Imaged capillary isoelectric focusing tandem high-resolution mass spectrometry using nano electrospray ionization (ESI) for protein heterogeneity characterization.

Recombinant monoclonal antibodies (mAbs) have been spurring the rapid growth of commercial biotherapeutics. During production their charge heterogeneity must be assessed as a critical quality attribute to ensure safety, efficacy, and potency. Although imaged capillary isoelectric focusing (icIEF) is a powerful tool for this process, it could be improved further with tandem high-resolution mass spectrometry (HRMS). In this work, a nano-electrospray ionization (nano-ESI) apparatus was constructed to directly couple icIEF to HRMS. The system was evaluated with the standard NISTmAb, as well as more complex mAb, bi-specific antibody, and fusion protein samples. NISTmAb concentrations as low as 0.25 mg/ml demonstrated excellent sensitivity. There were good repeatabilities at 1 mg/ml with 7.58% and 8.01% RSDs for intention time and MS intensity, respectively, and the HRMS signal showed a strong linearity (R = 0.9983) across different concentrations. Meanwhile, the fingerprinting of the complex samples illustrated the versatility and potential of icIEF-HRMS. icIEF-HRMS developed can provide a comprehensive understanding of the underlying structural modifications that impact protein charge heterogeneity. Compared to the traditional ESI, nano-ESI can significantly improve sensitivity while maintaining a reasonable repeatability and throughput. Furthermore, the interface is much easier to connect, and is compatible with many commercial HRMS instruments.

Investigating peak dispersion in free-flow counterflow gradient focusing due to electroosmotic flow.

Free-flow electrophoresis (FFE) has the ability to continuously separate charged solutes from complex biological mixtures. Recently, a free-flow counterflow gradient focusing mechanism has been introduced to FFE, and it offers the potential for improved resolution and versatility. However, further investigation is needed to understand the solute dispersion at the focal position. Therefore, the goal of this work is to model the impact of electroosmotic flow, which is found to produce a pressure-driven backflow to maintain the fixed counterflow inputs. Like the counterflow, this backflow has a parabolic velocity profile that must be considered when predicting the concentration distribution of a given solute. After the model is established, preliminary experimental results are presented for a qualitative comparison. Results demonstrate a reasonable agreement at low applied voltages and provide a strong framework for future experimental validation.

A protocol for analyzing repeated measures of online group behavior.

In this article, we feature a novel protocol that enables the analysis of repeated measures of online group behavior. The protocol accounts for (1) the nested hierarchy of the data with weeks nested in persons, and persons nested in weeks, and (2) the temporal nature of the behavior at the early, mid, and late periods of each week. To manage and analyze such data in a general way, we first give an illustration of the data structure. Thereafter, we propose a five-step Courtney-Fanguy-Costley protocol that (1) considers the data structure, (2) defines the levels of data, (3) considers variable variation, timing, and necessary aggregation, (4) ensures necessary variation, and (5) specifies null and mixed-effects models. We also provide exemplary R code for readers to replicate our approach.•A general five-step protocol for analyzing repeated measures of online group behavior is offered.•A description of the complex nested data structure is offered.•Users can simulate data in R to run through the protocol.

Investigating peak dispersion in free-flow counterflow gradient focusing.

Free-flow electrophoresis (FFE) enables the continuous separation and collection of charged solutes, and as a result, it has drawn interest as both a preparative and an analytical tool for biological applications. Recently, a free-flow counterflow gradient focusing (FF-CGF) mechanism has been proposed with the goal of improving the resolution and versatility of FFE. To realize this potential, the factors that influence solute dispersion deserve further attention, including the gradient strength and the parabolic profile of the counterflow. Therefore, the goal of this work is to develop a theoretical model to study the interplay between these factors and molecular diffusion. Overall, an asymmetric solute distribution emerges for a wide range of parameters, and this behavior can be characterized with an exponentially modified Gaussian function. Results show that FF-CGF can achieve high-resolution separations, with the potential for high-throughput protein purification. Moreover, this work provides a practical guide for optimizing experimental conditions, as well as a strong framework for understanding and developing FF-CGF further.

autopsych: An R Shiny tool for the reproducible Rasch analysis, differential item functioning, equating, and examination of group effects.

In this paper, we present autopsych, a novel online tool that allows school assessment experts, test developers, and researchers to perform routine psychometric analyses and equating of student test data and to examine the effect of student demographic and group conditions on student test performance. The app extends current open-source software by providing (1) extensive embedded result narration and summaries for written reports, (2) improved handling of partial credit data via customizable item-person Wright maps, (3) customizable item- and person-flagging systems, (4) item-response theory model constraints and controls, (5) many-facets Rasch analysis to examine item bias, (6) Rasch fixed item equating for mapping student ability across test forms, (7) tabbed spreadsheet outputs and immediate options for secondary data analysis, (8) customizable graphical color schemes, (9) extended ANOVA analysis for examining group differences, and (10) inter-rater reliability analyses for the verifying the consistency of rater scoring systems. We present the app's architecture and functionalities and test its performance with simulated and real-world small-, medium-, and large-scale assessment data. Implications and planned future developments are also discussed.

Counterflow Gradient Focusing in Free-Flow Electrophoresis for Protein Fractionation.

With its ability to continuously separate and collect charged analytes, free-flow electrophoresis (FFE) has become a useful tool for the purification and real-time analysis of biological mixtures. This work presents a new free-flow counterflow gradient focusing (FF-CGF) mechanism that uses a novel velocity gradient to counterbalance electrophoretic migration. This counterflow gradient is created by simply introducing fluid flow through the sidewalls of the FFE chamber. The theoretical foundation and device design for FF-CGF are provided in this work, followed by implementation and validation, with a detailed discussion on future opportunities and challenges. Initial results show promise, with the potential to improve FFE resolution and offer versatility. Compared to existing focusing techniques, such as free-flow isotachophoresis and isoelectric focusing, no complex buffer compositions are required.

Semi-automated on-demand control of individual droplets with a sample application to a drug screening assay.

Automated control of individual droplets in microfluidic channels offers tremendous potential for applications requiring high accuracy and minimal user involvement. The feasibility of active droplet control has been previously demonstrated with pressure-driven flow control and visual feedback, but the manual operation required to perform droplet manipulations limited the accuracy, repeatability, and throughput. The present study improves upon the aforementioned challenges with a higher-level algorithm capturing the dynamics of droplet motion for a semi-automated control system. With a simple T junction geometry, droplets can now be automatically and precisely controlled on-demand. Specifically, there is ±10% accuracy for droplet generation, ±1.3% monodispersity for 500 µm long droplets and ±4% accuracy for splitting ratios. On-demand merging, mixing, and sorting are also demonstrated as well as the application of a drug screening assay related to neurodegenerative disorders. Overall, this system serves as a foundation for a fully automated system that does not require valves, embedded electrodes, or complex multi-layer fabrication.

Counterflow gradient electrophoresis for focusing and elution.

Counterflow gradient electrofocusing uses the bulk flow of a liquid solution to counterbalance the electrophoretic migration of an analyte. When either the bulk velocity or the electrophoretic velocity of the analyte is made to vary across the length of the channel, there exists a unique zero-velocity point for the analyte. This focusing method enables simultaneous separation and concentration of different analytes. The high resolution and sensitivity achieved are similar to that of isoelectric focusing, which separates analytes based on their isoelectric points, but the key difference is that analytes will instead focus based on their electrophoretic mobility. Dynamically changing the applied voltage or the counterflow rate over time will shift the zero-velocity point, and therefore allows the focused analytes to pass through a fixed detection point, or elute from the separation channel. Throughout the review, a number of different counterflow gradient techniques will be discussed, along with their recent advancements and potential applications.

Engagement and Mentor Support as Drivers of Social Development in the Project K Youth Development Program.

Youth development programs can achieve positive social outcomes, however studies comparing the influence of different program components are rare. Structural equation modeling of longitudinal, multilevel data (N = 327) from Project K, a multi-component youth development program, assessed how experiences of engagement or support in each component affected social outcomes. Participants reported significant gains in social self-efficacy and sense of community after the program. Engagement in the outdoor adventure and support during the mentoring partnership components significantly contributed to observed social gains, while engagement in the community service component did not. Results confirm youth development programs can positively influence adolescent social development, while highlighting the importance of moving beyond "black box" investigations in order to maximize program impact and efficiency.

Droplet Microfluidic System with On-Demand Trapping and Releasing of Droplet for Drug Screening Applications.

96-Well plate has been the traditional method used for screening drug compounds libraries for potential bioactivity. Although this method has been proven successful in testing dose-response analysis, the microliter consumption of expensive reagents and hours of reaction and analysis time call for innovative methods for improvements. This work demonstrates a droplet microfluidic platform that has the potential to significantly reduce the reagent consumption and shorten the reaction and analysis time by utilizing nanoliter-sized droplets as a replacement of wells. This platform is evaluated by applying it to screen drug compounds that inhibit the tau-peptide aggregation, a phenomena related to Alzheimer's disease. In this platform, sample reagents are first dispersed into nanolitre-sized droplets by an immiscible carrier oil and then these droplets are trapped on-demand in the downstream of the microfluidic device. The relative decrease in fluorescence through drug inhibition is characterized using an inverted epifluorescence microscope. Finally, the trapped droplets are released on-demand after each test by manipulating the applied pressures to the channel network which allows continuous processing. The testing results agree well with that obtained from 96-well plates with much lower sample consumption (∼200 times lower than 96-well plate) and reduced reaction time due to increased surface volume ratio (2.5 min vs 2 h).

The Impact of the Project K Youth Development Program on Self-Efficacy: A Randomized Controlled Trial.

A key issue for youth development programs is whether the learning they provide is transferred to participants' daily lives. It is also important that they are effective for the diverse range of participants they attract. This study used a randomized controlled trial design to measure the impact of Project K, a New Zealand-based youth development program, on academic and social self-efficacy. Project K combines a 3-week wilderness adventure, a 10 day community service component, and 1 year of mentoring to promote positive growth in 14-15 year olds with low self-efficacy. At baseline, the evaluation included 600 Project K (46 % female) and 577 Control participants (48 % female) and revealed that Project K was effective in improving both social and academic self-efficacy from pre- to post-program with effects being sustained 1 year later. Parents' perceptions of changes in the participants' interpersonal skills supported these findings. Differential program effects were found across participant subgroups, particularly 1 year after program completion. The implications of these differences are discussed.