High-resolution Snapshot Research Articles

Abstract 7635: Orion™: 14-plex clinical sample imaging of liver cancer modalities using one-step staining and imaging

Abstract Background To better understand the complexities of the tumor microenvironment (TME), high-resolution imaging at a sub-cellular resolution and high-plex is needed. These serve as a basis for biomarker quantification and more downstream, may provide the ability to predict patient outcome. Here, we investigate three whole slide liver sections: fetal liver, hepatocellular carcinoma (HCC), and colorectal cancer (CRC) liver metastasis. Methods 5-micron sections were cut and mounted on Superfrost Plus slides prior to de-paraffinization and antigen retrieval using the BioGenex EZ-Retriever system. Autofluorescence was quenched using UV and white light and blocked with Image-iT™ FX Signal Enhancer. Whole slides were stained with the 14-plex antibody panel, coverslip mounted with ArgoFluor™ Mounting Medium and cured overnight. Whole slides were imaged at 20X using the Orion™ spatial biology platform. Coverslips were removed in aqueous solution prior to H&E staining and scanning. Results Sample specific differences were observed, likely stemming from the different tissue sources. Data revealed a distinction between hepatocytes highlighted in the autofluorescence channel, sinusoids filled with proliferating progenitor cell and macrophages, and epithelial cells lining the border of the fetal liver forming the ductal plate surrounding the portal vein. Whereas in HCC, imaging revealed autofluorescent hepatocytes obliterating the sinusoids with some hepatocytes dividing, an extensive capillary network, and red bloods cells also highlighted in the auto fluorescent channel. T cells and macrophages showed clustering within the capillary networks and as expected, hepatocytes expressed high levels of Albumin. In the CRC liver metastasis sample, hepatocytes were shown to be surrounded by T cells, neoplastic glands (PanCK), and CD163+FOLR2+ macrophages. Furthermore, the majority of the metastatic epithelial cancer cells were observed to be proliferating. Conclusions We were able to observe precise differences in the tissue architecture of liver samples from three divergent sources. Specifically, the ability to identify distinct cell subtypes and their spatial co-localization may provide mechanistic insights into the spatial immune landscape and its role in disease progression and treatment outcome. This exemplifies the need for dissecting the spatial heterogeneity of the TME using a technology that offers a high-resolution subcellular snapshot in time; exhibiting the potential for Orion to bridge the gap from bench-to-bedside. Citation Format: Selena Larkin, Jennifer Currenti, Rhea Pai, Soumi Chatterjee, Archita Mishra, Jacob George, Brady Gardner, Ankur Sharma. Orion™: 14-plex clinical sample imaging of liver cancer modalities using one-step staining and imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7635.

Comprehensive Gene Expression Profiling Analysis of Adipose Tissue in Male Individuals from Fat- and Thin-Tailed Sheep Breeds.

It has been shown that tail fat content varies significantly among sheep breeds and plays a significant role in meat quality. Recently, significant efforts have been made to understand the physiological, biochemical, and genomic regulation of fat deposition in sheep tails in order to unravel the mechanisms underlying energy storage and adipose tissue lipid metabolism. RNA-seq has enabled us to provide a high-resolution snapshot of differential gene expression between fat- and thin-tailed sheep breeds. Therefore, three RNA-seq datasets were meta-analyzed for the current work to elucidate the transcriptome profile differences between them. Specifically, we identified hub genes, performed gene ontology (GO) analysis, carried out enrichment analyses of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and validated hub genes using machine learning algorithms. This approach revealed a total of 136 meta-genes, 39 of which were not significant in any of the individual studies, indicating the higher statistical power of the meta-analysis. Furthermore, the results derived from the use of machine learning revealed POSTN, K35, SETD4, USP29, ANKRD37, RTN2, PRG4, and LRRC4C as substantial genes that were assigned a higher weight (0.7) than other meta-genes. Among the decision tree models, the Random Forest ones surpassed the others in adipose tissue predictive power fat deposition in fat- and thin-tailed breeds (accuracy > 0.85%). In this regard, combining meta-analyses and machine learning approaches allowed for the identification of three important genes (POSTN, K35, SETD4) related to lipid metabolism, and our findings could help animal breeding strategies optimize fat-tailed breeds' tail sizes.

New tools can propel research in lysosomal storage diseases

Historically, the clinical manifestations of lysosomal storage diseases offered an early glimpse into the essential digestive functions of the lysosome. However, it was only recently that the more subtle role of this organelle in the dynamic regulation of multiple cellular processes was appreciated. With the need for precise interrogation of lysosomal interplay in health and disease comes the demand for more sophisticated functional tools. This demand has recently been met with 1) induced pluripotent stem cell-derived models that recapitulate the disease phenotype in vitro, 2) methods for lysosome affinity purification coupled with downstream omics analysis that provide a high-resolution snapshot of lysosomal alterations, and 3) gene editing and CRISPR/Cas9-based functional genomic strategies that enable screening for genetic modifiers of the disease phenotype. These emerging methods have garnered much interest in the field of neurodegeneration, and their use in the field of metabolic disorders is now also steadily gaining momentum. Looking forward, these robust tools should accelerate basic science efforts to understand lysosomal dysfunction distal to substrate accumulation and provide translational opportunities to identify disease-modifying therapies.

Were climatic forcings the main driver for mid-holocene changes in settlement dynamics on the Varamin Plain (Central Iranian Plateau)?

Settlement crises in ancient cultures of Western Asia are commonly thought to be caused by climatic events such as severe droughts. However, the insufficient climate proxy situation in this region challenges the inference of clear relationships between climate and settlement dynamics. We investigate the Holocene climatic changes on the Varamin Plain in the context of the climatic history of Western Central Asia by using a transient comprehensive Earth System Model simulation (8 ka BP to pre-industrial), a high-resolution regional snapshot simulation and a synthesis of pollen-based climate reconstructions. In line with the reconstructions, the models reveal only slightly varying mean climatic conditions on the Varamin Plain but indicate substantial changes in seasonality during the Holocene. Increased precipitation during spring, combined with lower temperature and potentially stronger snow accumulation on the upstream Alborz mountains may have led to an increased water supply on the alluvial fan during the vegetation period and thus to more favourable conditions for agricultural production during the Mid-Holocene compared to modern times. According to the model, dry periods on the Central Iranian Plateau are related to particularly weak Westerly winds, fostering the subsidence in the mid-troposphere and hampering precipitation over the region. The model reveals that dry periods have spatially heterogenous manifestations, thus explaining why they do not appear in all proxy records in the wider study region. In fact, the climatic signal may depend on local environmental conditions. The interaction of the topography with the atmospheric circulation leads to additional spatial heterogeneity. Although our results provide several indications for a connection between climate and settlement dynamics, the small overall changes in moisture call into question whether climate is the main driver for settlement discontinuities on the Central Iranian Plateau. To shed further light on this issue, more high-resolution long-term proxy records are needed.

Crisis in motion: the final days of Iron Age I Megiddo

The destruction of Iron I Megiddo in the early 10th century BCE was a momentous event in the history of the southern Levant. It marked an abrupt break in the long cultural development of the Middle and Late Bronze Ages. Despite extensive field research, essential questions related to this event remain unanswered, especially regarding the processes that took place in the city immediately before its destruction. In this article, findings from recent excavations in the south-eastern sector of the mound, where a detailed Iron I stratigraphic sequence was explored, are reported. In addition, finds from two nearby areas previously excavated were re-evaluated, focusing mostly on contextual aspects of the osteological data. This study sheds light on the deterioration of the city in the decades preceding its final demise, and suggests that the event was caused by human agents rather than a natural disaster. It also hints that in its last days, Megiddo may have been besieged, which explains the peculiar re-appearance of intra-mural burials at the site. The case of Iron I Megiddo provides a high-resolution snapshot of actions taken by the inhabitants of a Near Eastern city on the eve of a major crisis.

Single-cell transcriptomics identify TNFRSF1B as a novel T-cell exhaustion marker for ovarian cancer.

Ovarian cancer (OC) patients routinely show poor immunotherapeutic response due to the complex tumour microenvironment (TME). It is urgent to explore new immunotherapeutic markers. Through the single-cell RNA sequencing (scRNA-seq) analyses on high-grade serous OC (HGSOC), moderate severity borderline tumour and matched normal ovary, we identified a novel exhausted T cells subpopulation that related to poor prognosis in OC. Histological staining, multiple immunofluorescences, and flow cytometry were applied to validate some results from scRNA-seq. Furthermore, a tumour-bearing mice model was constructed to investigate the effects of TNFRSF1B treatment on tumour growth in vivo. Highly immunosuppressive TME in HGSOC is displayed compared to moderate severity borderline tumour and matched normal ovary. Subsequently, a novel exhausted subpopulation of CD8+ TNFRSF1B+ T cells is identified, which is associated with poor survival. In vitro experiments demonstrate that TNFRSF1B is specifically upregulated on activated CD8+ T cells and suppressed interferon-γ secretion. The expression of TNFRSF1B on CD8+ T cells is closely related to OC clinical malignancy and is a marker of poor prognosis through 140 OC patients' verification. In addition, the blockade of TNFRSF1B inhibits tumour growth via profoundly remodeling the immune microenvironment in the OC mouse model. Our transcriptomic results analyzed by scRNA-seq delineate a high-resolution snapshot of the entire tumour ecosystem of OC TME. The major applications of our findings were an exhausted subpopulation of CD8+ TNFRSF1B+ T cells for predicting OC patient prognosis and the potential therapeutic value of TNFRSF1B. These findings demonstrated the clinical value of TNFRSF1B as a potential immunotherapy target and extended our understanding of factors contributing to immunotherapy failure in OC.

High-resolution multi-spectral snapshot 3D imaging with a SPAD array camera.

Currently, mainstream light detection and ranging (LiDAR) systems usually involve a mechanical scanner component, which enables large-scale, high-resolution and multi-spectral imaging, but is difficult to assemble and has a larger system size. Furthermore, the mechanical wear on the moving parts of the scanner reduces its usage lifetime. Here, we propose a high-resolution scan-less multi-spectral three-dimensional (3D) imaging system, which improves the resolution with a four-times increase in the pixel number and can achieve multi-spectral imaging in a single snapshot. This system utilizes a specially designed multiple field-of-view (multi-FOV) system to separate four-wavelength echoes carrying depth and spectral reflectance information with predetermined temporal intervals, such that one single pixel of the SPAD array can sample four adjacent positions through the four channels' FOVs with subpixel offset. The positions and reflectivity are thus mapped to wavelengths in different time-bins. Our results show that the system can achieve high-resolution multi-spectral 3D imaging in a single exposure without scanning component. This scheme is the first to realize scan-less single-exposure high-resolution and multi-spectral imaging with a SPAD array sensor.

Genomic analysis of 1710 surveillance-based Neisseria gonorrhoeae isolates from the USA in 2019 identifies predominant strain types and chromosomal antimicrobial-resistance determinants.

This study characterized high-quality whole-genome sequences of a sentinel, surveillance-based collection of 1710 Neisseria gonorrhoeae (GC) isolates from 2019 collected in the USA as part of the Gonococcal Isolate Surveillance Project (GISP). It aims to provide a detailed report of strain diversity, phylogenetic relationships and resistance determinant profiles associated with reduced susceptibilities to antibiotics of concern. The 1710 isolates represented 164 multilocus sequence types and 21 predominant phylogenetic clades. Common genomic determinants defined most strains' phenotypic, reduced susceptibility to current and historic antibiotics (e.g. bla TEM plasmid for penicillin, tetM plasmid for tetracycline, gyrA for ciprofloxacin, 23S rRNA and/or mosaic mtr operon for azithromycin, and mosaic penA for cefixime and ceftriaxone). The most predominant phylogenetic clade accounted for 21 % of the isolates, included a majority of the isolates with low-level elevated MICs to azithromycin (2.0 µg ml-1), carried a mosaic mtr operon and variants in PorB, and showed expansion with respect to data previously reported from 2018. The second largest clade predominantly carried the GyrA S91F variant, was largely ciprofloxacin resistant (MIC ≥1.0 µg ml-1), and showed significant expansion with respect to 2018. Overall, a low proportion of isolates had medium- to high-level elevated MIC to azithromycin ((≥4.0 µg ml-1), based on C2611T or A2059G 23S rRNA variants). One isolate carried the penA 60.001 allele resulting in elevated MICs to cefixime and ceftriaxone of 1.0 µg ml-1. This high-resolution snapshot of genetic profiles of 1710 GC sequences, through a comparison with 2018 data (1479 GC sequences) within the sentinel system, highlights change in proportions and expansion of select GC strains and the associated genetic mechanisms of resistance. The knowledge gained through molecular surveillance may support rapid identification of outbreaks of concern. Continued monitoring may inform public health responses to limit the development and spread of antibiotic-resistant gonorrhoea.

Bio-inspired Compact, High-resolution Snapshot Hyperspectral Imaging System with 3D Printed Glass Lightguide Array.

To address the major challenges to obtain high spatial resolution in snapshot hyperspectral imaging, 3D printed glass lightguide array has been developed to sample the intermediate image in high spatial resolution and redistribute the pixels in the output end to achieve high spectral resolution. Curved 3D printed lightguide array can significantly simplify the snapshot hyperspectral imaging system, achieve better imaging performance, and reduce the system complexity and cost. We have developed two-photon polymerization process to print glass lightguide array, and demonstrated the system performance with biological samples. This new snapshot technology will catalyze new hyperspectral imaging system development and open doors for new applications from UV to IR.

Structural basis for H2A–H2B recognitions by human Spt16

The human facilitates chromatin transcription (FACT) complex, consisting of Spt16 and SSRP1, is a versatile histone chaperone that can engage free H2A–H2B dimer and H3–H4 tetramer (or dimer), and partially unraveled nucleosome. The C-terminal domain of human Spt16 (hSpt16-CTD) is the decisive element for engaging H2A–H2B dimer and partially unraveled nucleosome. The molecular basis of the H2A–H2B dimer recognitions by hSpt16-CTD is not fully comprehended. Here, we present a high-resolution snapshot of the recognitions of the H2A–H2B dimer by hSpt16-CTD via an acidic intrinsically disordered (AID) segment, and reveal some distinct structural features of hSpt16-CTD as compared to the budding yeast Spt16-CTD.

Towards the outwelling hypothesis in a Patagonian estuary: First support from lipid markers and bacterial communities

Biogeochemical markers in combination with bacterial community composition were studied at two contrasting stations at the Río Negro (RN) estuary to assess the outwelling hypothesis in the Argentinian Patagonia. Inorganic nutrients and dissolved organic matter were exported clearly during the last hours of the ebb at the station Wetland. Moreover, a considerable outwelling of polyunsaturated fatty acids (PUFA), particulates and microalgae was inferred by this combined approach. The exported 22:6(n-3) and 20:5(n-3) contributed very likely to sustain higher trophic levels in the coasts of the Southwest Atlantic. The stable isotopes did not evidence clearly the outwelling; nevertheless, the combination of δ13C with fatty acid bacterial markers indicated organic matter degradation in the sediments. The dominance of Desulfobacterales and Desulfuromonadales suggested sulphate reduction in the sediments, a key mechanism for nutrient outwelling in salt marshes. Marivivens and other Rhodobacterales (Alphaproteobacteria) in the suspended particulate matter were clear indicators of the nutrient outwelling. The colonization of particles according to the island biogeography theory was a good hypothesis to explain the lower bacterial biodiversity at the wetland. The copiotrophic conditions of the RN estuary and particularly at the wetland were deduced also by the dynamic of some Actinobacteria, Bacteroidia and Gammaproteobacteria. This high-resolution snapshot combining isotopic, lipid and bacterial markers offers key pioneer insights into biogeochemical and ecological processes of the RN estuary.

Structural model of microtubule dynamics inhibition by kinesin-4 from the crystal structure of KLP-12 -tubulin complex.

Kinesin superfamily proteins are microtubule-based molecular motors driven by the energy of ATP hydrolysis. Among them, the kinesin-4 family is a unique motor that inhibits microtubule dynamics. Although mutations of kinesin-4 cause several diseases, its molecular mechanism is unclear because of the difficulty of visualizing the high-resolution structure of kinesin-4 working at the microtubule plus-end. Here, we report that KLP-12, a C. elegans kinesin-4 ortholog of KIF21A and KIF21B, is essential for proper length control of C. elegans axons, and its motor domain represses microtubule polymerization in vitro. The crystal structure of the KLP-12 motor domain complexed with tubulin, which represents the high-resolution structural snapshot of the inhibition state of microtubule-end dynamics, revealed the bending effect of KLP-12 for tubulin. Comparison with the KIF5B-tubulin and KIF2C-tubulin complexes, which represent the elongation and shrinking forms of microtubule ends, respectively, showed the curvature of tubulin introduced by KLP-12 is in between them. Taken together, KLP-12 controls the proper length of axons by modulating the curvature of the microtubule ends to inhibit the microtubule dynamics.

Multimodality in liquid biopsy: does a combination uncover insights undetectable in individual blood analytes?

Abstract The heterogeneity of each individual oncologic disease can be mirrored by molecular analysis of a simple blood draw in real time. Liquid biopsy testing has been shown useable for cancer detection, proof of minimal residual disease, therapy decision making and monitoring. However, an individual blood analyte does not present a comprehensive picture of the disease. It was recently shown that multi-modal/multi-parametric/multi-analyte liquid biopsy testing has the advantage of generating a high-resolution snapshot of the disease complexity. The different blood analytes such as circulating tumor cells, circulating immune cells, tumor-educated platelets, extracellular vesicles, cell-free DNA, cell-free RNA and circulating proteins complement each other and have additive value for clinical cancer management. We, here, like to review the studies leading to these promising conclusions and like to, at the end, mention that many challenges lie ahead before the translation into the clinic can be accomplished, including issues concerning clinical utility, method standardization, cost reimbursement and data management.

Substantial Sub-Surface Chlorophyll Patch Sustained by Vertical Nutrient Fluxes in Fram Strait Observed With an Autonomous Underwater Vehicle

We present results from a coordinated frontal survey in Fram Strait in summer 2016 using an autonomous underwater vehicle (AUV) combined with shipboard and zodiac-based hydrographic measurements. Based on satellite information, we identified a front between warm Atlantic Water and cold Polar Water. The AUV, equipped with oceanographic and biogeochemical sensors, profiled the upper 50 m along a 10 km-long cross-front oriented transect resulting in a high-resolution snapshot of the upper ocean. The transect was dominated by a 6 km-wide, 10 m-thick subsurface patch of high chlorophyll, located near the euphotic depth within a band of cold water. Nitrate was depleted in the surface, but abundant below the pycnocline. Potential vorticity and Richardson number estimates indicate conditions favorable for vertical mixing, which indicates that the high chlorophyll patch may have been sustained by upward nitrate fluxes. Our observations underline the complex hydrographic and biogeochemical structure in a region featuring fronts and meanders, and further underline the patchy and small-scale nature of subsurface phytoplankton blooms potentially fueled by submesoscale dynamics, which are easily missed by traditional surveys and satellite missions.

High-resolution snapshot of ketamine-receptor handshake may point to better antidepressants

High-resolution snapshot of ketamine-receptor handshake may point to better antidepressants

Genetic insights into the paternal admixture history of Chinese Mongolians via high-resolution customized Y-SNP SNaPshot panels

The Mongolian people, one of the Mongolic-speaking populations, are native to the Mongolian Plateau in North China and southern Siberia. Many ancient DNA studies recently reported extensive population transformations during the Paleolithic to historic periods in this region, while little is known about the paternal genetic legacy of modern geographically different Mongolians. Here, we genotyped 215 Y-chromosomal single nucleotide polymorphisms (Y-SNPs) and 37 Y-chromosomal short tandem repeats (Y-STRs) among 679 Mongolian individuals from Hohhot, Hulunbuir, and Ordos in North China using the AGCU Y37 kit and our developed eight Y-SNP SNaPshot panels (including two panels first reported herein). The C-M130 Y-SNP SNaPshot panel defines 28 subhaplogroups, and the N/O/Q complementary Y-SNP SNaPshot panel defines 30 subhaplogroups of N1b-F2930, N1a1a1a1a3-B197, Q-M242, and O2a2b1a1a1a4a-CTS4658, which improved the resolution our developed Y-SNP SNaPshot panel set and could be applied for dissecting the finer-scale paternal lineages of Mongolic speakers. We found a strong association between Mongolian-prevailing haplogroups and some observed microvariants among the newly generated Y-STR haplotype data, suggesting the possibility of haplogroup prediction based on the distribution of Y-STR haplotypes. We identified three main ancestral sources of the observed Mongolian-dominant haplogroups, including the local lineage of C2*-M217 and incoming lineages from other regions of southern East Asia (O2*-M122, O1b*-P31, and N1*-CTS3750) and western Eurasia (R1*-M173). We also observed DE-M145, D1*-M174, C1*-F3393, G*-M201, I-M170, J*-M304, L-M20, O1a*-M119, and Q*-M242 at relatively low frequencies (< 5.00%), suggesting a complex admixture history between Mongolians and other incoming Eurasians from surrounding regions. Genetic clustering analyses indicated that the studied Mongolians showed close genetic affinities with other Altaic-speaking populations and Sinitic-speaking Hui people. The Y-SNP haplotype/haplogroup-based genetic legacy not only revealed that the stratification among geographically/linguistically/ethnically different Chinese populations was highly consistent with the geographical division and language classification, but also demonstrated that patrilineal genetic materials could provide fine-scale genetic structures among geographically different Mongolian people, suggesting that our developed high-resolution Y-SNP SNaPshot panels have the potential for forensic pedigree searches and biogeographical ancestry inference.

Characterizing the Changes in Material Use due to Vehicle Electrification.

Modern automobiles are composed of more than 2000 different compounds comprising 76 different elements. Identifying supply risks across this palette of materials is important to ensure a smooth transition to more sustainable transportation technologies. This paper provides insight into how electrification is changing vehicle composition and how that change drives supply risk vulnerability by providing the first comprehensive, high-resolution (elemental and compound level) snapshot of material use in both conventional and hybrid electric vehicles (HEVs) using a consistent methodology. To make these contributions, we analyze part-level data of material use for seven current year models, ranging from internal combustion engine vehicles (ICEV) to plug-in hybrid vehicles (PHEVs). With this data set, we apply a novel machine learning algorithm to estimate missing or unreported composition data. We propose and apply a metric of vulnerability, referred to as exposure, which captures economic importance and susceptibility to price changes. We find that exposure increases from $874 per vehicle for ICEV passenger vehicles to $2344 per vehicle for SUV PHEVs. The shift to a PHEV fleet would double automaker exposure adding approximately $1 billion per year of supply risk to a hypothetical fleet of a million vehicles. The increase in exposure is largely not only due to the increased use of battery elements like cobalt, graphite, and nickel but also some more commonly used materials, most notably copper.

Abstract 2116: Promoter enrichment of subnucleosomes using breast cancer cell-free DNA

Abstract Breast cancer is the leading cancer diagnosis and the second major cause of cancer-related deaths in women worldwide. The use of non-invasive cell-free DNA (cfDNA) to detect and assess cancer dynamics has recently gained traction with a focus on targeted mutation panels. However, mutation panels have to contend with non-tumor origin of mutations like clonal hematopoiesis and the emergence of novel mutations in the tumor. A potential alternative is to harness the epigenomic, or non-genetic, features of cfDNA. In a healthy individual, blood cell turnover is the major contributor to the cfDNA pool. In cancer, tumor DNA adds to the cfDNA pool, which represents the end product of endogenous nucleases acting on accessible genomic DNA. Since genomic DNA is protected by histone proteins, cfDNA maps the chromatin structure of individual cells that turn over. Subnucleosomes (partially formed structures of nucleosomes that are the building blocks of chromatin) are by-products of transcription and thus reflect small protections enriched in active genes. The enrichment of subnucleosomes at promoters correlate with gene expression profiles of cfDNA tissues-of-origin and based on this knowledge, we hypothesized that we could reconstruct the transcriptional profiles of cancer cells by selectively targeting promoter regions. We first extracted cfDNA from 9 healthy female donors and 8 patient donors with various stages of breast cancer (Stage I, n=1; Stage III, n=1; Stage IV, n=4). We used a single-stranded DNA library protocol that robustly captures highly nicked cfDNA, enabling the genome-wide mapping of both nucleosomes and subnucleosomes. We performed targeted capture of promoter-proximal, nucleosome-protected sequences in cfDNA using non-overlapping tiling probes from transcription start sites to 300 bp downstream. After whole-genome sequencing of pooled libraries, we show that 76.4% (n=13/17) of samples exhibited &amp;gt;10X enrichment compared to unenriched samples, and 64.7% (n=11/17) of samples exhibited &amp;gt;100X enrichment. The enriched samples allowed us to profile &amp;gt;10,000 genes, whereas unenriched samples enabled profiling between 100-10,000 genes at an average sequencing depth of 40 million reads. Overall, we obtain higher depth per sequencing read after enrichment resulting in a high-resolution snapshot of promoter-nucleosome dynamics derived by probing only ~0.18% of the genome. We believe that this significant advancement will allow us not only to identify presence of cancer but also detect distinct tumor states. Citation Format: Alexis Zukowski, Amy Han, Peter Kabos, Srinivas Ramachandran. Promoter enrichment of subnucleosomes using breast cancer cell-free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2116.

A single snapshot multiplex immunoassay platform utilizing dense test lines based on engineered beads

Co-circulation of coronavirus disease 2019 (COVID-19) and dengue fever has been reported. Accurate and timely multiplex diagnosis is required to prevent future pandemics. Here, we developed an innovative microfluidic chip that enables a snapshot multiplex immunoassay for timely on-site response and offers unprecedented multiplexing capability with an operating procedure similar to that of lateral flow assays. An open microchannel assembly of individually engineered microbeads was developed to construct nine high-density test lines, which can be imaged in a 1 mm2 field-of-view. Thus, simultaneous detection of multiple antibodies would be achievable in a single high-resolution snapshot. Next, we developed a novel pixel intensity-based imaging process to distinguish effective and non-specific fluorescence signals, thereby improving the reliability of this fluorescence-based immunoassay. Finally, the chip specifically identified and classified random combinations of arbovirus (Zika, dengue, and chikungunya viruses) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies within 30 min. Therefore, we believe that this snapshot multiplex immunoassay chip is a powerful diagnostic tool to control current and future pandemics.

High-Speed Time-Resolved Tomographic Particle Shadow Velocimetry Using Smartphones

The video-capabilities of smartphones are rapidly improving both in pixel resolution and frame-rates. Herein we use four smartphones in the “slow-mo” option to perform time-resolved Tomographic Particle Shadow Velocimetry of a vortex ring, using 960 fps. We use background LED-illuminated diffusers, facing each camera, for shadow particle imaging. We discuss in-depth the challenges present in synchronizing the high-speed video capture on the smartphones and steps to overcome these challenges. The resulting 3-D velocity field is compared to an instantaneous, concurrent, high-resolution snapshot with four 4k-video cameras using dual-color to encode two time-steps on a single frame. This proof-of-concept demonstration, supports realistic low-cost alternatives to conventional 3-D experimental systems.