Individual Cells Research Articles

Integrated Single Camera µPTV And Florescence Imaging For Cell Tracking And Flow Investigation In Centrifugal Microfluidic Devices

A micro-hydrocyclone is investigated as a high throughput particle/cell sorting microfluidic device. A complex flow structure has been reported by limited numerical works that increases the importance of undertaking a comprehensive experimental investigation. In addition to the flow, utilizing damageable bio cells in a micro-hydrocyclone, requires a deep understanding of the interaction between the cells entering the device, the flow structure and its instabilities forming in different operational phases. In such cases the implementation of multiple measurement techniques can be challenging due to the small scale of devices, here < 5 mm diameter. Therefore, for this complex flow an experimental approach is introduced to capture the flow structure and the motion of cells. In this method, tracer particles and fluorescent stained cells are captured simultaneous by a single camera in the same flow field. A custom image processing scheme is used for partitioning cells and particles from the raw data. To determine velocity vectors, µPTV is employed on the segmented data sets to study the effect of flow on trajectory and velocity of each individual cell in the system. By a combined investigation in a vast range of Reynolds number (50<Re<800) the dynamics of cells are investigated in response to different flow structures and flow regimes.

Isolated cassava cells: Comparison of structure and physicochemical properties with starch and whole flour

Individual cells are the smallest units of the plant tissue structure, and their structure and physicochemical properties are essential for whole food processing. In this study, cassava cells were isolated using acid-alkali, hydrothermal, and pectinase methods, and the differences in microstructure and physicochemical properties among the cells, starch, and whole flour were investigated. Cassava cells isolated using pectinase showed intact individual cells with a higher isolation rate and less damage to the cell wall structure and intracellular composition. The presence of cell walls in intact individual cells inhibited the swelling and leaching of starch, resulting in a lower peak viscosity and higher gelatinization temperature than those of starch. The intact cell structure and non-starch composition enhanced the shear resistance of the gels in the sample. Heat treatment led to the gelatinization of intracellular starch and increased the permeability of the cell wall, destroying the physical barrier function of the cell wall; however, the compact cell matrix and non-starch components can inhibit starch hydrolysis. This study suggests that cells isolated using pectinase can be used to investigate the effect of cell walls on the functional properties of intracellular starch in cassava. The isolated cells provide new insights into the cassava industry.

Frequency of ABO and RhD Blood Groups in Patients with Celiac Disease

Celiac disease (CD) is a popular autoimmune disorder induced by gluten-containing foods and other environmental, and genetic factors. Every individual's red blood cell expresses over two million ABO blood-type antigens. Furthermore, the stomach and small intestine are major expression sites for the ABH antigens. This study was intended to assess the distribution of ABO and Rh blood types in Libyan patients with celiac disease as well as compare between CD and healthy control groups regarding complete blood count parameters. Samples from 250 CD patients and 45 healthy control groups represented by 80 males (27.1%) and 215 females (72.9%) were enrolled in the study. The samples of CD were collected from patients who attended the gastroenterology outpatient clinic at Tripoli University Hospital (TUH) for follow-up. Two hundred and fifty blood samples were serologically screened for ABO, and Rh antigens using a tube agglutination test. Another group of healthy subjects (n = 45) and CD patients (n = 45) were analyzed for CBC test. The findings showed that the most observed ABO blood group among celiac patients was blood group O 129 (51.6%), followed by blood group A 80 (32%), and Rh-positive 222 (88.8%). Furthermore, the heritable proportion was 33%, with 21% classified as first-degree and 12% as second-degree hereditary. Additionally, the result of the independent Sample (T) test to compare RBCs, HGB, HCT, and NUT levels in blood between CD patients and healthy control showed that there were significant differences in the RBCs, HGB, and HCT counts with p-value = 0.034, <0.001, and <0.001 respectively. In contrast, Mann-Whitney U test results revealed significant differences in the PLT, MCV, MCH, and MCHC counts with p-value = <0.001, and for LYM with p-value = 0.003. Future studies on these simple inflammatory markers can guide us in predicting the diagnosis and prognosis.

Diversity of visual inputs to Kenyon cells of the Drosophila mushroom body

The arthropod mushroom body is well-studied as an expansion layer representing olfactory stimuli and linking them to contingent events. However, 8% of mushroom body Kenyon cells in Drosophila melanogaster receive predominantly visual input, and their function remains unclear. Here, we identify inputs to visual Kenyon cells using the FlyWire adult whole-brain connectome. Input repertoires are similar across hemispheres and connectomes with certain inputs highly overrepresented. Many visual neurons presynaptic to Kenyon cells have large receptive fields, while interneuron inputs receive spatially restricted signals that may be tuned to specific visual features. Individual visual Kenyon cells randomly sample sparse inputs from combinations of visual channels, including multiple optic lobe neuropils. These connectivity patterns suggest that visual coding in the mushroom body, like olfactory coding, is sparse, distributed, and combinatorial. However, the specific input repertoire to the smaller population of visual Kenyon cells suggests a constrained encoding of visual stimuli.

Scanpro is a tool for robust proportion analysis of single-cell resolution data

In higher organisms, individual cells respond to signals and perturbations by epigenetic regulation and transcriptional adaptation. However, in addition to shifting the expression level of individual genes, the adaptive response of cells can also lead to shifts in the proportions of different cell types. Recent methods such as scRNA-seq allow for the interrogation of expression on the single-cell level, and can quantify individual cell type clusters within complex tissue samples. In order to identify clusters showing differential composition between different biological conditions, differential proportion analysis has recently been introduced. However, bioinformatics tools for robust proportion analysis of both replicated and unreplicated single-cell datasets are critically missing. In this manuscript, we present Scanpro, a modular tool for proportion analysis, seamlessly integrating into widely accepted frameworks in the Python environment. Scanpro is fast, accurate, supports datasets without replicates, and is intended to be used by bioinformatics experts and beginners alike.

A point cloud segmentation framework for image-based spatial transcriptomics

Recent progress in image-based spatial RNA profiling enables to spatially resolve tens to hundreds of distinct RNA species with high spatial resolution. It presents new avenues for comprehending tissue organization. In this context, the ability to assign detected RNA transcripts to individual cells is crucial for downstream analyses, such as in-situ cell type calling. Yet, accurate cell segmentation can be challenging in tissue data, in particular in the absence of a high-quality membrane marker. To address this issue, we introduce ComSeg, a segmentation algorithm that operates directly on single RNA positions and that does not come with implicit or explicit priors on cell shape. ComSeg is applicable in complex tissues with arbitrary cell shapes. Through comprehensive evaluations on simulated and experimental datasets, we show that ComSeg outperforms existing state-of-the-art methods for in-situ single-cell RNA profiling and in-situ cell type calling. ComSeg is available as a documented and open source pip package at https://github.com/fish-quant/ComSeg.

Space, the final frontier: The spatial component of phytoplankton-bacterial interactions.

Microscale interactions between marine phytoplankton and bacteria shape the microenvironment of individual cells, impacting their physiology and ultimately influencing global-scale biogeochemical processes like carbon and nutrient cycling. In dilute environments such as the ocean water column, metabolic exchange between microorganisms likely requires close proximity between partners. However, the biological strategies to achieve this physical proximity remain an understudied aspect of phytoplankton-bacterial associations. Understanding the mechanisms by which these microorganisms establish and sustain spatial relationships and the extent to which spatial proximity is necessary for interactions to occur, is critical to learning how spatial associations influence the ecology of phytoplankton and bacterial communities. Here, we provide an overview of current knowledge on the role of space in shaping interactions among ocean microorganisms, encompassing behavioural and metabolic evidence. We propose that characterising phytoplankton-bacterial interactions from a spatial perspective can contribute to a mechanistic understanding of the establishment and maintenance of these associations and, consequently, an enhanced ability to predict the impact of microscale processes on ecosystem-wide phenomena.

CrossMP: Enabling Cross-Modality Translation between Single-Cell RNA-Seq and Single-Cell ATAC-Seq through Web-Based Portal.

In recent years, there has been a growing interest in profiling multiomic modalities within individual cells simultaneously. One such example is integrating combined single-cell RNA sequencing (scRNA-seq) data and single-cell transposase-accessible chromatin sequencing (scATAC-seq) data. Integrated analysis of diverse modalities has helped researchers make more accurate predictions and gain a more comprehensive understanding than with single-modality analysis. However, generating such multimodal data is technically challenging and expensive, leading to limited availability of single-cell co-assay data. Here, we propose a model for cross-modal prediction between the transcriptome and chromatin profiles in single cells. Our model is based on a deep neural network architecture that learns the latent representations from the source modality and then predicts the target modality. It demonstrates reliable performance in accurately translating between these modalities across multiple paired human scATAC-seq and scRNA-seq datasets. Additionally, we developed CrossMP, a web-based portal allowing researchers to upload their single-cell modality data through an interactive web interface and predict the other type of modality data, using high-performance computing resources plugged at the backend.

ScMultiome analysis identifies a single caudal hindbrain compartment in the developing zebrafish nervous system

BackgroundA key step in nervous system development involves the coordinated control of neural progenitor specification and positioning. A long-standing model for the vertebrate CNS postulates that transient anatomical compartments – known as neuromeres – function to position neural progenitors along the embryonic anteroposterior neuraxis. Such neuromeres are apparent in the embryonic hindbrain – that contains six rhombomeres with morphologically apparent boundaries – but other neuromeres lack clear morphological boundaries and have instead been defined by different criteria, such as differences in gene expression patterns and the outcomes of transplantation experiments. Accordingly, the caudal hindbrain (CHB) posterior to rhombomere (r) 6 has been variably proposed to contain from two to five ‘pseudo-rhombomeres’, but the lack of comprehensive molecular data has precluded a detailed definition of such structures.MethodsWe used single-cell Multiome analysis, which allows simultaneous characterization of gene expression and chromatin state of individual cell nuclei, to identify and characterize CHB progenitors in the developing zebrafish CNS.ResultsWe identified CHB progenitors as a transcriptionally distinct population, that also possesses a unique profile of accessible transcription factor binding motifs, relative to both r6 and the spinal cord. This CHB population can be subdivided along its dorsoventral axis based on molecular characteristics, but we do not find any molecular evidence that it contains multiple pseudo-rhombomeres. We further observe that the CHB is closely related to r6 at the earliest embryonic stages, but becomes more divergent over time, and that it is defined by a unique gene regulatory network.ConclusionsWe conclude that the early CHB represents a single neuromere compartment that cannot be molecularly subdivided into pseudo-rhombomeres and that it may share an embryonic origin with r6.

Addressing variability in cell electrorotation through holographic imaging and correction factors

This study addresses the variations observed in electrorotation measurements due to cell positioning and movement. Electrorotation provides a non-disruptive method for inferring the electrical properties of individual cells. However, its widespread adoption is hindered by significant variation in the observed speed. By mitigating the impact of positional dependencies and other influencing factors, our methodology opens avenues for broader applications of electrorotation in single-cell analysis without the need for complex setups to trap and retain the cell in place. Our novel approach combines multi-plane imaging with mathematical treatment of rotation data. This method uses a conventional quadrupole chip and lens-free imaging to track cell movement, resulting in a simpler design and set-up. Through numerical simulations incorporating cell coordinates, chip design, and experimental parameters, we calculate the variation in torque for each position. These values serve as the basis for the correction factors. Validation experiments with T-lymphocytes and fibroblasts show that the correction factors reduce electrorotation speed variation due to cell movement, with an average reduction to 21% and 18%, respectively. These corrections also revealed previously concealed changes in cell properties, in response to external stimuli, thereby enhancing the reliability of measurements and enabling broader applications in single-cell analysis.

Toxin Dynamics among Populations of the Bioluminescent HAB Species Pyrodinium bahamense from the Indian River Lagoon, FL.

Dinoflagellate species that form some of the most frequent toxic blooms are also bioluminescent, yet the two traits are rarely linked when studying bloom development and persistence. P. bahamense is a toxic, bioluminescent dinoflagellate that previously bloomed in Florida with no known record of saxitoxin (STX) production. Over the past 20 years, STX was identified in P. bahamense populations. The goal of this study was to examine toxin dynamics and associated molecular mechanisms in spatially and temporally distinct P. bahamense populations from the Indian River Lagoon, FL. SxtA4 is a key gene required for toxin biosynthesis. SxtA4 genotype analysis was performed on individual cells from multiple sites. Cell abundance, toxin quota cell-1, and sxtA4 and RubisCo (rbcL) transcript abundance were also measured. There was a significant negative correlation between cell abundance and toxin quota cell-1. While the sxtA4+ genotype was dominant at all sites, its frequency varied, but it occurred at 90-100% in many samples. The underlying mechanism for toxin decrease with increased cell abundance remains unknown. However, a strong, statistically significant negative correlation was found between stxA4 transcripts and the sxtA4/rbcL ratio, suggesting cells make fewer sxtA4 transcripts as a bloom progresses. However, the influence of sxtA4- cells must also be considered. Future plans include bioluminescence measurements, normalized to a per cell basis, at sites when toxicity is measured along with concomitant quantification of sxtA4 gene and transcript copy numbers as a means to elucidate whether changes in bloom toxicity are driven more at the genetic (emergence of sxtA4- cells) or transcriptional (repression of sxtA4 in sxtA4+ cells) level. Based on the results of this study, a model is proposed that links the combined traits of toxicity and bioluminescence in P. bahamense bloom development.

Shaping Drosophila eggs: unveiling the roles of Arpc1 and cpb in morphogenesis.

The Drosophila egg chamber (EC) starts as a spherical tissue at the beginning. With maturation, the outer follicle cells of EC collectively migrate in a direction perpendicular to the anterior-posterior axis, to shape EC from spherical to ellipsoidal. Filamentous actin (F-actin) plays a significant role in shaping individual migratory cells to the overall EC shape, like in every cell migration. The primary focus of this article is to unveil the function of different Actin Binding Proteins (ABPs) in regulating mature Drosophila egg shape. We have screened 66 ABPs, and the genetic screening data revealed that individual knockdown of Arp2/3 complex genes and the "capping protein β" (cpb) gene have severely altered the egg phenotype. Arpc1 and cpb RNAi mediated knockdown resulted in the formation of spherical eggs which are devoid of dorsal appendages. Studies also showed the role of Arpc1 and cpb on the number of laid eggs and follicle cell morphology. Furthermore, the depletion of Arpc1 and cpb resulted in a change in F-actin quantity. Together, the data indicate that Arpc1 and cpb regulate Drosophila egg shape, F-actin management, egg-laying characteristics and dorsal appendages formation.

Transition to Cellular Nutrition as a Solution to Reduce Threats to Environmental Stability

Nutrition is the process of supplying the organism with substances that have certain useful characteristics for the organism. It is through the process of nutrition that the organism receives the necessary amount of energy required for normal life activity. The cell is a structural and functional unit of the human body, and it is from a set of similar formations that tissue, organ, and organ systems are formed. Consequently, each cell of the body must receive the necessary supply of nutrients in order to continue its normal functioning. Cellular nutrition is the ability of each cell of the human body to consume nutrient molecules in the amount they need. In this case, the object of the study is just that nutrition, which begins with the nourishment of an individual cell. This article presents the results obtained by analyzing the data regarding the process of making the cell's consumption of the right nutrients while taking into account how the transition to cellular nutrition affects the environment and its constituents.

Study on the Influence of the Cell Structure on the Pressure Drop of Gasoline Particulate Filter

The cell structure of a gasoline particulate filter (GPF) is made up of thousands of individual cells. Although the symmetric square cell structure of the GPF is commonly used internationally, several cell designs have been proposed to reduce the pressure drop in the GPF trapping process. The aim of this paper was to use AVL-Fire software to establish GPF models of different cell structures, mainly including the symmetric square cell structure, asymmetric square cell structure, and symmetric hexagonal cell structure, and analyze the GPF pressure drop characteristics of different cell structures according to the carrier structural parameters and altitude. The results show that compared with the pressure drop of the symmetric square cell structure, the pressure drop of the asymmetric cell structure with inlet/outlet side length ratios ranging from 1.1 to 1.4 is decreased by 4.61%, 9.07%, 12.19%, and 13.22%, respectively, and the pressure drop of the symmetric hexagonal cell structure is decreased by 33.17%. Both asymmetric and symmetric hexagonal cell structure GPFs can decrease the pressure drop during trapping by increasing the cell density. From 200 CPSI to 300 CPSI, the pressure drop of the asymmetric cell structure with inlet/outlet side length ratios ranging from 1.1 to 1.4 is decreased by 20.43%, 20.53%, 20.39%, and 18.56%, respectively, and the pressure drop of the symmetric hexagonal cell structure is decreased by 18.70%. The pressure drop values of GPFs with asymmetric and symmetric hexagonal cell structures show an increasing trend with an increasing filter wall thickness and inlet/outlet plug length. The pressure drop values of GPFs with asymmetric and symmetric hexagonal cell structures show an increasing trend with an increasing altitude, and the larger the inlet/outlet ratio, the more significant the increase in the pressure drop.

Adult-neurogenesis allows for representational stability and flexibility in early olfactory system.

In the early olfactory system, adult-neurogenesis, a process of neuronal replacement results in the continuous reorganization of synaptic connections and network architecture throughout the animal's life. This poses a critical challenge: How does the olfactory system maintain stable representations of odors and therefore allow for stable sensory perceptions amidst this ongoing circuit instability? Utilizing a detailed spiking network model of early olfactory circuits, we uncovered dual roles for adult-neurogenesis: one that both supports representational stability to faithfully encode odor information and also one that facilitates plasticity to allow for learning and adaptation. In the main olfactory bulb, adult-neurogenesis affects neural codes in individual mitral and tufted cells but preserves odor representations at the neuronal population level. By contrast, in the olfactory piriform cortex, both individual cell responses and overall population dynamics undergo progressive changes due to adult-neurogenesis. This leads to representational drift, a gradual alteration in sensory perception. Both processes are dynamic and depend on experience such that repeated exposure to specific odors reduces the drift due to adult-neurogenesis; thus, when the odor environment is stable over the course of adult-neurogenesis, it is neurogenesis that actually allows the representations to remain stable in piriform cortex; when those olfactory environments change, adult-neurogenesis allows the cortical representations to track environmental change. Whereas perceptual stability and plasticity due to learning are often thought of as two distinct, often contradictory processing in neuronal coding, we find that adult-neurogenesis serves as a shared mechanism for both. In this regard, the quixotic presence of adult-neurogenesis in the mammalian olfactory bulb that has been the focus of considerable debate in chemosensory neuroscience may be the mechanistic underpinning behind an array of complex computations.

Active Methods for the Equalization of a Serially Connected Lithium-Ion Battery Pack: A Review

Traditional fuel vehicles are currently still the main means of transportation when people travel. It brings convenience to their travels, but it also causes energy shortages and environmental pollution. With the development of science and technology and the popularization of green environmental protection, electric vehicles have gradually entered people’s lives, greatly alleviating these problems. As a power supply device for electric vehicles, the performance of batteries directly affects various indicators of vehicles. Due to their long lifespan and high energy density, lithium-ion batteries are now the preferred source of power for electric vehicles. However, due to various factors in the manufacturing and operation of lithium-ion batteries, there are often differences among individual cells. The power balance and performance of a battery pack are closely related. Thus, battery equalization is an important standard for a battery management system to work normally, and it is also one of the various battery management application problems. This paper reviews battery equalization systems and various active equalization circuits and summarizes the working principle and research progress of each active equalization circuit. Then, various active equalization circuits are analyzed and compared, and dynamic equalization for a second-life battery is introduced to enrich this review of equalization technology. Finally, the above contents are summarized and prospected. In order to obtain the best outcomes, different equalization circuits need to be chosen for various situations.

Monitoring European anthropogenic NOx emissions from space

Abstract. Since the launch of TROPOMI on the Sentinel-5 Precursor (S5P) satellite, NO2 observations have become available with a resolution of 3.5× 5 km, which makes monitoring NOx emissions possible at the scale of city districts and industrial facilities. For Europe, emissions are reported on an annual basis for country totals and large industrial facilities and made publicly available via the European Environment Agency (EEA). Satellite observations can provide independent and more timely information on NOx emissions. A new version of the inversion algorithm DECSO (Daily Emissions Constrained by Satellite Observations) has been developed for deriving emissions for Europe on a daily basis, averaged to monthly mean maps. The estimated precision of these monthly emissions is about 25 % for individual grid cells. These satellite-derived emissions from DECSO have been compared to the officially reported European emissions and spatial–temporal disaggregated emission inventories. The country total DECSO NOx emissions are close to the reported emissions and the emissions compiled by the Copernicus Atmosphere Monitoring Service (CAMS). Comparison of the spatially distributed NOx emissions of DECSO and CAMS showed that the satellite-derived emissions are often higher in cities, while they are similar for large power plants and slightly lower in rural areas.

Identifying antibiotic-resistant strains via cell sorting and elastic-light-scatter phenotyping

The proliferation and dissemination of antimicrobial-resistant bacteria is an increasingly global challenge and is attributed mainly to the excessive or improper use of antibiotics. Currently, the gold-standard phenotypic methodology for detecting resistant strains is agar plating, which is a time-consuming process that involves multiple subculturing steps. Genotypic analysis techniques are fast, but they require pure starting samples and cannot differentiate between viable and non-viable organisms. Thus, there is a need to develop a better method to identify and prevent the spread of antimicrobial resistance. This work presents a novel method for detecting and identifying antibiotic-resistant strains by combining a cell sorter for bacterial detection and an elastic-light-scattering method for bacterial classification. The cell sorter was equipped with safety mechanisms for handling pathogenic organisms and enabled precise placement of individual bacteria onto an agar plate. The patterning was performed on an antibiotic-gradient plate, where the growth of colonies in sections with high antibiotic concentrations confirmed the presence of a resistant strain. The antibiotic-gradient plate was also tested with an elastic-light-scattering device where each colony’s unique colony scatter pattern was recorded and classified using machine learning for rapid identification of bacteria. Sorting and patterning bacteria on an antibiotic-gradient plate using a cell sorter reduced the number of subculturing steps and allowed direct qualitative binary detection of resistant strains. Elastic-light-scattering technology is a rapid, label-free, and non-destructive method that permits instantaneous classification of pathogenic strains based on the unique bacterial colony scatter pattern.Key points• Individual bacteria cells are placed on gradient agar plates by a cell sorter• Laser-light scatter patterns are used to recognize antibiotic-resistant organisms• Scatter patterns formed by colonies correspond to AMR-associated phenotypes

P-349 Impact of an Aurora kinase inhibitor on AML cells and mouse ovarian follicles in vitro

Abstract Study question Can an aurora kinase B/C inhibitor suppress AML cell line proliferation while not impacting on mouse follicles in vitro? Summary answer Exposure to an aurora kinase inhibitor at a predetermined cytotoxic concentration suppressed AML cell proliferation but had no impact on subsequent follicle health in vitro. What is known already There is a risk that cryopreserved ovarian tissue from young women with a leukaemia diagnosis harbours leukemic cells, which on grafting could transfer disease. Novel chemotherapy agents, specifically aurora kinase inhibitors [ e.g. GSK 1070916 (GSK) ], target proteins involved in chromosomal alignment and segregation during mitosis and meiosis. It is anticipated that aurora kinase inhibitors would have an impact on the development of all somatic cells, including granulosa cells proliferating during follicle growth, however a previous study using ovarian tissue pieces indicated no impact on follicles. Study design, size, duration Primary and secondary follicles (diameter ≤ 100 µm) were manually dissected from adult mouse ovaries and embedded in an extracellular scaffold together with >1,000 AML cells. Individual follicles and AML cells were cultured for 7 days followed by exposure to GSK for 24 hours. GSK was removed by washing and the culture continued for another two days at which time cells were assessed for normal morphology and survival with live/dead staining (Calcein AM/ Ethidium Homodimer-1). Participants/materials, setting, methods The leukemic cell line OC1-AML-3 (DSMZ) was cultured in RPMI-1640 +10% FCS and routinely passaged as recommended. Cell proliferation and cytotoxicity were assessed using the Alamar Blue assay. 20% DMSO was used as a positive control in the cytotoxicity assays and live/dead evaluation. Follicles were cultured in αMEM, ITS, FSH, ascorbic acid and 10% FCS under oil in 5%CO2 : 95% air. Survival and growth were evaluated in two extracellular scaffolds; UltiMatrix and Hystem™-HP. Main results and the role of chance Follicle survival and growth was better (p < 0.05) in UltiMatrix (84.7%; 61/72) compared to Hystem™-HP (72.0%;77/107) with an average increase of 20 µm in diameter at day 7. When OCI-AML-3 cells (0.7 x105/ml) were exposed to a range of concentrations of GSK (10nM to 10µM) for 24hr in an in vitro cytotoxicity assessment, survival was reduced to 0% at a concentration of 10µM. The 20% DMSO positive control also resulted in 0% survival. However, in the 3- dimensional scaffold culture, exposure to 10 µM GSK for 24hr resulted in a mean survival of OCI-AML-3 cells of 27% (100 cells counted/ well) which was similar to the 20% DMSO positive control (32%) and significantly different to the negative (no GSK) control (95%; p < 0.01). Follicles exposed to GSK appeared alive, emitting a strong green fluorescence for both oocyte and granulosa cells, although some of the surface follicular cells were dead. Live/dead staining of GSK exposed follicles was similar to follicles not exposed to GSK. In contrast, the majority of the follicular cells were dead following exposure to 20% DMSO. Limitations, reasons for caution Due to whole mount assessment of the follicles for live/dead staining, it is difficult to determine whether the oocyte is alive. In scaffold culture some of the free cells present may be of follicular origin, potentially resulting in overestimation of the proportion of AML cells alive after GSK treatment. Wider implications of the findings This 3- dimensional culture system, incorporating a follicle and a leukemic cell line within a scaffold provides a good model to assess the impact of chemotherapy on both cell types and could be applicable for human follicles. Trial registration number Not applicable

O-095 Assessment of genome editing outcomes in human preimplantation embryos subjected to CRISPR-Cas9 – most loss of heterozygosity (LOH) events are caused by DNA repair deficiency

Abstract Study question What is the cause of loss of heterozygosity, frequently observed in embryos subjected to CRISPR-Cas9, an interhomolog-homologous recombination repair (IH-HR) or deficiency in DNA repair? Summary answer Vast majority of LOH observed in human zygotes subjected to CRISPR-based editing is explained by deficiency in DNA repair prior to genome activation, not IH-HR. What is known already Genome editing (GE) at the preimplantation stage, intended to correct disease-causing mutations, is controversial due to safety and ethical concerns. It has been suggested that a high frequency of LOH seen in zygotes subjected to GE could be explained by resolution of the double-stranded DNA breaks (DSBs) created using CRISPR-Cas9 by interhomolog-homologous recombination repair (IH-HR), a process with potential to correct deleterious heterozygous mutations. However, such hypotheses are controversial and it is unclear whether embryos at this stage development can utilise IH-HR. CRISPR-Cas9 targeted sites are difficult to interrogate with traditional methods, risking a misinterpretation of editing outcomes. Study design, size, duration We hypothesised that the frequently observed LOH affecting the CRISPR-targeted sites in human zygotes is caused by the deficiency in DNA repair at this developmental stage, resulting in complex genotypes which traditional molecular methods have failed to detect, and not IH-HR which uses the second un-cleaved parental allele as a template for repair. DSBs were induced at three genomic sites in embryos microinjected at the time of fertilisation (n = 27) in this IRB-approved study. Participants/materials, setting, methods Research embryos were cultured for 60 hours, then disaggregated. Individual cells underwent an array of genetic tests, including NGS, allowing determination of the repair pathway utilised and revealing any cytogenetic abnormalities. A novel state-of-the-art analytical workflow was developed to interrogate 25kb around CRISPR cleavage sites. This approach provided a means of detecting complex genomic rearrangements as well as unresolved DNA damage, which would appear as LOH when employing traditional PCR-based approaches for assessment. Main results and the role of chance Induction of DSBs using CRISPR-Cas9 was 100% efficient when applied at the time of fertilisation. LOH was confirmed to be common at targeted sites, affecting 69/216 (32%) loci examined in 108 blastomeres. Remarkably, the great majority of LOH events (>90%) were a direct consequence of inappropriate/failed DNA repair (unresolved DNA breaks or complex genomic rearrangements). Strikingly, not a single instance of IH-HR was observed when CRISPR reagents were introduced at fertilisation. This may be explained by the physical separation of the two parental genomes in distinct pronuclei at that time. Allele-drop out (ADO), a technical problem, accounted for <10% of all LOH events. In total, 46% of DSBs failed to undergo appropriate repair, resulting in segmental aneuploidy (21%), large-scale deletions (10%), inversions/translocations (10%), or loss of the targeted chromosome (5%). 45% of DSBs were resolved using error-prone non-homologous end joining (NHEJ), while only 9% utilised the homology directed repair (HDR) pathway required for correction of mutations. Application of CRISPR-Cas9 to embryos on day-3 of development (after embryonic genome activation - EGA) was less efficient, only producing a DSB in 6/37 embryos (16%). However, the mode of repair differed markedly, 100% employing HDR (p = 0.0001). In 4 cases, this involved IH-HR. Limitations, reasons for caution This data provides strong evidence that LOH recorded in earlier studies was incorrectly attributed to IH-HR and was actually due to abnormal/failed repair. CRISPR appears more effective after EGA, with efficient DNA repair dominated by desirable HDR. However, a larger number of embryos is required to corroborate this finding. Wider implications of the findings The use of CRISPR-Cas9 at the time of fertilisation is frequently associated with problematic, unintended outcomes, rendering the technique clinically inapplicable in the current format. Careful study design which allows determination of all possible editing outcomes is essential and advances our understanding of the underlying mechanisms of DNA repair. Trial registration number not applicable