Biophysical Profiling Research Articles

Decipher 'Em All: A Profiling Study on the Effects of Acyl Groups in O-Acyl-ω-hydroxy Fatty Acids.

The O-acyl-ω-hydroxy fatty acids (OAHFAs) are an intriguing class of surface-active lipids which can be found in the human tear film lipid layer (TFLL). Recent studies have suggested that OAHFAs exist in the polar lipid layer and play a central role in TFLL function. Surprisingly, biophysical profiling studies have only shed light on the properties of OAHFAs bearing an oleate acyl group and insights on species with other acyl groups are scarce. Herein, we seek to address this issue through (1) focusing on the synthesis and characterization of a representative library of OAHFA analogues bearing a palmitate, palmitoleate, stearate, and linoleate acyl group, and (2) performing an in-depth mapping of their biophysical properties. Our results indicate that NMR-spectroscopic techniques can be utilized for rough estimation of the amounts of distinct acyl groups in a sample and more importantly, how the subtle variations in both parent chains and acyl groups influence the core properties of the OAHFAs. We reach the conclusion that the correlation between melting points and film properties is not as clear-cut as previously thought. Nevertheless, grouping of OAHFA species into three separate categories which display distinct behavior seems to be possible utilizing the melting points as a guiding parameter. Altogether, our study suggests that the properties of OAHFAs need to be assessed from a viewpoint which combines both the parent chain and acyl group instead of independent analysis based on either fragment alone.

Biophysical profiling of red blood cells from thin-film blood smears using deep learning

Microscopic inspection of thin-film blood smears is widely used to identify red blood cell (RBC) pathologies, including malaria parasitism and hemoglobinopathies, such as sickle cell disease and thalassemia. Emerging research indicates that non-pathologic changes in RBCs can also be detected in images, such as deformability and morphological changes resulting from the storage lesion. In transfusion medicine, cell deformability is a potential biomarker for the quality of donated RBCs. However, a major impediment to the clinical translation of this biomarker is the difficulty associated with performing this measurement. To address this challenge, we developed an approach for biophysical profiling of RBCs based on cell images in thin-film blood smears. We hypothesize that subtle cellular changes are evident in blood smear images, but this information is inaccessible to human expert labellers. To test this hypothesis, we developed a deep learning strategy to analyze Giemsa-stained blood smears to assess the subtle morphologies indicative of RBC deformability and storage-based degradation. Specifically, we prepared thin-film blood smears from 27 RBC samples (9 donors evaluated at 3 storage time points) and imaged them using high-resolution microscopy. Using this dataset, we trained a convolutional neural network to evaluate image-based morphological features related to cell deformability. The prediction of donor deformability is strongly correlated to the microfluidic scores and can be used to categorize images into specific deformability groups with high accuracy. We also used this model to evaluate differences in RBC morphology resulting from cold storage. Together, our results demonstrate that deep learning models can detect subtle cellular morphology differences resulting from deformability and cold storage. This result suggests the potential to assess donor blood quality from thin-film blood smears, which can be acquired ubiquitously in clinical workflows.

Biophysical profiling and in vitro study of apocarotenoids with calf thymus DNA to explore their efficacy in adjuvant cancer therapy

DNA is one of the important targets for therapeutic drugs. Investigating the interaction between these drug molecules and DNA yields valuable insights for the further advancement of medication formulations. In this study, we explore the interaction between calf thymus-DNA and temozolomide with apocarotenoids such as bixin and crocin, which are known for their therapeutic activities. The combinatory effect of the compounds on ct-DNA was studied through diverse spectroscopic methods including UV-visible, circular dichroism studies coupled with viscosity and cell cycle analysis. UV-visible spectroscopy evaluation indicates that apocarotenoids and the temozolomide combination display a non-intercalative binding mechanism. The circular dichroism study revealed that the interaction between DNA and apocarotenoids, as well as their combinations with temozolomide, did not present any significant alterations in the structure of DNA suggesting a possible groove binding interaction. The viscosity analyses verified that ct-DNA interacts with apocarotenoids along with their temozolomide combination through a groove-binding mechanism. This study offers a more comprehensive understanding of the potential mechanism of interaction between ct-DNA and natural compounds with the addition of temozolomide.

Another Brick in the Wall of Tear Film Insights Added Through the Total Synthesis and Biophysical Profiling of anteiso-Branched Wax and Cholesteryl Esters.

The tear film lipid layer (TFLL) plays a vital part in maintenance of ocular health and represents a unique biological barrier comprising unusual and specialized lipid classes and species. The wax and cholesteryl esters (WEs and CEs) constitute roughly 80-90% of the TFLL. The majority of species in these lipid classes are branched and it is therefore surprising that the synthesis and properties of the second largest category of species, i.e., the anteiso-branched species, remain poorly characterized. In this study, we have developed a total synthesis route and completed a detailed NMR spectroscopic characterization of two common anteiso-branched species, namely: (22S)-22-methyltetracosanyl oleate and cholesteryl (22'S)-22'-methyltetracosanoate. In addition, we have studied their structural properties in the bulk state by wide-angle and small-angle X-ray scattering and their behavior at the aqueous interface using Langmuir monolayer techniques. A comparison to the properties displayed by iso-branched and straight-chain analogues indicate that branching patterns lead to distinct properties in the CE and WE lipid classes. Overall, this study complements the previous work in the field and adds another important brick in the tear film insights wall.

A Stratum corneum lipid model as a platform for biophysical profiling of bioactive chemical interactions at the skin level

Stratum Corneum (SC), the outermost layer of mammalian skin, provides the first and most challenging barrier to skin permeation of cosmetic and pharmaceutical compounds. SC barrier function relies primarily on the complex structure and organization of the intercellular lipid matrix. This matrix consists mainly of ceramides (CER), free fatty acids (FFA), and cholesterol (Chol) in an equimolar ratio, forming a multilamellar structure with short- and long-periodicity phases. Along with permeation studies, it becomes paramount the molecular investigation of the interaction between the lipid matrix and the compounds, towards a comprehensive picture of their biophysical impact, including lipid packing impairment, which can result in skin damage, and in consequences for their permeation and mechanism of action. In this study, a lipid-based model (CER[EOS]:CER[AP]:Chol:FFA) was developed to accurately resemble the barrier properties of the intercellular lipid matrix. In a monolayer assembly, this SC lipid model was characterized and applied as an in vitro platform to explore interactions with model compounds – caffeine and testosterone – following OECD recommendations. The SC model successfully mimicked different barrier properties of the SC’s lipid matrix, and its interactions with the model compounds were discussed closely aligned with existing data, including in vitro permeation studies using human and animal membranes. This SC model can be a step forward towards the development of new in vitro platforms for investigating the skin barrier function and the interactions with external molecules. Moreover, the composition of these platforms can be easily modified to mimic impaired lipid matrices to study different skin conditions, providing valuable insights for both cosmetic and pharmaceutical applications.

Hepatic Morphometric Correlation with Biophysical Profiling of Height

Hepatic Morphometric Correlation with Biophysical Profiling of Height

255. Borrelia-specific antibody profiling in joint fluid distinguishes antibiotic-refractory Lyme arthritis from antibiotic-responsive arthritis

Abstract Background Lyme disease, caused by the spirochete Borrelia burgdorferi, is the most common vector-borne disease in the United States. It drives a multisystem disorder, of which Lyme arthritis is the most common feature of late disseminated disease, seen in approximately 60% of untreated individuals. While most Lyme arthritis resolves with oral or IV antibiotics, termed “antibiotic-responsive” arthritis, a small percentage of individuals develop progressive synovitis despite both oral and IV antibiotic therapy, called “antibiotic refractory” Lyme arthritis (LA), requiring treatment with immunosuppressive, disease modifying antirheumatic drugs (DMARDs). The primary drivers behind antibiotic refractory disease are likely multifactorial and remain incompletely understood. More specifically, it remains unclear whether antibodies, specific for Borrelia or autoantibodies, may act as biomarkers or play a mechanistic role in driving pathogenesis in the joint. Methods We performed a matched, cross-compartmental comparison of antibody profiles from blood and joint fluid of individuals with antibiotic responsive (n = 11) or antibiotic refractory arthritis (n= 31). We used a multiplexed luminex assay to perform biophysical profiling of Borrelia burgdorferi -specific antibodies to B67 lysate, CRASP1, CRASP2, DbpA, DbpB, Arp37, Flagellin, OspA, OspB, OspC, OspE, p27, p35, p39, VlsE, and autoantigen Apolipoprotein B100. Results While serum antibody profiles poorly discriminated responsive from refractory LA patients, a discrete profile of B.burgdorferi-specific antibodies in the joint fluid was able to discriminate antibiotic responsive from refractory LA patients. Cross compartmental comparison of antibody glycosylation, IgA1, and antibody-dependent complement deposition (ADCD) revealed differences across compartments, with more poorly coordinated Lyme-specific humoral responses and increased antibody-dependent complement deposition in antibiotic-refractory arthritis. Conclusion These data demonstrate B.burgdorferi-specific serological markers that may support the early stratification and clinical management of LA, but also point to immune complex driven complement activation as a key mechanism underlying persistent Lyme arthritis. Disclosures Christine Wiggins, B.S., Takeda Pharmaceutical Company: Stocks/Bonds Elizabeth Deriso, PhD, Takeda: Employee Klemen Strle, PhD, Takeda: I am an employee of Takeda. My work at Takeda does not overlap with my academic efforts in Lyme disease. John A. Branda, M.D, Analog Devices Inc.: Grant/Research Support|DiaSorin: Grant/Research Support|Gold Standard Diagnostics: Grant/Research Support|Pfizer Inc: Grant/Research Support|Zeus Scientific: Grant/Research Support Douglas A. Lauffenburger, PhD, Sanofi: Board Member|Sanofi: Honoraria|Sanofi: Honoraria Galit Alter, PhD, Leyden Labs: Ownership Interest|Moderna Therapeutics: Employee|Seromyx Systems: Ownership Interest

Biophysical Profiling of Sickle Cell Disease Using Holographic Cytometry and Deep Learning.

Sickle cell disease (SCD) is an inherited hematological disorder associated with high mortality rates, particularly in sub-Saharan Africa. SCD arises due to the polymerization of sickle hemoglobin, which reduces flexibility of red blood cells (RBCs), causing blood vessel occlusion and leading to severe morbidity and early mortality rates if untreated. While sickle solubility tests are available to sub-Saharan African population as a means for detecting sickle hemoglobin (HbS), the test falls short in assessing the severity of the disease and visualizing the degree of cellular deformation. Here, we propose use of holographic cytometry (HC), a high throughput, label-free imaging modality, for comprehensive morphological profiling of RBCs as a means to detect SCD. For this study, more than 2.5 million single-cell holographic images from normal and SCD patient samples were collected using the HC system. We have developed an approach for specially defining training data to improve machine learning classification. Here, we demonstrate the deep learning classifier developed using this approach can produce highly accurate classification, even on unknown patient samples.

Nav1.7 P610T mutation in two siblings with persistent ocular pain after corneal axon transection: impaired slow inactivation and hyperexcitable trigeminal neurons.

Despite extensive study, the mechanisms underlying pain after axonal injury remain incompletely understood. Pain after corneal refractive surgery provides a model, in humans, of the effect of injury to trigeminal afferent nerves. Axons of trigeminal ganglion neurons that innervate the cornea are transected by laser-assisted in situ keratomileusis (LASIK). Although most patients do not experience postoperative pain, a small subgroup develop persistent ocular pain. We previously carried out genomic analysis and determined that some patients with persistent pain after axotomy of corneal axons during refractive surgery carry mutations in genes that encode the electrogenisome of trigeminal ganglion neurons, the ensemble of ion channels and receptors that regulate excitability within these cells, including SCN9A, which encodes sodium channel Nav1.7, a threshold channel abundantly expressed in sensory neurons that has been implicated in a number of pain-related disorders. Here, we describe the biophysical and electrophysiological profiling of the P610T Nav1.7 mutation found in two male siblings with persistent ocular pain after refractive surgery. Our results indicate that this mutation impairs the slow inactivation of Nav1.7. As expected from this proexcitatory change in channel function, we also demonstrate that this mutation produces increased spontaneous activity in trigeminal ganglion neurons. These findings suggest that this gain-of-function mutation in Nav1.7 may contribute to pain after injury to the axons of trigeminal ganglion neurons.NEW & NOTEWORTHY Mechanisms underlying pain after axonal injury remain elusive. A small subgroup of patients experience pain after corneal refractive surgery, providing a human pain model after well-defined injury to axons. Here we analyze a mutation (P610T) in Nav1.7, a threshold sodium channel expressed in nociceptors, found in two siblings with persistent ocular pain after refractive surgery. We show that it impairs channel slow inactivation, thereby triggering inappropriate repetitive activity in trigeminal ganglion axons that signal eye pain.

Biophysical profiling of synthetic ultra-long tear film lipids

The tear film lipid layer (TFLL) is a unique biological membrane of importance to the maintenance of ocular surface health. The underlying factors at play, e.g. the ability to retard evaporation and offer protection from the environment, are all closely connected to the properties of individual lipid components and their interplay. The TFLL contains unique ultra-long polar lipid species such as O-acyl-ω-hydroxy fatty acids, type I-St diesters and type II diesters, which are considered important for its proper function. Herein, we have synthesized model compounds from these categories and studied their biophysical and surface rheological properties at the aqueous interface. Altogether, we provide insights on the distinct biophysical profiles of these lipid classes and discuss how their interplay may affect the structure and function of the TFLL.

Advancing ASMS with LC-MS/MS for the discovery of novel PDCL2 ligands from DNA-encoded chemical library selections.

A safe, effective, and reversible nonhormonal male contraceptive drug is greatly needed for male contraception as well as for circumventing the side effects of female hormonal contraceptives. Phosducin-like 2 (PDCL2) is a testis-specific phosphoprotein in mice and humans. We recently found that male PDCL2 knockout mice are sterile due to globozoospermia caused by impaired sperm head formation, indicating that PDCL2 is a potential target for male contraception. Herein, our study for the first time developed a biophysical assay for PDCL2 allowing us to screen a series of small molecules, to study structure-activity relationships, and to discover two PDCL2 binders with novel chemical structure. To identify a PDCL2 ligand for therapeutic male contraception, we performed DNA-encoded chemical library (DECL) screening and off-DNA hit validation using a unique affinity selection mass spectrometry (ASMS) biophysical profiling strategy. We employed the screening process of DECL, which contains billions of chemically unique DNA-barcoded compounds generated through individual sequences of reactions and different combinations of functionalized building blocks. The structures of the PDCL2 binders are proposed based on the sequencing analysis of the DNA barcode attached to each individual DECL compound. The proposed structure is synthesized through multistep reactions. To confirm and determine binding affinity between the DECL identified molecules and PDCL2, we developed an ASMS assay that incorporates liquid chromatography with tandem mass spectrometry (LC-MS/MS). After a screening process of PDCL2 with DECLs containing>440 billion compounds, we identified a series of hits. The selected compounds were synthesized as off-DNA small molecules, characterized by spectroscopy data, and subjected to our ASMS/LC-MS/MS binding assay. By this assay, we discovered two novel compounds, which showed good binding affinity for PDCL2 in comparison to other molecules generated in our laboratory andwhich were further confirmed by a thermal shift assay. With the ASMS/LC-MS/MS assay developed in this paper, we successfully discovered a PDCL2 ligand that warrants further development as a male contraceptive.

Biophysical, Molecular and Proteomic Profiling of Human Retinal Organoid-Derived Exosomes.

There is a growing interest in extracellular vesicles (EVs) for ocular applications as therapeutics, biomarkers, and drug delivery vehicles. EVs secreted from mesenchymal stem cells (MSCs) have shown to provide therapeutic benefits in ocular conditions. However, very little is known about the properties of bioreactor cultured-3D human retinal organoids secreted EVs. This study provides a comprehensive morphological, nanomechanical, molecular, and proteomic characterization of retinal organoid EVs and compares it with human umbilical cord (hUC) MSCs. The morphology and nanomechanical properties of retinal organoid EVs were assessed using Nanoparticle tracking analysis (NTA) and Atomic force microscopy (AFM). Gene expression analysis of exosome biogenesis of early and late retinal organoids were compared using qPCR. The protein profile of the EVs were analyzed with proteomic tools. NTA indicated the average size of EV as 100-250nm. A high expression of exosome biogenesis genes was observed in late retinal organoids EVs. Immunoblot analysis showed highly expressed exosomal markers in late retinal organoids EVs compared to early retinal organoids EVs. Protein profiling of retinal organoid EVs displayed a higher differential expression of retinal function-related proteins and EV biogenesis proteins than hUCMSC EVs, implicating that the use of retinal organoid EVs may have a superior therapeutic effect on retinal disorders. This study provides supplementary knowledge on the properties of retinal organoid EVs and suggests their potential use in the diagnostic and therapeutic treatments for ocular diseases.

Anandamide-Modulated Changes in Metabolism, Glycosylation Profile and Migration of Metastatic Melanoma Cells.

Simple SummaryAnandamide (AEA) belongs to the group of endocannabinoids and possesses various regulatory properties in physiological as well as pathological processes occurring in the organism. In this research some basic biological tests were applied to investigate AEA-induced changes in cell metabolism and motility, as well as advanced biophysical methods for the determination of the differences in the cell glycosylation profile on a highly dangerous model of melanoma skin cancer, for which an effective therapy is not yet available. Our research suggests that anandamide treatment of metastatic melanoma cells increases the cell metabolism which leads to the reduction in the metastatic potential of cells in terms of the cell glycosylation profile and cell migration. In the view of our research, it can be presumed that anandamide usage in the combined therapy of advanced melanoma would be an advantage for the patient.An effective therapy for advanced melanoma, a skin cancer with the highest mortality, has not yet been developed. The endocannabinoid system is considered to be an attractive target for cancer treatment. The use of endocannabinoids, such as anandamide (AEA), is considered to be much greater than as a palliative agent. Thus, we checked its influence on various signaling pathways in melanoma cells. Our investigation was performed on four commercial cell lines derived from different progression stages (radial WM35 and vertical WM115 growth phases, lymph node WM266-4 metastasis, solid tumor A375-P metastasis). Cell viability, glucose uptake, quantification of reactive oxygen species production, expression of selected genes encoding glycosyltransferases, quantification of glycoproteins production and changes in the glycosylation profile and migration, as well as in cell elastic properties were analyzed. The cell glycosylation profile was investigated using the biophysical profiling method—the quartz crystal microbalance with dissipation monitoring (QCM-D). Anandamide treatment of only metastatic cells resulted in: an increase in the cell metabolism, a decrease in GFAT-1 and DPM1 expression, followed by a decrease in L1-CAM glycoprotein production, which further influenced the reduction in the cell glycosylation profile and migration. Considering our results, AEA usage is highly recommended in the combined therapy of advanced melanoma.

Evaluation of Homocysteine and Gamma-Glutamyl Transferase Concentrations As Markers of Chronic Kidney Disease: An Indian Perspective.

BackgroundChronic kidney disease (CKD) involves a gradual loss of kidney function over months to years. Oxidative stress plays a critical role in the pathogenesis of CKD. Homocysteine (Hcy), an amino acid derivative, is a known risk factor for oxidative stress and endothelial damage. Gamma-glutamyl transferase (GGT), an enzyme abundant on the cell surface of liver and kidney cells, is raised during oxidative stress. The objectives of this study were to estimate the concentrations of serum Hcy and GGT among CKD patients and healthy controls and to determine whether there is an association between serum Hcy and GGT levels in CKD.MethodologyA total of 246 participants were needed to meet the calculated sample size. A total of 123 CKD patients meeting the inclusion and exclusion criteria were recruited as cases from the Nephrology outpatient department of our institute. Equal numbers of age- and sex-matched healthy volunteers were recruited as controls. Biophysical profiling of participants was done. Baseline investigations were recorded. A blood sample was collected from each participant and analyzed for GGT and Hcy along with other routine parameters.ResultsHcy and GGT concentrations were significantly high in CKD patients compared to healthy controls. There was a significant positive correlation between serum GGT and Hcy levels (r = 0.357).ConclusionsElevated levels of GGT and Hcy in CKD patients compared to healthy controls demonstrated the oxidative stress associated with the disease. GGT and Hcy can be used as prognostic markers of the disease.

Novel diagnostic and prognostic factors for the advanced melanoma based on the glycosylation-related changes studied by biophysical profiling methods

Melanoma is a life-threatening disease due to the early onset of metastasis and frequent resistance to the applied treatment. For now, no single histological, immunohistochemical or serological biomarker was able to provide a precise predictive value for the aggressive behavior in melanoma patients. Thus, the search for quantifying methods allowing a simultaneous diagnosis and prognosis of melanoma patients is highly desirable. By investigating specific molecular interactions with some biosensor-based techniques, one can determine novel prognostic factors for this tumor. In our previous study, we have shown the possibility of a qualitative in vitro distinguishing the commercially available melanoma cells at different progression stages based on the measurements of the lectin Concanavalin A interacting with surface glycans present on cells.Here, we present the results of the quantitative diagnostic and prognostic study of both commercial and patient-derived melanoma cells based on the evaluation of two novel factors: lectin affinity and glycan viscoelastic index obtained from the quartz crystal microbalance with dissipation monitoring (QCM-D) measurements. Two approaches to the QCM-D measurements were applied, the first uses the ability of melanoma cells to grow as a monolayer of cells on the sensor (cell-based sensors), and the second shortens the time of the analysis (suspension cell based-sensors). The results were confirmed by the complementary label-free (atomic force microscopy, AFM; and surface plasmon resonance, SPR) and labeling (lectin-ELISA; and microscale thermophoresis, MST) techniques. This new approach provides additional quantitative diagnosis and a personalized prognosis which can be done simultaneously to the traditional histopathological analysis.

Diverse antiviral IgG effector activities are predicted by unique biophysical antibody features

BackgroundThe critical role of antibody Fc-mediated effector functions in immune defense has been widely reported in various viral infections. These effector functions confer cellular responses through engagement with innate immune cells. The precise mechanism(s) by which immunoglobulin G (IgG) Fc domain and cognate receptors may afford protection are poorly understood, however, in the context of HIV/SHIV infections. Many different in vitro assays have been developed and utilized to measure effector functions, but the extent to which these assays capture distinct antibody activities has not been fully elucidated.ResultsIn this study, six Fc-mediated effector function assays and two biophysical antibody profiling assays were performed on a common set of samples from HIV-1 infected and vaccinated subjects. Biophysical antibody profiles supported robust prediction of diverse IgG effector functions across distinct Fc-mediated effector function assays. While a number of assays showed correlated activities, supervised machine learning models indicated unique antibody features as primary contributing factors to the associated effector functions. Additional experiments established the mechanistic relevance of relationships discovered using this unbiased approach.ConclusionsIn sum, this study provides better resolution on the diversity and complexity of effector function assays, offering a clearer perspective into this family of antibody mechanisms of action to inform future HIV-1 treatment and vaccination strategies.

Tensor‐structured decomposition improves systems serology analysis

Systems serology provides a broad view of humoral immunity by profiling both the antigen‐binding and Fc properties of antibodies. These studies contain structured biophysical profiling across disease‐relevant antigen targets, alongside additional measurements made for single antigens or in an antigen‐generic manner. Identifying patterns in these measurements helps guide vaccine and therapeutic antibody development, improve our understanding of diseases, and discover conserved regulatory mechanisms. Here, we report that coupled matrix–tensor factorization (CMTF) can reduce these data into consistent patterns by recognizing the intrinsic structure of these data. We use measurements from two previous studies of HIV‐ and SARS‐CoV‐2‐infected subjects as examples. CMTF outperforms standard methods like principal components analysis in the extent of data reduction while maintaining equivalent prediction of immune functional responses and disease status. Under CMTF, model interpretation improves through effective data reduction, separation of the Fc and antigen‐binding effects, and recognition of consistent patterns across individual measurements. Data reduction also helps make prediction models more replicable. Therefore, we propose that CMTF is an effective general strategy for data exploration in systems serology.

Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols towards Downstream Isolation and Analysis.

Since the outbreak of the COVID-19 crisis, the handling of biological samples from confirmed or suspected SARS-CoV-2-positive individuals demanded the use of inactivation protocols to ensure laboratory operators’ safety. While not standardized, these practices can be roughly divided into two categories, namely heat inactivation and solvent-detergent treatments. These routine procedures should also apply to samples intended for Extracellular Vesicles (EVs) analysis. Assessing the impact of virus-inactivating pre-treatments is therefore of pivotal importance, given the well-known variability introduced by different pre-analytical steps on downstream EVs isolation and analysis. Arguably, shared guidelines on inactivation protocols tailored to best address EVs-specific requirements will be needed among the analytical community, yet deep investigations in this direction have not yet been reported. We here provide insights into SARS-CoV-2 inactivation practices to be adopted prior to serum EVs analysis by comparing solvent/detergent treatment vs. heat inactivation. Our analysis entails the evaluation of EVs recovery and purity along with biochemical, biophysical and biomolecular profiling by means of a set of complementary analytical techniques: Nanoparticle Tracking Analysis, Western Blotting, Atomic Force Microscopy, miRNA content (digital droplet PCR) and tetraspanin assessment by microarrays. Our data suggest an increase in ultracentrifugation (UC) recovery following heat treatment; however, it is accompanied by a marked enrichment in EVs-associated contaminants. On the other hand, solvent/detergent treatment is promising for small EVs (<150 nm range), yet a depletion of larger vesicular entities was detected. This work represents a first step towards the identification of optimal serum inactivation protocols targeted to EVs analysis.

Inflammation Biomarkers: Label‐Free Biophysical Markers from Whole Blood Microfluidic Immune Profiling Reveal Severe Immune Response Signatures (Small 12/2021)

Host immune response plays a critical role in disease manifestation and severity. In article number 2006123, Win Sen Kuan, Jongyoon Han, and co-workers present a whole blood biophysical immune response profiling tool using deterministic lateral displacement microfluidics. The speed and accuracy of immune response stratification demonstrated in an emergency department has a clear impact in enabling deployable point-of-care systems for rapid clinical triage.

Abstract IA20: Aberrant leukemic developmental hierarchies and MRD-specific targeting informed by single-cell biophysical and molecular profiling

Abstract Targeted inhibitors of essential oncogenic kinases induce high rates of clinical response but cure few patients due to the persistence of minimal residual disease (MRD). BCR-ABL mutant leukemias are a classic example of this paradigm where patients usually achieve deep remissions followed by near inevitable relapses. Multiple factors have been shown to influence how an individual patient’s leukemic cells will navigate treatment including differentiation state, mutational background, and communication with the microenvironment. Here, we use BCR-ABL-rearranged acute lymphoblastic leukemia (BCR-ABL ALL) to interrogate cell-autonomous features leading to therapeutic resistance using low-input single-cell assays. Specifically, we use a combination of primary samples and PDX models to dissect aberrant developmental hierarchies and monitor leukemic cell transcriptional and biophysical phenotype at pretreatment, MRD, and relapse. Using machine learning, we relate malignant B cells to normal development, allowing us to define leukemic developmental programs and demonstrate that these have consequences for the time to progression as well as the genetic alterations seen at relapse. Further, we determine that there are unique biophysical features tied to leukemic developmental states and that these integrative properties co-evolve with transcriptional state over the course of treatment. Finally, we demonstrate in PDX studies that it may be possible to intercept relapse by targeting specific features of MRD cells. Together, these data suggest that significant developmental hierarchies exist in ALL, tumor subpopulations can be identified directly within MRD, and their phenotypic and molecular characterization can be exploited to therapeutic effect. Citation Format: Peter S. Winter, Andrew Navia, Haley Strouf, Mahnoor Mirza, Jennyfer Galvez-Reyes, Nolawit Mulugeta, Laura Bilal, Nezha Senhaji, Peter Dennis, Catharine S. Leahy, Kay Shigemori, Foster Powers, Alejandro Gupta, Nicholas Calistri, Alex Van Scoyk, Kristen Jones, Huiyun Liu, Kristen E. Stevenson, Robert Kimmerling, Mark Stevens, David M. Weinstock, Scott R. Manalis, Mark A. Murakami, Alex K. Shalek. Aberrant leukemic developmental hierarchies and MRD-specific targeting informed by single-cell biophysical and molecular profiling [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA20.