Nanoparticle-Supported, Rapid, Digital Quantification of Neutralizing Antibodies Against SARS-CoV-2 Variants.

The measurement of neutralizing immune responses to viral infection is essential, given the heterogeneity of human immunity and the emergence of new virus strains. However, neutralizing antibody (nAb) assays often require high-level biosafety containment, sophisticated instrumentation, and long detection times. Here, as a proof-of-principle, we designed a nanoparticle-supported, rapid, electronic detection (NasRED) assay to assess the neutralizing potency of monoclonal antibodies (mAbs) against SARS-CoV-2.

Predicting monoclonal antibody binding sequences from a sparse sampling of all possible sequences.

Previous work has shown that binding of target proteins to a sparse, unbiased sample of all possible peptide sequences is sufficient to train a machine learning model that can then predict, with statistically high accuracy, target binding to any possible peptide sequence of similar length. Here, highly sequence-specific molecular recognition is explored by measuring binding of 8 monoclonal antibodies (mAbs) with specific linear cognate epitopes to an array containing 121,715 near-random sequences about 10 residues in length. Network models trained on resulting sequence-binding values are used to predict the binding of each mAb to its cognate sequence and to an in silico generated one million random sequences.

Modeling the sequence dependence of differential antibody binding in the immune response to infectious disease.

Past studies have shown that incubation of human serum samples on high density peptide arrays followed by measurement of total antibody bound to each peptide sequence allows detection and discrimination of humoral immune responses to a variety of infectious diseases. This is true even though these arrays consist of peptides with near-random amino acid sequences that were not designed to mimic biological antigens. This "immunosignature" approach, is based on a statistical evaluation of the binding pattern for each sample but it ignores the information contained in the amino acid sequences that the antibodies are binding to.

Integrating fluorescence computed tomography with optical sheet illumination for imaging of live single cells.

We present a new approach for three-dimensional (3D) live single-cell imaging with isotropic sub-micron spatial resolution using fluorescence computed tomography (fCT). A thin, highly inclined and laminated optical (HILO) sheet of light is used for fluorescence excitation in live single cells that are rotated around an axis perpendicular to the optical axis. During a full rotation, 400-500 two-dimensional (2D) projection images of the cell are acquired from multiple viewing perspectives by rapidly scanning the HILO light sheet along the optical axis.

Simultaneous Multiparameter Cellular Energy Metabolism Profiling of Small Populations of Cells.

Functional and genomic heterogeneity of individual cells are central players in a broad spectrum of normal and disease states. Our knowledge about the role of cellular heterogeneity in tissue and organism function remains limited due to analytical challenges one encounters when performing single cell studies in the context of cell-cell interactions. Information based on bulk samples represents ensemble averages over populations of cells, while data generated from isolated single cells do not account for intercellular interactions.

Optical computed tomography for spatially isotropic four-dimensional imaging of live single cells.

Quantitative three-dimensional (3D) computed tomography (CT) imaging of living single cells enables orientation-independent morphometric analysis of the intricacies of cellular physiology. Since its invention, x-ray CT has become indispensable in the clinic for diagnostic and prognostic purposes due to its quantitative absorption-based imaging in true 3D that allows objects of interest to be viewed and measured from any orientation. However, x-ray CT has not been useful at the level of single cells because there is insufficient contrast to form an image.

A versatile method for dynamically controlled patterning of small populations of epithelial cells on substrates via non-contact piezoelectric inkjet printing.

Intercellular interactions play a central role at the tissue and whole organism level modulating key cellular functions in normal and disease states. Studies of cell-cell communications are challenging due to ensemble averaging effects brought about by intrinsic heterogeneity in cellular function which requires such studies to be conducted with small populations of cells. Most of the current methods for producing and studying such small cell populations are complex to implement and require skilled personnel limiting their widespread utility in biomedical research labs.

A platform for high-throughput bioenergy production phenotype characterization in single cells.

Driven by an increasing number of studies demonstrating its relevance to a broad variety of disease states, the bioenergy production phenotype has been widely characterized at the bulk sample level. Its cell-to-cell variability, a key player associated with cancer cell survival and recurrence, however, remains poorly understood due to ensemble averaging of the current approaches. We present a technology platform for performing oxygen consumption and extracellular acidification measurements of several hundreds to 1,000 individual cells per assay, while offering simultaneous analysis of cellular communication effects on the energy production phenotype.

Platform for combined analysis of functional and biomolecular phenotypes of the same cell.

Functional and molecular cell-to-cell variability is pivotal at the cellular, tissue and whole-organism levels. Yet, the ultimate goal of directly correlating the function of the individual cell with its biomolecular profile remains elusive. We present a platform for integrated analysis of functional and transcriptional phenotypes in the same single cells.

Transcriptional regulation by normal epithelium of premalignant to malignant progression in Barrett's esophagus.

In carcinogenesis, intercellular interactions within and between cell types are critical but remain poorly understood. We present a study on intercellular interactions between normal and premalignant epithelial cells and their functional relevance in the context of premalignant to malignant progression in Barrett's esophagus. Using whole transcriptome profiling we found that in the presence of normal epithelial cells, dysplastic cells but not normal cells, exhibit marked down-regulation of a number of key signaling pathways, including the transforming growth factor beta (TGFβ) and epithelial growth factor (EGF).

Modulating short wavelength fluorescence with long wavelength light.

Two molecules in which the intensity of shorter-wavelength fluorescence from a strong fluorophore is modulated by longer-wavelength irradiation of an attached merocyanine-spirooxazine reverse photochromic moiety have been synthesized and studied. This unusual fluorescence behavior is the result of quenching of fluorophore fluorescence by the thermally stable, open, zwitterionic form of the spirooxazine, whereas the photogenerated closed, spirocyclic form has no effect on the fluorophore excited state. The population ratio of the closed and open forms of the spirooxazine is controlled by the intensity of the longer-wavelength modulated light.

A minimally invasive method for retrieving single adherent cells of different types from cultures.

The field of single-cell analysis has gained a significant momentum over the last decade. Separation and isolation of individual cells is an indispensable step in almost all currently available single-cell analysis technologies. However, stress levels introduced by such manipulations remain largely unstudied.

Single-cell analysis reveals early manifestation of cancerous phenotype in pre-malignant esophageal cells.

Cellular heterogeneity plays a pivotal role in a variety of functional processes in vivo including carcinogenesis. However, our knowledge about cell-to-cell diversity and how differences in individual cells manifest in alterations at the population level remains very limited mainly due to the lack of appropriate tools enabling studies at the single-cell level. We present a study on changes in cellular heterogeneity in the context of pre-malignant progression in response to hypoxic stress.

A physical sciences network characterization of non-tumorigenic and metastatic cells.

To investigate the transition from non-cancerous to metastatic from a physical sciences perspective, the Physical Sciences-Oncology Centers (PS-OC) Network performed molecular and biophysical comparative studies of the non-tumorigenic MCF-10A and metastatic MDA-MB-231 breast epithelial cell lines, commonly used as models of cancer metastasis. Experiments were performed in 20 laboratories from 12 PS-OCs. Each laboratory was supplied with identical aliquots and common reagents and culture protocols.

Method for physiologic phenotype characterization at the single-cell level in non-interacting and interacting cells.

Intercellular heterogeneity is a key factor in a variety of core cellular processes including proliferation, stimulus response, carcinogenesis, and drug resistance. However, cell-to-cell variability studies at the single-cell level have been hampered by the lack of enabling experimental techniques. We present a measurement platform that features the capability to quantify oxygen consumption rates of individual, non-interacting and interacting cells under normoxic and hypoxic conditions.

Isotropic 3D nuclear morphometry of normal, fibrocystic and malignant breast epithelial cells reveals new structural alterations.

Background: Grading schemes for breast cancer diagnosis are predominantly based on pathologists' qualitative assessment of altered nuclear structure from 2D brightfield microscopy images. However, cells are three-dimensional (3D) objects with features that are inherently 3D and thus poorly characterized in 2D. Our goal is to quantitatively characterize nuclear structure in 3D, assess its variation with malignancy, and investigate whether such variation correlates with standard nuclear grading criteria.

A statistical framework for multiparameter analysis at the single-cell level.

Phenotypic characterization of individual cells provides crucial insights into intercellular heterogeneity and enables access to information that is unavailable from ensemble averaged, bulk cell analyses. Single-cell studies have attracted significant interest in recent years and spurred the development of a variety of commercially available and research-grade technologies. To quantify cell-to-cell variability of cell populations, we have developed an experimental platform for real-time measurements of oxygen consumption (OC) kinetics at the single-cell level.

Quantitative single-cell gene expression measurements of multiple genes in response to hypoxia treatment.

Cell-to-cell heterogeneity in gene transcription plays a central role in a variety of vital cell processes. To quantify gene expression heterogeneity patterns among cells and to determine their biological significance, methods to measure gene expression levels at the single-cell level are highly needed. We report an experimental technique based on the DNA-intercalating fluorescent dye SYBR green for quantitative expression level analysis of up to ten selected genes in single mammalian cells.

Quantitative characterization of preneoplastic progression using single-cell computed tomography and three-dimensional karyometry.

The development of morphological biosignatures to precisely characterize preneoplastic progression necessitates high-resolution three-dimensional (3D) cell imagery and robust image processing algorithms. We report on the quantitative characterization of nuclear structure alterations associated with preneoplastic progression in human esophageal epithelial cells using single-cell optical tomography and fully automated 3D karyometry. We stained cultured cells with hematoxylin and generated 3D images of individual cells by mathematically reconstructing 500 projection images acquired using optical absorption tomographic imaging.

Intrinsic promoter nucleosome stability/dynamics variations support a novel targeting mechanism.

Genomic processes like transcription initiation typically involve the alteration of nucleosome structure, to expose DNA elements for regulatory factor binding. Nucleosome altering/modifying complexes have been identified, but precisely how these complexes find their specific targets remains unclear. We have shown that nucleosomes can exhibit significant DNA sequence-dependent stability and dynamics variations and have suggested that these inherent variations could facilitate nucleosome recognition and thus aid in specific targeting.

DNA sequence-dependent variation in nucleosome structure, stability, and dynamics detected by a FRET-based analysis.

Förster resonance energy transfer (FRET) techniques provide powerful and sensitive methods for the study of conformational features in biomolecules. Here, we review FRET-based studies of nucleosomes, focusing particularly on our work comparing the widely used nucleosome standard, 5S rDNA, and 2 promoter-derived regulatory element-containing nucleosomes, mouse mammary tumor virus (MMTV)-B and GAL10. Using several FRET approaches, we detected significant DNA sequence-dependent structure, stability, and dynamics differences among the three.

Nucleosomal stability and dynamics vary significantly when viewed by internal versus terminal labels.

Nucleosomes are a major impediment to regulatory factor activities and therefore to the operation of genomic processes in eukaryotes. One suggested mechanism for overcoming in vivo nucleosomal repression is factor-mediated removal of H2A/H2B from nucleosomes. Using nucleosomes labeled internally with FRET fluorophores, we previously observed significant, DNA sequence-dependent variation in stability and dynamics under conditions (subnanomolar concentrations) reported to produce H2A/H2B release from nucleosomes.

Prolonged irradiation of enhanced cyan fluorescent protein or Cerulean can invalidate Forster resonance energy transfer measurements.

Since its discovery, green fluorescent protein (GFP) and its variants have proven to be a good and convenient fluorescent label for proteins: GFP and other visible fluorescent proteins (VFPs) can be fused selectively to the protein of interest by simple cloning techniques and develop fluorescence without additional cofactors. Among the steadily growing collection of VFPs, several pairs can be chosen that can serve as donor and acceptor fluorophores in Forster resonance energy transfer (FRET) experiments. Among them, the cyan fluorescent proteins (ECFP/Cerulean) and the enhanced yellow fluorescent protein (EYFP) are most commonly used.

Sequence-dependent variations associated with H2A/H2B depletion of nucleosomes.

Mechanisms that can alter nucleosome structure to enhance DNA accessibility are of great interest because of their potential involvement in genomic processes. One such mechanism is H2A/H2B release from nucleosomes; it occurs in vivo and is involved in the in vitro activities of several transcription-associated complexes. Using fluorescence approaches based on Förster resonance energy transfer, we previously detected sequence-dependent structure/stability variations between 5S and two types of promoter nucleosomes (from yeast GAL10 or mouse mammary tumor virus promoters).