Infectivity assays are the key analytical technology for the development and manufacturing of virus-based therapeutics. Here, we introduce a novel assay format that utilizes label-free bright-field images to determine the kinetics of infection-dependent changes in cell morphology. In particular, cell rounding is directly proportional to the amount of infectious virus applied, enabling rapid determination of viral titers in relation to a standard curve. Our kinetic infectious virus titer (KIT) assay is stability-indicating and, due to its sensitive readout method, provides results within 24 h post-infection. Compared to traditional infectivity assays, which depend on a single readout of an infection endpoint, cumulated analysis of kinetic data by a fit model results in precise results (CV < 20%) based on only three wells per sample. This approach allows for a high throughput with ~400 samples processed by a single operator per week. We demonstrate the applicability of the KIT assay for the genetically engineered oncolytic VSV-GP, Newcastle disease virus (NDV), and parapoxvirus ovis (ORFV), but it can potentially be extended to a wide range of viruses that induce morphological changes upon infection. The versatility of this assay, combined with its independence from specific instruments or software, makes it a promising solution to overcome the analytical bottleneck in infectivity assays within the pharmaceutical industry and as a routine method in academic research.

Developments in droplet microfluidics have facilitated an era of high-throughput, sensitive single-cell, or single-molecule measurements capable of tackling the heterogeneity present in biological systems. Relying on single emulsion (SE) compartments, droplet assays achieve absolute quantification of nucleic acids, massively parallel single-cell profiling, and more. Double emulsions (DEs) have seen recent interest for their potential to build upon SE techniques. DEs are compatible with flow cytometry enabling high-throughput multi-parameter drop screening and eliminate content mixing due to coalescence during lengthy workflows. Despite these strengths, DEs lack important technical functions that exist in SEs such as methods for adding reagents to droplets on demand. Consequently, DEs cannot be used for multistep workflows which has limited their adoption in assay development. Here, strategies to enable reagent addition and other active manipulations on DEs are reported by converting DE inputs to SEs on chip. After conversion, drops are manipulated using existing SE techniques, including reagent addition, before reforming a DE at the outlet. Device designs and operation conditions achieving drop-by-drop reagent addition to DEs are identified and used as part of a multi-step aptamer screening assay performed entirely in DE drops. This work enables the further development of multistep DE droplet assays.

Recent decades have seen a dramatic increase in the commercial use of biocatalysts, transitioning from energy-intensive traditional chemistries to more sustainable methods. Current enzyme engineering techniques, such as directed evolution, require the generation and testing of large mutant libraries to identify optimized variants. Unfortunately, conventional screening methods are unable to screen such large libraries in a robust and timely manner. Droplet-based microfluidic systems have emerged as a powerful high-throughput tool for library screening at kilohertz rates. Unfortunately, almost all reported systems are based on fluorescence detection, restricting their use to a limited number of enzyme types that naturally convert fluorogenic substrates or require the use of surrogate substrates. To expand the range of enzymes amenable to evolution using droplet-based microfluidic systems, we present an absorbance-activated droplet sorter that allows of droplet sorting at kilohertz rates without the need for optical monitoring of the microfluidic system. To demonstrate the utility of the sorter, we rapidly screen a 105-member aldehyde dehydrogenase library towards D-glyceraldehyde using a NADH mediated coupled assay that generates WST-1 formazan as the colorimetric product. We successfully identify a variant with a 51% improvement in catalytic efficiency and a significant increase in overall activity across a broad substrate spectrum.

Pathogenic microorganisms play a crucial role in the global disease burden due to their ability to cause various diseases and spread through multiple transmission routes. Immunity tests identify antigens related to these pathogens, thereby confirming past infections and monitoring the host\'s immune response. Traditional pathogen detection methods, including enzyme-linked immunosorbent assays (ELISAs) and chemiluminescent immunoassays (CLIAs), are often labor-intensive, slow, and reliant on sophisticated equipment and skilled personnel, which can be limiting in resource-poor settings. In contrast, the development of microfluidic technologies presents a promising alternative, offering automation, miniaturization, and cost efficiency. These advanced methods are poised to replace traditional assays by streamlining processes and enabling rapid, high-throughput immunity testing for pathogens. This review highlights the latest advancements in microfluidic systems designed for rapid and high-throughput immunity testing, incorporating immunosensors, single molecule arrays (Simoas), a lateral flow assay (LFA), and smartphone integration. It focuses on key pathogenic microorganisms such as SARS-CoV-2, influenza, and the ZIKA virus (ZIKV). Additionally, the review discusses the challenges, commercialization prospects, and future directions to advance microfluidic systems for infectious disease detection.

BACKGROUND: Proper hydration is essential for maintaining health and supports various biological processes, including temperature regulation, immune function, nutrient delivery, and organ function. Visual assessment has traditionally been used to quantify liquid intake, although technological advances in optical and electrical sensors now offer higher accuracy and larger potential for automatic operation with millisecond precision and individual lick resolution.
METHODS: We describe an inexpensive electronic sensor board to monitor mouse licking behavior. The system is equipped with integrated filtering and data preprocessing steps. It measures lick count, frequency, width and interlick intervals with high resolution, allowing the real-time monitoring of complex licking patterns in several mice in their respective home cages over prolonged periods.
RESULTS: Our lickometer provides two-millisecond resolution, efficiently detecting variations in licking behaviors in mice. The system is adapted to monitor licking behaviors in up to 12 mice simultaneously. Lick count, duration and interlick intervals, along with preference for sweet water were monitored over two days, revealing variations in licking patterns across light and dark phases extended over prolonged periods.
METHODS: Our lickometer allows for monitoring licking behaviors and dynamics. It can be adapted to conventional mouse cages using electrical circuits. It is open-source, cost-effective, efficient, and can be utilized in real-time for large cohorts, representing an ideal tool for studying ingestive dynamics in different environmental and pathological contexts.
CONCLUSIONS: We have developed a novel, cost-effective, and efficient device to monitor ingestive behaviors in mice. The throughput of our device allows for monitoring several mice simultaneously while it can be applied directly to a conventional mouse cage, simplifying its implementation into pre-existing experimental setups.

CRISPR derived base editing techniques tend to edit multiple bases in the targeted region, which impedes precise reversion of disease-associated single nucleotide variations (SNVs). We designed an imperfect gRNA (igRNA) editing strategy to achieve bystander-less single-base editing. To predict the performance and provide ready-to-use igRNAs, we employed a high-throughput method to edit 5000 loci, each with approximate 19 systematically designed ABE igRNAs. Through deep learning of the relationship of editing efficiency, original gRNA sequence and igRNA sequence, AI models were constructed and tested, designated igRNA Prediction and Selection AI models (igRNA-PS). The models have three functions, First, they can identify the major editing site from the bystanders on a gRNA protospacer with a near 90% accuracy. second, a modified single-base editing efficiency (SBE), considering both single-base editing efficiency and product purity, can be predicted for any given igRNAs. Third, for an editing locus, a set of 64 igRNAs derived from a gRNA can be generated, evaluated through igRNA-PS to select for the best performer, and provided to the user. In this work, we overcome one of the most significant obstacles of base editors, and provide a convenient and efficient approach for single-base bystander-less ABE base editing.

BACKGROUND: Study of synaptic integrity using conventional electrophysiology is a gold standard for quantitative assessment of neurodegeneration. Fluorescence assisted single-synapse long-term potentiation (FASS-LTP) provides a high throughput method to assess the synaptic integrity of neurotransmission within and between different brain regions as a measure of pharmacological efficacy in translational models.
METHODS: We adapted the existing method to our purpose by adding a step during the thawing of frozen samples, by an extra step of placing them on a rocker at room temperature for 30 minutes immediately following thawing with constant mixing on a shaker. This allowed for gradual, uniform thawing, effectively separating the synaptosomes. Our study demonstrates FASS-LTP on four brain regions at 6- and 12-month periods in the 3xTg-AD mouse model, treating sibling cohorts with VU0155069 (a small molecule inhibitor) or vehicle (0.9 % saline).
RESULTS: Our findings demonstrate the robust ability of the FASS-LTP technique to characterize the functional synaptic integrity maintained by disease-treatment therapies in multiple brain regions longitudinally using frozen brain tissue.
METHODS: By providing a detailed, user-friendly protocol for this well-known analysis and including a recovery step improved the ability to robustly replicate the FASS-LTP between different brain regions. This may be extrapolated to a translational use on human clinical samples to improve understanding of the therapeutic impact on synaptic performance related to glutamate neurotransmission.
CONCLUSIONS: FASS-LTP method offers a robust analysis of synaptosomes isolated from frozen tissue samples, demonstrating greater reproducibility in rodent and human synapses in physiological and pathological states.

Noninvasive detection of protein biomarkers in plasma is crucial for clinical purposes. Liquid chromatography-mass spectrometry (LC-MS) is the gold standard technique for plasma proteome analysis, but despite recent advances, it remains limited by throughput, cost, and coverage. Here, we introduce a new hybrid method that integrates direct infusion shotgun proteome analysis (DISPA) with nanoparticle (NP) protein corona enrichment for high-throughput and efficient plasma proteomic profiling. We realized over 280 protein identifications in 1.4 min collection time, which enables a potential throughput of approximately 1000 samples daily. The identified proteins are involved in valuable pathways, and 44 of the proteins are FDA-approved biomarkers. The robustness and quantitative accuracy of this method were evaluated across multiple NPs and concentrations with a mean coefficient of variation of 17%. Moreover, different protein corona profiles were observed among various NPs based on their distinct surface modifications, and all NP protein profiles exhibited deeper coverage and better quantification than neat plasma. Our streamlined workflow merges coverage and throughput with precise quantification, leveraging both DISPA and NP protein corona enrichment. This underscores the significant potential of DISPA when paired with NP sample preparation techniques for plasma proteome studies.

To detect and analyze the changes of microorganisms in expressed prostatic secretion (EPS) of patients with IIIB prostatitis before and after low-intensity pulsed ultrasound (LIPUS) treatment, and to explore the mechanism of LIPUS in the treatment of chronic prostatitis (CP). 25 patients (study power was estimated using a Dirichlet-multinomial approach and reached 96.5% at α = 0.05 using a sample size of 25) with IIIB prostatitis who were effective in LIPUS treatment were divided into two groups before and after LIPUS treatment. High throughput second-generation sequencing technique was used to detect and analyze the relative abundance of bacterial 16 s ribosomal variable regions in EPS before and after treatment. The data were analyzed by bioinformatics software and database, and differences with P < 0.05 were considered statistically significant. Beta diversity analysis showed that there was a significant difference between groups (P = 0.046). LEfSe detected four kinds of characteristic microorganisms in the EPS of patients with IIIB prostatitis before and after LIPUS treatment. After multiple comparisons among groups by DESeq2 method, six different microorganisms were found. LIPUS may improve patients\' clinical symptoms by changing the flora structure of EPS, stabilizing and affecting resident bacteria or opportunistic pathogens.

Chimeric antigen receptors (CARs) are synthetic fusion proteins that can reprogram immune cells to target specific antigens. CAR-expressing T cells have emerged as an effective treatment method for hematological cancers; despite this success, the mechanisms and structural properties that govern CAR responses are not fully understood. Here, we provide a simple assay to assess cellular avidity using a standard flow cytometer. This assay measures the interaction kinetics of CAR-expressing T cells and targets antigen-expressing target cells. By co-culturing stably transfected CAR Jurkat cells with target positive and negative cells for short periods of time in a varying effector-target gradient, we were able to observe the formation of CAR-target cell doublets, providing a readout of actively bound cells. When using the optimized protocol reported here, we observed unique cellular binding curves that varied between CAR constructs with differing antigen binding domains. The cellular binding kinetics of unique CARs remained consistent, were dependent on specific target antigen expression, and required active biological signaling. While existing literature is not clear at this time whether higher or lower CAR cell binding is beneficial to CAR therapeutic activity, the application of this simplified protocol for assessing CAR binding could lead to a better understanding of the proximal signaling events that regulate CAR functionality. Key features • Determines CAR receptor cellular interaction kinetics using a Jurkat cell model. • Can be used for a wide variety of CAR target antigens, including both hematological and solid tumor targets. • Experiments can be performed in under two hours with no staining using a standard flow cytometer. • Requires stable CAR Jurkat cells and target cells with stable fluorescent marker expression for optimal results.