes and pathways involved in carbohydrate transport and source–sink relationships in tomato fruit. Of note, at the time of fruit ripening studied here, we identified differentially abundant proteins throughout the pericarp related to chlorophyll biogenesis, photosynthesis, and especially transport.
Ambient mass spectrometry (MS) has revolutionized the way of MS analysis and broadened its application in various fields. This paper describes the use of microfluidic techniques to simplify the setup ...and improve the functions of ambient MS by integrating the sampling probe, electrospray emitter probe, and online mixer on a single glass microchip. Two types of sampling probes, including a parallel-channel probe and a U-shaped channel probe, were designed for dry-spot and liquid-phase droplet samples, respectively. We demonstrated that the microfabrication techniques not only enhanced the capability of ambient MS methods in analysis of dry-spot samples on various surfaces, but also enabled new applications in the analysis of nanoliter-scale chemical reactions in an array of droplets. The versatility of the microchip-based ambient MS method was demonstrated in multiple different applications including evaluation of residual pesticide on fruit surfaces, sensitive analysis of low-ionizable analytes using postsampling derivatization, and high-throughput screening of Ugi-type multicomponent reactions.
Background
Many healthcare facilities and providers prohibit blenderized tube feeding (BTF) for patients who request it due to concerns of high microbial load. The current project compared microbial ...loads of a standard ready‐to‐feed polymeric commercial formula (CF), a BTF made using baby food (BTF‐BF), and a BTF prepared from blending whole food (BTF‐WF), following food safety standards expected of U.S. hospitals.
Methods
Three tube‐feeding formulas (CF, BTF‐BF, BTF‐WF) were prepared in a U.S. hospital and delivered in vitro to an unoccupied patient room. Samples were collected at zero hour, 2 hours, and 4 hours and compared for growth of aerobic microorganisms, Staphylococus aureus, coliforms, and Escherichia coli. The experiment was conducted in triplicate, 1 week apart.
Results
No S. aureus or coliform/E. coli were detected at any time point following preparation, and total bacterial count was well below acceptable limits. All 3 feeding formulas at zero hour, 2 hours, and 4 hours for each of the 3 sampling dates were acceptable for human consumption.
Conclusion
Judicious BTF recipe selection and adherence to safe food handling provide a safe feeding substrate equivalent to CF in the hospital setting. Due to increased use and interest in BTF by patients and their caregivers, healthcare facilities may need to reexamine their policies prohibiting BTF use.
Packaged, transferred, and delivered: A method has been developed that automatically transfers the contents of oil‐encapsulated aqueous plugs to a co‐flowing aqueous stream, which enables ...dilution‐free electrospray ionization (ESI) mass spectrometric analysis for droplet‐based microfluidics.
A microfluidic platform was developed to perform online electrokinetic sample preconcentration and rapid hydrodynamic sample injection for zone electrophoresis using a single microvalve. The ...polydimethylsiloxane microchip comprises a separation channel, a side channel for sample introduction, and a control channel which is used as a pneumatic microvalve aligned at the intersection of the two flow channels. The closed microvalve, created by multilayer soft lithography, serves as a nanochannel preconcentrator under an applied electric potential, enabling current to pass through while preventing bulk flow. Once analytes are concentrated, the valve is briefly opened and the stacked sample is pressure injected into the separation channel for electrophoretic separation. Fluorescently labeled peptides were enriched by a factor of ∼450 in 230 s. This method enables both rapid analyte concentration and controlled injection volume for high sensitivity, high‐resolution CE.
Single-cell measurements are uniquely capable of characterizing cell-to-cell heterogeneity and have been used to explore the large diversity of cell types and physiological functions present in ...tissues and other complex cell assemblies. An intriguing application of single-cell proteomics is the characterization of proteome dynamics during biological transitions, like cellular differentiation or disease progression. Time-course experiments, which regularly take measurements during state transitions, rely on the ability to detect dynamic trajectories in a data series. However, in a single-cell proteomics experiment, cell-to-cell heterogeneity complicates the confident identification of proteome dynamics as measurement variability may be higher than expected. Therefore, a critical question for these experiments is how many data points need to be acquired during the time course to enable robust statistical analysis. We present here an analysis of the most important variables that affect statistical confidence in the detection of proteome dynamics: fold change, measurement variability, and the number of cells measured during the time course. Importantly, we show that datasets with less than 16 measurements across the time domain suffer from low accuracy and also have a high false-positive rate. We also demonstrate how to balance competing demands in experimental design to achieve a desired result.
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•Detecting temporal change in proteins depends on fold change and variability.•Replicate time courses improve reliability of detecting temporal dynamics.•Temporal experiments require a dense sampling of cells to track gradual transitions.•Time-course trajectory experiments require more samples than two-state comparisons.
Cellular development and disease progression are gradual transitions between phenotypic stages. Time-course measurements that explicitly measure this transition are important to discover proteome dynamics. Single-cell measurements are a powerful tool for understanding heterogeneity, especially during phenotypic transitions. Single-cell proteomics measurements are emerging as an available tool to characterize the cellular state. We created a statistical method that predicts the success of an experimental design for temporal dynamics.
Sample preparation for single-cell proteomics is generally performed in a one-pot workflow with multiple dispensing and incubation steps. These hours-long processes can be labor intensive and lead to ...long sample-to-answer times. Here we report a sample preparation method that achieves cell lysis, protein denaturation, and digestion in 1 h using commercially available high-temperature-stabilized proteases with a single reagent dispensing step. Four different one-step reagent compositions were evaluated, and the mixture providing the highest proteome coverage was compared to the previously employed multistep workflow. The one-step preparation increases proteome coverage relative to the previous multistep workflow while minimizing labor input and the possibility of human error. We also compared sample recovery between previously used microfabricated glass nanowell chips and injection-molded polypropylene chips and found the polypropylene provided improved proteome coverage. Combined, the one-step sample preparation and the polypropylene substrates enabled the identification of an average of nearly 2400 proteins per cell using a standard data-dependent workflow with Orbitrap mass spectrometers. These advances greatly simplify sample preparation for single-cell proteomics and broaden accessibility with no compromise in terms of proteome coverage.
The goal of proteomics is to identify and quantify the complete set of proteins in a biological sample. Single-cell proteomics specializes in the identification and quantitation of proteins for ...individual cells, often used to elucidate cellular heterogeneity. The significant reduction in ions introduced into the mass spectrometer for single-cell samples could impact the features of MS2 fragmentation spectra. As all peptide identification software tools have been developed on spectra from bulk samples and the associated ion-rich spectra, the potential for spectral features to change is of great interest. We characterize the differences between single-cell spectra and bulk spectra by examining three fundamental spectral features that are likely to affect peptide identification performance. All features show significant changes in single-cell spectra, including the loss of annotated fragment ions, blurring signal and background peaks due to diminishing ion intensity, and distinct fragmentation pattern, compared to bulk spectra. As each of these features is a foundational part of peptide identification algorithms, it is critical to adjust algorithms to compensate for these losses.
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