Evaluation of serological lateral flow assays for severe acute respiratory syndrome coronavirus-2.

BACKGROUND: COVID-19 has resulted in significant morbidity and mortality worldwide. Lateral flow assays can detect anti-Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) antibodies to monitor transmission. However, standardized evaluation of their accuracy and tools to aid in interpreting results are needed. METHODS: We evaluated 20 IgG and IgM assays selected from available tests in April 2020. We evaluated the assays' performance using 56 pre-pandemic negative and 56 SARS-CoV-2-positive plasma samples, collected 10-40 days after symptom onset, confirmed by a molecular test and analyzed by an ultra-sensitive immunoassay. Finally, we developed a user-friendly web app to extrapolate the positive predictive values based on their accuracy and local prevalence. RESULTS: Combined IgG + IgM sensitivities ranged from 33.9 to 94.6%, while combined specificities ranged from 92.6 to 100%. The highest sensitivities were detected in Lumiquick for IgG (98.2%), BioHit for both IgM (96.4%), and combined IgG + IgM sensitivity (94.6%). Furthermore, 11 LFAs and 8 LFAs showed perfect specificity for IgG and IgM, respectively, with 15 LFAs showing perfect combined IgG + IgM specificity. Lumiquick had the lowest estimated limit-of-detection (LOD) (0.1 µg/mL), followed by a similar LOD of 1.5 µg/mL for CareHealth, Cellex, KHB, and Vivachek. CONCLUSION: We provide a public resource of the accuracy of select lateral flow assays with potential for home testing. The cost-effectiveness, scalable manufacturing process, and suitability for self-testing makes LFAs an attractive option for monitoring disease prevalence and assessing vaccine responsiveness. Our web tool provides an easy-to-use interface to demonstrate the impact of prevalence and test accuracy on the positive predictive values.

Proteomic and biological profiling of extracellular vesicles from Alzheimer's disease human brain tissues.

INTRODUCTION: Extracellular vesicles (EVs) from human Alzheimer's disease (AD) biospecimens contain amyloid beta (Aß) peptide and tau. While AD EVs are known to affect brain disease pathobiology, their biochemical and molecular characterizations remain ill defined. METHODS: EVs were isolated from the cortical gray matter of 20 AD and 18 control brains. Tau and Aß levels were measured by immunoassay. Differentially expressed EV proteins were assessed by quantitative proteomics and machine learning. RESULTS: Levels of pS396 tau and Aß1-42 were significantly elevated in AD EVs. High levels of neuron- and glia-specific factors are detected in control and AD EVs, respectively. Machine learning identified ANXA5, VGF, GPM6A, and ACTZ in AD EV compared to controls. They distinguished AD EVs from controls in the test sets with 88% accuracy. DISCUSSION: In addition to Aß and tau, ANXA5, VGF, GPM6A, and ACTZ are new signature proteins in AD EVs.

Ultra-fast Cycling for Multiplexed Cellular Fluorescence Imaging.

Rapid analysis of single and scant cell populations is essential in modern diagnostics, yet existing methods are often limited and slow. Herein, we describe an ultra-fast, highly efficient cycling method for the analysis of single cells based on unique linkers for tetrazine (Tz)/trans-cyclooctene (TCO)-mediated quenching. Surprisingly, the quenching reaction rates were more than 3 orders of magnitude faster (t1/2 <1 s) than predicted. This allowed multi-cycle staining and immune cell profiling within an hour, leveraging the accelerated kinetics to open new diagnostic possibilities for rapid cellular analyses.

Detection and isolation of circulating exosomes and microvesicles for cancer monitoring and diagnostics using micro-/nano-based devices.

In the last several years, nanoscale vesicles that originate from tumor cells and which can be found circulating in the blood (i.e. exosomes and microvesicles) have been discovered to contain a wealth of proteomic and genetic information to monitor cancer progression, metastasis, and drug efficacy. However, the use of exosomes and microvesicles as biomarkers to improve patient care has been limited by their small size (30 nm-1 µm) and the extensive sample preparation required for their isolation and measurement. In this Critical Review, we explore the emerging use of micro and nano-technology to isolate and detect exosomes and microvesicles in clinical samples and the application of this technology to the monitoring and diagnosis of cancer.

Chalcones isolated from Angelica keiskei inhibit cysteine proteases of SARS-CoV.

Two viral proteases of severe acute respiratory syndrome coronavirus (SARS-CoV), a chymotrypsin-like protease (3CL(pro)) and a papain-like protease (PL(pro)) are attractive targets for the development of anti-SARS drugs. In this study, nine alkylated chalcones (1-9) and four coumarins (10-13) were isolated from Angelica keiskei, and the inhibitory activities of these constituents against SARS-CoV proteases (3CL(pro) and PL(pro)) were determined (cell-free/based). Of the isolated alkylated chalcones, chalcone 6, containing the perhydroxyl group, exhibited the most potent 3CL(pro) and PL(pro) inhibitory activity with IC50 values of 11.4 and 1.2 µM. Our detailed protein-inhibitor mechanistic analysis of these species indicated that the chalcones exhibited competitive inhibition characteristics to the SARS-CoV 3CL(pro), whereas noncompetitive inhibition was observed with the SARS-CoV PL(pro).

Adsorption of CMIT/MIT on the Model Pulmonary Surfactant Monolayers.

Polyhexamethylene guanidine (PHMG) is a guanidine-based chemical that has long been used as an antimicrobial agent. However, recently raised concerns regarding the pulmonary toxicity of PHMG in humans and aquatic organisms have led to research in this area. Along with PHMG, there are concerns about the safety of non-guanidine 5-chloro-2-methylisothiazol-3(2H)-one/2-methylisothiazol-3(2H)-one (CMIT/MIT) in human lungs; however, the safety of such chemicals can be affected by many factors, and it is difficult to rationalize their toxicity. In this study, we investigated the adsorption characteristics of CMIT/ MIT on a model pulmonary surfactant (lung surfactant, LS) using a Langmuir trough attached to a fluorescence microscope. Analysis of the π-A isotherms and lipid raft morphology revealed that CMIT/MIT exhibited minimal adsorption onto the LS monolayer deposited at the air/water interface. Meanwhile, PHMG showed clear signs of adsorption to LS, as manifested by the acceleration of the L o phase growth with increasing surface pressure. Consequently, in the presence of CMIT/MIT, the interfacial properties of the model LS monolayer exhibited significantly fewer changes than PHMG.

Anti-cancer bioactivity of sweet basil leaf derived extracellular vesicles on pancreatic cancer cells.

Most living organisms secrete tiny lipid bilayer particles encapsulating various biomolecular entities, including nucleic acids and proteins. These secreted extracellular vesicles (EVs) are shown to aid in communication between cells and their environment. EVs are mainly involved in the signalling and manipulation of physiological processes. Plant EVs display similar functional activity as seen in mammalian EVs. Medicinal plants have many bioactive constituents with potential applications in cancer treatment. Particularly, Basil (Ocimum basilicum), has wide therapeutic properties including anti-inflammatory, anti-cancer, and anti-infection, among others. In this study, we focused on using EVs purified from Apoplast Washing Fluid (AWF) of Basil plant leaves as a biological therapeutic agent against cancer. Characterization of Basil EVs revealed a size range of 100-250 nm, which were later assessed for their cell uptake and apoptosis inducing abilities in pancreatic cancer cells. Basil plant EVs (BasEVs) showed a significant cytotoxic effect on pancreatic cancer cell line MIA PaCa-2 at a concentration of 80 and 160 µg/mL in cell viability, as well as clonogenic assays. Similarly, RT-PCR and western blot analysis has shown up regulation in apoptotic gene and protein expression of Bax, respectively, in BasEV treatment groups compared to untreated controls of MIA PaCa-2. Overall, our results suggest that EVs from basil plants have potent anti-cancer effects in pancreatic cancer cells and can serve as a drug delivery system, demanding an investigation into the therapeutic potential of other medicinal plant EVs.

Optimizing cell therapy by sorting cells with high extracellular vesicle secretion.

Critical challenges remain in clinical translation of extracellular vesicle (EV)-based therapeutics due to the absence of methods to enrich cells with high EV secretion. Current cell sorting methods are limited to surface markers that are uncorrelated to EV secretion or therapeutic potential. Here, we utilize a nanovial technology for enrichment of millions of single cells based on EV secretion. This approach is applied to select mesenchymal stem cells (MSCs) with high EV secretion as therapeutic cells for improving treatment. The selected MSCs exhibit distinct transcriptional profiles associated with EV biogenesis and vascular regeneration and maintain high levels of EV secretion after sorting and regrowth. In a mouse model of myocardial infarction, treatment with high-secreting MSCs improves heart functions compared to treatment with low-secreting MSCs. These findings highlight the therapeutic importance of EV secretion in regenerative cell therapies and suggest that selecting cells based on EV secretion could enhance therapeutic efficacy.

Live Organoid Cyclic Imaging.

Organoids are becoming increasingly relevant in biology and medicine for their physiological complexity and accuracy in modeling human disease. To fully assess their biological profile while preserving their spatial information, spatiotemporal imaging tools are warranted. While previously developed imaging techniques, such as four-dimensional (4D) live imaging and light-sheet imaging have yielded important clinical insights, these technologies lack the combination of cyclic and multiplexed analysis. To address these challenges, bioorthogonal click chemistry is applied to display the first demonstration of multiplexed cyclic imaging of live and fixed patient-derived glioblastoma tumor organoids. This technology exploits bioorthogonal click chemistry to quench fluorescent signals from the surface and intracellular of labeled cells across multiple cycles, allowing for more accurate and efficient molecular profiling of their complex phenotypes. Herein, the versatility of this technology is demonstrated for the screening of glioblastoma markers in patient-derived human glioblastoma organoids while conserving their viability. It is anticipated that the findings and applications of this work can be broadly translated into investigating physiological developments in other organoid systems.

Characterization of a novel steviol-producing ß-glucosidase from Penicillium decumbens and optimal production of the steviol.

This study aimed to develop an economically viable enzyme for the optimal production of steviol (S) from stevioside (ST). Of 9 commercially available glycosidases tested, S-producing ß-glucosidase (SPGase) was selected and purified 74-fold from Penicillium decumbens naringinase by a three-step column chromatography procedure. The 121-kDa protein was stable at pH 2.3-6.0 and at 40-60 °C. Hydrolysis of ST by SPGase produced rubusoside (R), steviolbioside (SteB), steviol mono-glucoside (SMG), and S, as determined by HPLC, HPLC-MS, and (1)H- and (13)C-nuclear magnetic resonance. SPGase showed higher activity toward steviol mono-glucosyl ester, ST, R, and SMG than other ß-linked glucobioses. The optimal conditions for S production (30 mM, 64 % yield) were 47 mM ST and 43 µl of SPGase at pH 4.0 and 55 °C. This is the first report detailing the production of S from ST hydrolysis by a novel ß-glucosidase, which may be useful for the pharmaceutical and agricultural areas.

Single Extracellular Vesicle Analysis Using Droplet Microfluidics.

Extracellular vesicles (EVs) are lipid-bound nanometer-sized vesicles released by all cell types that contain molecular payload such as proteins and/or nucleic acids. EVs are a key facet of cell-to-cell communication and have the potential to be used in the diagnosis of numerous diseases, chief among them being cancer. However, most methods of EV analysis struggle to identify the rare, malformed proteins indicative of tumor cells as tumor EVs represent only a tiny fraction of the bulk EVs present in the bloodstream. Here, we present a method of single EV analysis, utilizing droplet microfluidics to encapsulate EVs, which are labeled with DNA barcodes linked to antibodies, in droplets with the DNA extension used to amplify the signals associated with each EV. The amplified DNA can then be sequenced to assess the protein content of individual EVs, enabling the detection of rare proteins and EV subpopulations within a bulk EV sample.

Optimizing Cell Therapy by Sorting Cells with High Extracellular Vesicle Secretion.

Critical challenges remain in clinical translation of extracellular vesicle (EV)-based therapeutics due to the absence of methods to enrich cells with high EV secretion. Current cell sorting methods are limited to surface markers that are uncorrelated to EV secretion or therapeutic potential. We developed a nanovial technology for enrichment of millions of single cells based on EV secretion. This approach was applied to select mesenchymal stem cells (MSCs) with high EV secretion as therapeutic cells for improving treatment. The selected MSCs exhibited distinct transcriptional profiles associated with EV biogenesis and vascular regeneration and maintained high levels of EV secretion after sorting and regrowth. In a mouse model of myocardial infarction, treatment with high-secreting MSCs improved heart functions compared to treatment with low-secreting MSCs. These findings highlight the therapeutic importance of EV secretion in regenerative cell therapies and suggest that selecting cells based on EV secretion could enhance therapeutic efficacy.

Highly parallel, wash-free, and ultrasensitive centrifugal droplet digital protein detection in sub-microliter blood.

The ability to efficiently detect low-abundance protein biomarkers in tiny blood samples is a significant challenge in clinical and laboratory settings. Currently, high-sensitivity approaches require specialized instrumentation, involve multiple washing steps, and lack the ability to parallelize, preventing their widespread implementation. Herein, we developed a parallelized, wash-free, and ultrasensitive centrifugal droplet digital protein detection (CDPro) technology that achieves a femtomolar limit of detection (LoD) of target proteins with sub-microliters of plasma. The CDPro combines two techniques, namely a centrifugal microdroplet generation device and a digital immuno-PCR assay. Miniaturized centrifugal devices enable emulsification of hundreds of samples within 3 minutes using a common centrifuge. The bead-free digital immuno-PCR assay not only eliminates the need for multistep washing, but also possesses ultra-high detection sensitivity and accuracy. We characterized the performance of CDPro using recombinant interleukins (IL-3 and IL-6) as example targets and reported a LoD of 0.0128 pg mL-1. We also quantified IL-6 from 7 human clinical blood samples using the CDPro with only 0.5 µL plasma, which showed excellent agreement with an existing clinical protein diagnostic system with 25 µL plasma from those samples (R2 = 0.98).

Double Digital Assay for Single Extracellular Vesicle and Single Molecule Detection.

Extracellular vesicles (EVs) have emerged as a promising source of biomarkers for disease diagnosis. However, current diagnostic methods for EVs present formidable challenges, given the low expression levels of biomarkers carried by EV samples, as well as their complex physical and biological properties. Herein, a highly sensitive double digital assay is developed that allows for the absolute quantification of individual molecules from a single EV. Because the relative abundance of proteins is low for a single EV, tyramide signal amplification (TSA) is integrated to increase the fluorescent signal readout for evaluation. With the integrative microfluidic technology, the technology's ability to compartmentalize single EVs is successfully demonstrated, proving the technology's digital partitioning capacity. Then the device is applied to detect single PD-L1 proteins from single EVs derived from a melanoma cell line and it is discovered that there are ≈2.7 molecules expressed per EV, demonstrating the applicability of the system for profiling important prognostic and diagnostic cancer biomarkers for therapy response, metastatic status, and tumor progression. The ability to accurately quantify protein molecules of rare abundance from individual EVs will shed light on the understanding of EV heterogeneity and discovery of EV subtypes as new biomarkers.

Equine mesenchymal stem cell derived extracellular vesicle immunopathology biomarker discovery.

The use of mesenchymal stem cells (MSCs) in human and veterinary clinical applications has become a subject of increasing importance due to their roles in immunomodulation and regenerative processes. MSCs are especially relevant in equine medicine because they may have the ability to treat prevalent musculoskeletal disorders, among other conditions. However, recent evidence suggests that the components secreted by MSCs, particularly extracellular vesicles (EVs), are responsible for these properties. EVs contain proteins and nucleic acids, which possess an active role in intercellular communication and can be used as therapeutics. However, because the intersection of equine veterinary medicine with EVs remains a relatively new field, there is a demand to identify biomarkers that can discern and enrich for therapeutic EVs, progressing their clinical efficacy. In this study, we identified and characterized 84 miRNAs, between three equine donors involved in immunomodulation in cell and EV subjects. We discovered distinct groups of shared miRNAs, like miR-21-5p and miR-451a, that are abundant and enriched between the donors' EVs, respectively. By mapping and comparing the MSC-EV miRNA expression, we discovered many pathways that are involved in immunomodulation and tissue regenerative processes related to equine clinical applications. Therefore, the miRNAs highlighted in this article can be used as valuable biomarkers for screening MSC-derived EVs for potential equine therapy.

Patient-derived melanoma organoid models facilitate the assessment of immunotherapies.

BACKGROUND: Only a minority of melanoma patients experience durable responses to immunotherapies due to inter- and intra-tumoral heterogeneity in melanoma. As a result, there is a pressing need for suitable preclinical models to investigate resistance mechanisms and enhance treatment efficacy. METHODS: Here, we report two different methods for generating melanoma patient-derived organoids (MPDOs), one is embedded in collagen gel, and the other is inlaid in Matrigel. MPDOs in Matrigel are used for assessing the therapeutic effects of anti-PD-1 antibodies (αPD-1), autochthonous tumor infiltrating lymphocytes (TILs), and small molecule compounds. MPDOs in collagen gel are used for evaluating the chemotaxis and migratory capacity of TILs. FINDING: The MPDOs in collagen gel and Matrigel have similar morphology and immune cell composition to their parental melanoma tissues. MPDOs show inter- and intra-tumoral heterogeneity and contain diverse immune cells such as CD4+, CD8+ T, Treg, CD14+ monocytic, CD15+, and CD11b+ myeloid cells. The tumor microenvironment (TME) in MPDOs is highly immunosuppressive, and the lymphoid and myeloid lineages express similar levels of PD-1, PD-L1, and CTLA-4 as their parental melanoma tissues. Anti-PD-1 antibodies (αPD-1) reinvigorate CD8+ T cells and induce melanoma cell death in the MPDOs. TILs expanded by IL-2 and αPD-1 show significantly lower expression of TIM-3, better migratory capacity and infiltration of autochthonous MPDOs, and more effective killing of melanoma cells than TILs expanded by IL-2 alone or IL-2 with αCD3. A small molecule screen discovers that Navitoclax increases the cytotoxicity of TIL therapy. INTERPRETATION: MPDOs may be used to test immune checkpoint inhibitors and cellular and targeted therapies. FUNDING: This work was supported by the NIH grants CA114046, CA261608, CA258113, and the Tara Miller Melanoma Foundation.

Future of Digital Assays to Resolve Clinical Heterogeneity of Single Extracellular Vesicles.

Extracellular vesicles (EVs) are complex lipid membrane vehicles with variable expressions of molecular cargo, composed of diverse subpopulations that participate in the intercellular signaling of biological responses in disease. EV-based liquid biopsies demonstrate invaluable clinical potential for overhauling current practices of disease management. Yet, EV heterogeneity is a major needle-in-a-haystack challenge to translate their use into clinical practice. In this review, existing digital assays will be discussed to analyze EVs at a single vesicle resolution, and future opportunities to optimize the throughput, multiplexing, and sensitivity of current digital EV assays will be highlighted. Furthermore, this review will outline the challenges and opportunities that impact the clinical translation of single EV technologies for disease diagnostics and treatment monitoring.

A Customizable and Low-Cost Ultraviolet Exposure System for Photolithography.

For microfluidic device fabrication in the research, industry, and commercial areas, the curing and transfer of patterns on photoresist relies on ultraviolet (UV) light. Often, this step is performed by commercial mask aligner or UV lamp exposure systems; however, these machines are often expensive, large, and inaccessible. To find an alternative solution, we present an inexpensive, customizable, and lightweight UV exposure system that is user-friendly and readily available for a homemade cleanroom. We fabricated a portable UV exposure system that costs under $200. The wafer holder's adjustable height enabled for the selection of the appropriate curing distance, demonstrating our system's ability to be easily tailored for different applications. The high light uniformity across a 4" diameter wafer holder (light intensity error ~2.9%) was achieved by adding a light diffusing film to the apparatus. These values are comparable to the light uniformity across a 5" diameter wafer holder from a commercial mask aligner (ABM 3000HR Mask Aligner), that has a light intensity error of ~4.0%. We demonstrated the ability to perform photolithography with high quality by fabricating microfluidic devices and generating uniform microdroplets. We achieved comparable quality to the wafer patterns, microfluidic devices, and droplets made from the ABM 3000HR Mask Aligner.

Advancing microfluidic diagnostic chips into clinical use: a review of current challenges and opportunities.

Microfluidic diagnostic (µDX) technologies miniaturize sensors and actuators to the length-scales that are relevant to biology: the micrometer scale to interact with cells and the nanometer scale to interrogate biology's molecular machinery. This miniaturization allows measurements of biomarkers of disease (cells, nanoscale vesicles, molecules) in clinical samples that are not detectable using conventional technologies. There has been steady progress in the field over the last three decades, and a recent burst of activity catalyzed by the COVID-19 pandemic. In this time, an impressive and ever-growing set of technologies have been successfully validated in their ability to measure biomarkers in clinical samples, such as blood and urine, with sensitivity and specificity not possible using conventional tests. Despite our field's many accomplishments to date, very few of these technologies have been successfully commercialized and brought to clinical use where they can fulfill their promise to improve medical care. In this paper, we identify three major technological trends in our field that we believe will allow the next generation of µDx to have a major impact on the practice of medicine, and which present major opportunities for those entering the field from outside disciplines: 1. the combination of next generation, highly multiplexed µDx technologies with machine learning to allow complex patterns of multiple biomarkers to be decoded to inform clinical decision points, for which conventional biomarkers do not necessarily exist. 2. The use of micro/nano devices to overcome the limits of binding affinity in complex backgrounds in both the detection of sparse soluble proteins and nucleic acids in blood and rare circulating extracellular vesicles. 3. A suite of recent technologies that obviate the manual pre-processing and post-processing of samples before they are measured on a µDX chip. Additionally, we discuss economic and regulatory challenges that have stymied µDx translation to the clinic, and highlight strategies for successfully navigating this challenging space.