Protocol for optimizing surface expression and speed in coaggregation assays using K562 cells and subsequent analysis with a CoAg Index.

Coaggregation assays using K562 cells have been extensively employed to study how cell adhesion molecules mediate specificity between different populations. Here we describe how to prepare K562 cells, optimize electroporation conditions, calibrate antibodies used for protein detection, determine the surface expression of desired adhesion molecules, and considerations for the rotational force to be applied during the assay. We also detail procedures for analyzing coaggregates using our established CoAggregation (CoAg) Index. For complete details on the use and execution of this protocol, please refer to Bisogni et al.1.

BIDCell: Biologically-informed self-supervised learning for segmentation of subcellular spatial transcriptomics data.

Recent advances in subcellular imaging transcriptomics platforms have enabled high-resolution spatial mapping of gene expression, while also introducing significant analytical challenges in accurately identifying cells and assigning transcripts. Existing methods grapple with cell segmentation, frequently leading to fragmented cells or oversized cells that capture contaminated expression. To this end, we present BIDCell, a self-supervised deep learning-based framework with biologically-informed loss functions that learn relationships between spatially resolved gene expression and cell morphology. BIDCell incorporates cell-type data, including single-cell transcriptomics data from public repositories, with cell morphology information. Using a comprehensive evaluation framework consisting of metrics in five complementary categories for cell segmentation performance, we demonstrate that BIDCell outperforms other state-of-the-art methods according to many metrics across a variety of tissue types and technology platforms. Our findings underscore the potential of BIDCell to significantly enhance single-cell spatial expression analyses, enabling great potential in biological discovery.

Pcdh19 mediates olfactory sensory neuron coalescence during postnatal stages and regeneration.

The mouse olfactory system regenerates constantly throughout life. While genes critical for the initial projection of olfactory sensory neurons (OSNs) to the olfactory bulb have been identified, what genes are important for maintaining the olfactory map during regeneration are still unknown. Here we show a mutation in Protocadherin 19 (Pcdh19), a cell adhesion molecule and member of the cadherin superfamily, leads to defects in OSN coalescence during regeneration. Surprisingly, lateral glomeruli were more affected and males in particular showed a more severe phenotype. Single cell analysis unexpectedly showed OSNs expressing the MOR28 odorant receptor could be subdivided into two major clusters. We showed that at least one protocadherin is differentially expressed between OSNs coalescing on the medial and lateral glomeruli. Moreover, females expressed a slightly different complement of genes from males. These features may explain the differential effects of mutating Pcdh19 on medial and lateral glomeruli in males and females.

Scalable workflow for characterization of cell-cell communication in COVID-19 patients.

COVID-19 patients display a wide range of disease severity, ranging from asymptomatic to critical symptoms with high mortality risk. Our ability to understand the interaction of SARS-CoV-2 infected cells within the lung, and of protective or dysfunctional immune responses to the virus, is critical to effectively treat these patients. Currently, our understanding of cell-cell interactions across different disease states, and how such interactions may drive pathogenic outcomes, is incomplete. Here, we developed a generalizable and scalable workflow for identifying cells that are differentially interacting across COVID-19 patients with distinct disease outcomes and use this to examine eight public single-cell RNA-seq datasets (six from peripheral blood mononuclear cells, one from bronchoalveolar lavage and one from nasopharyngeal), with a total of 211 individual samples. By characterizing the cell-cell interaction patterns across epithelial and immune cells in lung tissues for patients with varying disease severity, we illustrate diverse communication patterns across individuals, and discover heterogeneous communication patterns among moderate and severe patients. We further illustrate patterns derived from cell-cell interactions are potential signatures for discriminating between moderate and severe patients. Overall, this workflow can be generalized and scaled to combine multiple scRNA-seq datasets to uncover cell-cell interactions.

Uncovering cell identity through differential stability with Cepo.

The use of single-cell RNA-sequencing (scRNA-seq) allows observation of different cells at multi-tiered complexity in the same microenvironment. To get insights into cell identity using scRNA-seq data, we present Cepo, which generates cell-type-specific gene statistics of differentially stable genes from scRNA-seq data to define cell identity. When applied to multiple datasets, Cepo outperforms current methods in assigning cell identity and enhances several cell identification applications such as cell-type characterisation, spatial mapping of single cells and lineage inference of single cells.

scClassify: sample size estimation and multiscale classification of cells using single and multiple reference.

Automated cell type identification is a key computational challenge in single-cell RNA-sequencing (scRNA-seq) data. To capitalise on the large collection of well-annotated scRNA-seq datasets, we developed scClassify, a multiscale classification framework based on ensemble learning and cell type hierarchies constructed from single or multiple annotated datasets as references. scClassify enables the estimation of sample size required for accurate classification of cell types in a cell type hierarchy and allows joint classification of cells when multiple references are available. We show that scClassify consistently performs better than other supervised cell type classification methods across 114 pairs of reference and testing data, representing a diverse combination of sizes, technologies and levels of complexity, and further demonstrate the unique components of scClassify through simulations and compendia of experimental datasets. Finally, we demonstrate the scalability of scClassify on large single-cell atlases and highlight a novel application of identifying subpopulations of cells from the Tabula Muris data that were unidentified in the original publication. Together, scClassify represents state-of-the-art methodology in automated cell type identification from scRNA-seq data.

Evaluating stably expressed genes in single cells.

BACKGROUND: Single-cell RNA-seq (scRNA-seq) profiling has revealed remarkable variation in transcription, suggesting that expression of many genes at the single-cell level is intrinsically stochastic and noisy. Yet, on the cell population level, a subset of genes traditionally referred to as housekeeping genes (HKGs) are found to be stably expressed in different cell and tissue types. It is therefore critical to question whether stably expressed genes (SEGs) can be identified on the single-cell level, and if so, how can their expression stability be assessed? We have previously proposed a computational framework for ranking expression stability of genes in single cells for scRNA-seq data normalization and integration. In this study, we perform detailed evaluation and characterization of SEGs derived from this framework. RESULTS: Here, we show that gene expression stability indices derived from the early human and mouse development scRNA-seq datasets and the "Mouse Atlas" dataset are reproducible and conserved across species. We demonstrate that SEGs identified from single cells based on their stability indices are considerably more stable than HKGs defined previously from cell populations across diverse biological systems. Our analyses indicate that SEGs are inherently more stable at the single-cell level and their characteristics reminiscent of HKGs, suggesting their potential role in sustaining essential functions in individual cells. CONCLUSIONS: SEGs identified in this study have immediate utility both for understanding variation and stability of single-cell transcriptomes and for practical applications such as scRNA-seq data normalization. Our framework for calculating gene stability index, "scSEGIndex," is incorporated into the scMerge Bioconductor R package (https://sydneybiox.github.io/scMerge/reference/scSEGIndex.html) and can be used for identifying genes with stable expression in scRNA-seq datasets.

Tuning of delta-protocadherin adhesion through combinatorial diversity.

The delta-protocadherins (δ-Pcdhs) play key roles in neural development, and expression studies suggest they are expressed in combination within neurons. The extent of this combinatorial diversity, and how these combinations influence cell adhesion, is poorly understood. We show that individual mouse olfactory sensory neurons express 0-7 δ-Pcdhs. Despite this apparent combinatorial complexity, K562 cell aggregation assays revealed simple principles that mediate tuning of δ-Pcdh adhesion. Cells can vary the number of δ-Pcdhs expressed, the level of surface expression, and which δ-Pcdhs are expressed, as different members possess distinct apparent adhesive affinities. These principles contrast with those identified previously for the clustered protocadherins (cPcdhs), where the particular combination of cPcdhs expressed does not appear to be a critical factor. Despite these differences, we show δ-Pcdhs can modify cPcdh adhesion. Our studies show how intra- and interfamily interactions can greatly amplify the impact of this small subfamily on neuronal function.

A Subset of Dogs with Presumptive Idiopathic Epilepsy Show Hippocampal Asymmetry: A Volumetric Comparison with Non-Epileptic Dogs Using MRI.

MRI-acquired volumetric measurements from 100 dogs with presumptive idiopathic epilepsy (IE) and 41 non-epileptic (non-IE) dogs were used to determine if hippocampal asymmetry exists in the IE as compared to the non-IE dogs. MRI databases from three institutions were searched for dogs that underwent MRI of the brain and were determined to have IE and those that were considered non-IE dogs. Volumes of the right and left hippocampi were measured using Mimics® software. Median hippocampal volumes of IE and non-IE dogs were 0.47 and 0.53 cm3, respectively. There was no significant difference in overall hippocampal volume between IE and non-IE dogs; however, IE dogs had greater hippocampal asymmetry than non-IE dogs (P < 0.012). A threshold value of 1.16 from the hippocampal ratio had an 85% specificity for identifying IE-associated asymmetry. Thirty five percent of IE dogs had a hippocampal ratio >1.16. Asymmetry was not associated with any particular hemisphere (P = 0.67). Our study indicates that hippocampal asymmetry occurs in a subset of dogs with presumptive idiopathic/genetic epilepsy, suggesting a structural etiology to some cases of IE.

Integrated single cell data analysis reveals cell specific networks and novel coactivation markers.

BACKGROUND: Large scale single cell transcriptome profiling has exploded in recent years and has enabled unprecedented insight into the behavior of individual cells. Identifying genes with high levels of expression using data from single cell RNA sequencing can be useful to characterize very active genes and cells in which this occurs. In particular single cell RNA-Seq allows for cell-specific characterization of high gene expression, as well as gene coexpression. RESULTS: We offer a versatile modeling framework to identify transcriptional states as well as structures of coactivation for different neuronal cell types across multiple datasets. We employed a gamma-normal mixture model to identify active gene expression across cells, and used these to characterize markers for olfactory sensory neuron cell maturity, and to build cell-specific coactivation networks. We found that combined analysis of multiple datasets results in more known maturity markers being identified, as well as pointing towards some novel genes that may be involved in neuronal maturation. We also observed that the cell-specific coactivation networks of mature neurons tended to have a higher centralization network measure than immature neurons. CONCLUSION: Integration of multiple datasets promises to bring about more statistical power to identify genes and patterns of interest. We found that transforming the data into active and inactive gene states allowed for more direct comparison of datasets, leading to identification of maturity marker genes and cell-specific network observations, taking into account the unique characteristics of single cell transcriptomics data.

Inferring data-specific micro-RNA function through the joint ranking of micro-RNA and pathways from matched micro-RNA and gene expression data.

MOTIVATION: In practice, identifying and interpreting the functional impacts of the regulatory relationships between micro-RNA and messenger-RNA is non-trivial. The sheer scale of possible micro-RNA and messenger-RNA interactions can make the interpretation of results difficult. RESULTS: We propose a supervised framework, pMim, built upon concepts of significance combination, for jointly ranking regulatory micro-RNA and their potential functional impacts with respect to a condition of interest. Here, pMim directly tests if a micro-RNA is differentially expressed and if its predicted targets, which lie in a common biological pathway, have changed in the opposite direction. We leverage the information within existing micro-RNA target and pathway databases to stabilize the estimation and annotation of micro-RNA regulation making our approach suitable for datasets with small sample sizes. In addition to outputting meaningful and interpretable results, we demonstrate in a variety of datasets that the micro-RNA identified by pMim, in comparison to simpler existing approaches, are also more concordant with what is described in the literature. AVAILABILITY AND IMPLEMENTATION: This framework is implemented as an R function, pMim, in the package sydSeq available from http://www.ellispatrick.com/r-packages. CONTACT: jean.yang@sydney.edu.au SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Effects of a multimodality blood conservation schema toward improvement of intraoperative hemoglobin levels and off-pump transfusions in coronary artery bypass graft surgery.

BACKGROUND: Cardiothoracic surgery places significant demands on blood bank resources. Measures aimed at reducing intraoperative hemodilution were initiated as part of a blood conservation program. STUDY DESIGN AND METHODS: We initiated a series of measures aimed at reducing hemodilution volume: 1) reduction of intravenous fluid (IVF) volume, 2) reduction of circuit size, and 3) use of autologous priming techniques. All sources and volumes of IVF were obtained from the medical record. Intraoperative hematocrit (Hct) measurements were performed at the following intervals: first in operating room (OR), lowest on-pump, last on-pump, after protamine reversal, and immediately before discharge from OR. Red blood cell (RBC) transfusions were recorded. Intraoperative IVF, Hct levels, and transfusions were analyzed by cardiopulmonary bypass phase (prepump, on-pump, and off-pump), comparing preimplementation and postimplementation periods. RESULTS: Total intraoperative IVF volume was reduced by 973.7 mL (95% confidence interval, 671.6-1275.9 mL; p < 0.001) leading to a mean on-pump Hct improvement of more than 2% (p < 0.004). This contributed to a reduction in off-pump RBC transfusions by 20.6% (p = 0.014). A significant degree of heterogeneity in transfusion practice was noted between anesthesiologists. CONCLUSIONS: Blood conservation efforts in cardiac surgery should include efforts aimed at reducing hemodilution. Potential improvements are blunted by variation in transfusion practice.

Measuring trade-offs that matter: assessing the impact of a new electronic cross-match policy on the turnaround time and the cross-match workload efficiency.

BACKGROUND: Our traditional cross-match (XM) policy generated a significant number of XM units that were never issued. To minimize the unnecessary XM workload, we proposed a new policy where orders eligible for the electronic XM (EXM) are pended until orders to issue red blood cells (RBCs) are received. To address concerns that this new policy might unduly delay blood availability, we conducted a study to assess whether the new policy was noninferior to the traditional policy with regard to the turnaround time (TAT). STUDY DESIGN AND METHODS: We monitored the TAT and XM workload efficiency (XM-to-issue [C : I] ratio) for a total of 8 weeks split between the two policies' periods. The primary outcome was the proportion of RBC issue requests that was turned around in less than 12 minutes. RESULTS: Fifty percent (1133 of 2265) of issue requests were turned around in 12 minutes or less under the traditional policy compared to 43.9% (975 of 2223) under the new policy (absolute difference of 6.1%; 95% confidence interval [CI], 3.2%-9.1%; p < 0.001). The adjusted overall median TAT was slower by 1 minute (13 min vs. 14 min, p < 0.001) but the adjusted C : I ratio was better (1.00 vs. 1.15; p < 0.001) under the new policy. CONCLUSION: Our study showed that the impact of the new policy on the TAT was not inferior to the traditional policy. Since the median TAT of 14 minutes under the new policy met the published benchmarks, the trade-off between delays in the TAT and efficiency gains in the XM workload remained acceptable for patient care.

Lack of fear response in mice (Mus musculus) exposed to human urine odor.

A goal of the Guide for the Care and Use of Laboratory Animals is to improve animal welfare by minimizing sources of fear, anxiety, and stress. As a result, it includes recommendations on overcrowding, frequency of cage changes, enrichment, and group housing. However, human odorants are a potential but unexplored source of fear, anxiety, and stress. Although mice have been maintained for decades for animal research, whether mice perceive humans as predators is unknown. If so, this would necessitate changes in animal care and use procedures to minimize this source of chronic fear, anxiety, and stress. Odorants from predator urine are well known to elicit strong fear responses in mice, leading to modification of animal behavior and elevated levels of stress. To begin asking whether human odors influence mouse behavior, we tested the effect of human urine odor on fear response in mice. We assessed mouse behavior by using a modified shuttle cage to record various parameters of mouse exposure to odorants. We found that mice displayed fear responses to 2,4,5-trimethylthiazoline, a synthetic analog of red fox feces, but no fear response to DMSO, the diluent for 2,4,5-trimethylthiazoline. In contrast, mice exposed to human urine samples showed no significant fear response.

Efficacy and safety of erythropoietin and intravenous iron in perioperative blood management: a systematic review.

The use of erythropoietin (EPO) and intravenous (IV) iron as bloodless therapeutic modalities is being explored in the current era of restrictive transfusion strategies and perioperative blood management. It is unclear, however, whether the evidence in the literature supports their safety and efficacy in reducing perioperative red cell transfusions. Adhering to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines, we conducted a systematic review to evaluate their use in a variety of perioperative settings. We performed a literature search of English articles published between July 1997 and July 2012 in MEDLINE via PubMed, The Cochrane Library, and CINAHL. Only studies with a comparator group were eligible for inclusion. Twenty-four randomized controlled trials (RCTs) and 15 nonrandomized studies were included in the final review. Using the Cochrane risk of bias tool, 8 RCTs were assessed to be at low risk for methodological bias. Of these, however, only 4 RCTs were adequately powered to detect a reduction in transfusion rates. Patients with preoperative iron deficiency anemia may have an earlier and more robust hemoglobin recovery with preoperative IV iron therapy than with oral iron supplementation. A short preoperative regimen of EPO, or a single dose of EPO plus IV iron in the preoperative or intraoperative period, may significantly reduce transfusion rates (number needed to treat to avoid any transfusion ranged from 3 to 6). With regard to the safety of erythropoietin-stimulating agent therapy, IV iron appears to be as well tolerated as oral iron; however, the incidence of severe anaphylactic-type reactions attributable to IV iron is difficult to estimate in prospective trials because of its relatively infrequent occurrence. Furthermore, EPO may increase the risk of thromboembolism in spinal surgery patients who receive mechanical antithrombotic prophylaxis in the perioperative period so pharmacological thromboprophylaxis is advised. Future low risk of bias, adequately powered prospective efficacy, and safety trials in various surgical settings that traditionally require red cell transfusions would be required to make evidenced-based conclusions about the clinical significance of erythropoietin-stimulating agent as a transfusion avoidance strategy in perioperative blood management.

Use of prothrombin complex concentrates and fibrinogen concentrates in the perioperative setting: a systematic review.

The use of prothrombin complex concentrates (PCCs) and fibrinogen concentrates (FIBCs) to achieve hemostasis in the perioperative setting as alternatives to allogeneic blood products remains controversial. To examine the efficacy and safety of PCCs and FIBCs, we conducted a systematic review-in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis statement-to compare the use of these transfusion alternatives in bleeding surgical patients. We performed a literature search of English articles published between July 1997 and July 2012 in MEDLINE via PubMed, The Cochrane Library, and CINAHL. Five randomized trials and 15 nonrandomized studies with a comparator group were included in the final review. Studies were sorted into 1 of the following 3 clinical settings: cardiac surgery, non-cardiac surgery, and reversal of warfarin anticoagulation. Risk of bias was assessed using the Cochrane risk of bias tool. With the exception of 2 randomized controlled trials, the existing body of literature on the use of PCCs and FIBCs in the perioperative setting was assessed to have a high degree of methodological bias. Overall, prospective studies in the cardiac surgery grouping suggested that patients receiving FIBC and/or PCCs required less allogeneic blood transfusion and had less chest tube drainage. In studies of warfarin reversal, PCCs more rapidly corrected the International Normalized Ratio compared to plasma; however, in the setting of intracranial hemorrhage, functional outcomes were poor regardless of the reversal strategy. With regards to safety outcomes, reporting was not uniform and raises concerns of underreporting. Adequately powered, methodologically sound trials would be required for more definitive conclusions to be drawn about the efficacy of PCCs and FIBC over conventional blood components for the treatment of perioperative coagulopathy in bleeding patients.

Delta Protocadherin 10 is Regulated by Activity in the Mouse Main Olfactory System.

Olfactory sensory neurons (OSNs) are thought to use activity-dependent and independent mechanisms to regulate the expression of axon guidance genes. However, defining the molecular mechanisms that underlie activity-dependent OSN guidance has remained elusive. Only a handful of genes have been identified whose expression is regulated by activity. Interestingly, all of these genes have been shown to play a role in OSN axon guidance, underscoring the importance of identifying other genes regulated by activity. Furthermore, studies suggest that more than one downstream mechanism regulates target gene expression. Thus, both the number of genes regulated by activity and how many total mechanisms control this expression are not well understood. Here we identify delta protocadherin 10 (pcdh10) as a gene regulated by activity. Delta protocadherins are members of the cadherin superfamily, and pcdh10 is known to be important for axon guidance during development. We show pcdh10 is expressed in the nasal epithelium and olfactory bulb in patterns consistent with providing guidance information to OSNs. We use naris occlusion, genetic manipulations, and pharmacological assays to show pcdh10 can be regulated by activity, consistent with activation via the cyclic nucleotide-gated channel. Transgenic analysis confirms a potential role for pcdh10 in OSN axon guidance.

Shmt1 heterozygosity impairs folate-dependent thymidylate synthesis capacity and modifies risk of Apc(min)-mediated intestinal cancer risk.

Folate-mediated one-carbon metabolism is required for the de novo synthesis of purines, thymidylate, and S-adenosylmethionine, the primary cellular methyl donor. Impairments in folate metabolism diminish cellular methylation potential and genome stability, which are risk factors for colorectal cancer (CRC). Cytoplasmic serine hydroxymethyltransferase (SHMT1) regulates the partitioning of folate-activated one-carbons between thymidylate and S-adenosylmethionine biosynthesis. Therefore, changes in SHMT1 expression enable the determination of the specific contributions made by thymidylate and S-adenosylmethionine biosynthesis to CRC risk. Shmt1 hemizygosity was associated with a decreased capacity for thymidylate synthesis due to downregulation of enzymes in its biosynthetic pathway, namely thymidylate synthase and cytoplasmic thymidine kinase. Significant Shmt1-dependent changes to methylation capacity, gene expression, and purine synthesis were not observed. Shmt1 hemizygosity was associated with increased risk for intestinal cancer in Apc(min)(/+) mice through a gene-by-diet interaction, indicating that the capacity for thymidylate synthesis modifies susceptibility to intestinal cancer in Apc(min)(/+) mice.

Mthfd1 is a modifier of chemically induced intestinal carcinogenesis.

The causal metabolic pathways underlying associations between folate and risk for colorectal cancer (CRC) have yet to be established. Folate-mediated one-carbon metabolism is required for the de novo synthesis of purines, thymidylate and methionine. Methionine is converted to S-adenosylmethionine (AdoMet), the major one-carbon donor for cellular methylation reactions. Impairments in folate metabolism can modify DNA synthesis, genomic stability and gene expression, characteristics associated with tumorigenesis. The Mthfd1 gene product, C1-tetrahydrofolate synthase, is a trifunctional enzyme that generates one-carbon substituted tetrahydrofolate cofactors for one-carbon metabolism. In this study, we use Mthfd1(gt/+) mice, which demonstrate a 50% reduction in C1-tetrahydrofolate synthase, to determine its influence on tumor development in two mouse models of intestinal cancer, crosses between Mthfd1(gt/+) and Apc(min)(/+) mice and azoxymethane (AOM)-induced colon cancer in Mthfd1(gt/+) mice. Mthfd1 hemizygosity did not affect colon tumor incidence, number or load in Apc(min/+) mice. However, Mthfd1 deficiency increased tumor incidence 2.5-fold, tumor number 3.5-fold and tumor load 2-fold in AOM-treated mice. DNA uracil content in the colon was lower in Mthfd1(gt/+) mice, indicating that thymidylate biosynthesis capacity does not play a significant role in AOM-induced colon tumorigenesis. Mthfd1 deficiency-modified cellular methylation potential, as indicated by the AdoMet: S-adenosylhomocysteine ratio and gene expression profiles, suggesting that changes in the transcriptome and/or decreased de novo purine biosynthesis and associated mutability cause cellular transformation in the AOM CRC model. This study emphasizes the impact and complexity of gene-nutrient interactions with respect to the relationships among folate metabolism and colon cancer initiation and progression.