Genome engineering with Cas9 and AAV repair templates generates frequent concatemeric insertions of viral vectors.

CRISPR-Cas9 paired with adeno-associated virus serotype 6 (AAV6) is among the most efficient tools for producing targeted gene knockins. Here, we report that this system can lead to frequent concatemeric insertions of the viral vector genome at the target site that are difficult to detect. Such errors can cause adverse and unreliable phenotypes that are antithetical to the goal of precision genome engineering. The concatemeric knockins occurred regardless of locus, vector concentration, cell line or cell type, including human pluripotent and hematopoietic stem cells. Although these highly abundant errors were found in more than half of the edited cells, they could not be readily detected by common analytical methods. We describe strategies to detect and thoroughly characterize the concatemeric viral vector insertions, and we highlight analytical pitfalls that mask their prevalence. We then describe strategies to prevent the concatemeric inserts by cutting the vector genome after transduction. This approach is compatible with established gene editing pipelines, enabling robust genetic knockins that are safer, more reliable and more reproducible.

Engineering heterothallic strains in fission yeast.

In poor nitrogen conditions, fission yeast cells mate, undergo meiosis and form spores that are resistant to deleterious environments. Natural isolates of Schizosaccharomyces pombe are homothallic. This allows them to naturally switch between the two h- and h+ mating types with a high frequency, thereby ensuring the presence of both mating partners in a population of cells. However, alteration of the mating type locus can abolish mating type switching or reduce it to a very low frequency. Such heterothallic strains have been isolated and are common in research laboratories due to the simplicity of their use for Mendelian genetics. In addition to the standard laboratory strains, a large collection of natural S. pombe isolates is now available, representing a powerful resource for investigating the genetic diversity and biology of fission yeast. However, most of these strains are homothallic, and only tedious or mutagenic strategies have been described to obtain heterothallic cells from a homothallic parent. Here, we describe a simple approach to generate heterothallic strains. It takes advantage of an alteration of the mating type locus that was previously identified in a mating type switching-deficient strain and the CRISPR-Cas9 editing tool, allowing for a one-step engineering of heterothallic cells with high efficiency.

Mapping the cellular and molecular landscape of cardiac non-myocytes in murine diabetic cardiomyopathy.

Diabetes is associated with a significantly elevated risk of heart failure. However, despite extensive efforts to characterize the phenotype of the diabetic heart, the molecular and cellular protagonists that underpin cardiac pathological remodeling in diabetes remain unclear, with a notable paucity of data regarding the impact of diabetes on non-myocytes within the heart. Here we aimed to define key differences in cardiac non-myocytes between spontaneously type-2 diabetic (db/db) and healthy control (db/h) mouse hearts. Single-cell transcriptomic analysis revealed a concerted diabetes-induced cellular response contributing to cardiac remodeling. These included cell-specific activation of gene programs relating to fibroblast hyperplasia and cell migration, and dysregulation of pathways involving vascular homeostasis and protein folding. This work offers a new perspective for understanding the cellular mediators of diabetes-induced cardiac pathology, and pathways that may be targeted to address the cardiac complications associated with diabetes.

Multi-omic analysis of the cardiac cellulome defines a vascular contribution to cardiac diastolic dysfunction in obese female mice.

Coronary microvascular dysfunction (CMD) is associated with cardiac dysfunction and predictive of cardiac mortality in obesity, especially in females. Clinical data further support that CMD associates with development of heart failure with preserved ejection fraction and that mineralocorticoid receptor (MR) antagonism may be more efficacious in obese female, versus male, HFpEF patients. Accordingly, we examined the impact of smooth muscle cell (SMC)-specific MR deletion on obesity-associated coronary and cardiac diastolic dysfunction in female mice. Obesity was induced in female mice via western diet (WD) feeding alongside littermates fed standard diet. Global MR blockade with spironolactone prevented coronary and cardiac dysfunction in obese females and specific deletion of SMC-MR was sufficient to prevent obesity-associated coronary and cardiac diastolic dysfunction. Cardiac gene expression profiling suggested reduced cardiac inflammation in WD-fed mice with SMC-MR deletion independent of blood pressure, aortic stiffening, and cardiac hypertrophy. Further mechanistic studies utilizing single-cell RNA sequencing of non-cardiomyocyte cell populations revealed novel impacts of SMC-MR deletion on the cardiac cellulome in obese mice. Specifically, WD feeding induced inflammatory gene signatures in non-myocyte populations including B/T cells, macrophages, and endothelium as well as increased coronary VCAM-1 protein expression, independent of cardiac fibrosis, that was prevented by SMC-MR deletion. Further, SMC-MR deletion induced a basal reduction in cardiac mast cells and prevented WD-induced cardiac pro-inflammatory chemokine expression and leukocyte recruitment. These data reveal a central role for SMC-MR signaling in obesity-associated coronary and cardiac dysfunction, thus supporting the emerging paradigm of a vascular origin of cardiac dysfunction in obesity.

In Vitro Human Haematopoietic Stem Cell Expansion and Differentiation.

The haematopoietic system plays an essential role in our health and survival. It is comprised of a range of mature blood and immune cell types, including oxygen-carrying erythrocytes, platelet-producing megakaryocytes and infection-fighting myeloid and lymphoid cells. Self-renewing multipotent haematopoietic stem cells (HSCs) and a range of intermediate haematopoietic progenitor cell types differentiate into these mature cell types to continuously support haematopoietic system homeostasis throughout life. This process of haematopoiesis is tightly regulated in vivo and primarily takes place in the bone marrow. Over the years, a range of in vitro culture systems have been developed, either to expand haematopoietic stem and progenitor cells or to differentiate them into the various haematopoietic lineages, based on the use of recombinant cytokines, co-culture systems and/or small molecules. These approaches provide important tractable models to study human haematopoiesis in vitro. Additionally, haematopoietic cell culture systems are being developed and clinical tested as a source of cell products for transplantation and transfusion medicine. This review discusses the in vitro culture protocols for human HSC expansion and differentiation, and summarises the key factors involved in these biological processes.

Fragment Coupling Approach to Diaporthein B.

Pimarane diterpenes are produced by a diverse array of plants, fungi, and bacteria. Many members of this family possess antimicrobial and antiproliferative activities. The pimarane diterpenes are characterized by a tricyclic carbon scaffold comprising three fused six-membered rings and at least three quaternary centers. Here, we describe two convergent, fragment-based strategies toward the synthesis of diaporthein B (3), one of the most highly oxidized pimarane diterpenes. The first approach provided access to the tricyclic carbon scaffold of the target and featured a highly diastereoselective fragment coupling, a novel carbonylative Stille cross-coupling to directly access an α-hydroxyketone from a vinyl iodide, and a tandem aldol cyclization-deprotection cascade. The second route utilized a diastereoselective 1,4-addition of a silyloxyfuran to an unsaturated ketone, followed by an epoxidation-ring opening sequence, to access a highly oxidized intermediate containing two elaborated cyclohexane rings. The chemistry developed herein may ultimately be useful in an eventual synthesis of this class of natural products.

Physioxia improves the selectivity of hematopoietic stem cell expansion cultures.

Hematopoietic stem cells (HSCs) are a rare type of hematopoietic cell that can entirely reconstitute the blood and immune system after transplantation. Allogeneic HSC transplantation (HSCT) is used clinically as a curative therapy for a range of hematolymphoid diseases; however, it remains a high-risk therapy because of its potential side effects, including poor graft function and graft-versus-host disease (GVHD). Ex vivo HSC expansion has been suggested as an approach to improve hematopoietic reconstitution in low-cell dose grafts. Here, we demonstrate that the selectivity of polyvinyl alcohol (PVA)-based mouse HSC cultures can be improved using physioxic culture conditions. Single-cell transcriptomic analysis helped confirm the inhibition of lineage-committed progenitor cells in physioxic cultures. Long-term physioxic expansion also afforded culture-based ex vivo HSC selection from whole bone marrow, spleen, and embryonic tissues. Furthermore, we provide evidence that HSC-selective ex vivo cultures deplete GVHD-causing T cells and that this approach can be combined with genotoxic-free antibody-based conditioning HSCT approaches. Our results offer a simple approach to improve PVA-based HSC cultures and the underlying molecular phenotype, and highlight the potential translational implications of selective HSC expansion systems for allogeneic HSCT.

Multiplexed Assembly and Annotation of Synthetic Biology Constructs Using Long-Read Nanopore Sequencing.

Recombinant DNA is a fundamental tool in biotechnology and medicine. These DNA sequences are often built, replicated, and delivered in the form of plasmids. Validation of these plasmid sequences is a critical and time-consuming step, which has been dominated for the last 35 years by Sanger sequencing. As plasmid sequences grow more complex with new DNA synthesis and cloning techniques, we need new approaches that address the corresponding validation challenges at scale. Here we prototype a high-throughput plasmid sequencing approach using DNA transposition and Oxford Nanopore sequencing. Our method, Circuit-seq, creates robust, full-length, and accurate plasmid assemblies without prior knowledge of the underlying sequence. We demonstrate the power of Circuit-seq across a wide range of plasmid sizes and complexities, generating full-length, contiguous plasmid maps. We then leverage our long-read data to characterize epigenetic marks and estimate plasmid contamination levels. Circuit-seq scales to large numbers of samples at a lower per-sample cost than commercial Sanger sequencing, accelerating a key step in synthetic biology, while low equipment costs make it practical for individual laboratories.

Streamlined and quantitative detection of chimerism using digital PCR.

Animal chimeras are widely used for biomedical discoveries, from developmental biology to cancer research. However, the accurate quantitation of mixed cell types in chimeric and mosaic tissues is complicated by sample preparation bias, transgenic silencing, phenotypic similarity, and low-throughput analytical pipelines. Here, we have developed and characterized a droplet digital PCR single-nucleotide discrimination assay to detect chimerism among common albino and non-albino mouse strains. In addition, we validated that this assay is compatible with crude lysate from all solid organs, drastically streamlining sample preparation. This chimerism detection assay has many additional advantages over existing methods including its robust nature, minimal technical bias, and ability to report the total number of cells in a prepared sample. Moreover, the concepts discussed here are readily adapted to other genomic loci to accurately measure mixed cell populations in any tissue.

Immunological barriers to haematopoietic stem cell gene therapy.

Cell and gene therapies using haematopoietic stem cells (HSCs) epitomize the transformative potential of regenerative medicine. Recent clinical successes for gene therapies involving autologous HSC transplantation (HSCT) demonstrate the potential of genetic engineering in this stem cell type for curing disease. With recent advances in CRISPR gene-editing technologies, methodologies for the ex vivo expansion of HSCs and non-genotoxic conditioning protocols, the range of clinical indications for HSC-based gene therapies is expected to significantly expand. However, substantial immunological challenges need to be overcome. These include pre-existing immunity to gene-therapy reagents, immune responses to neoantigens introduced into HSCs by genetic engineering, and unique challenges associated with next-generation and off-the-shelf HSC products. By synthesizing these factors in this Review, we hope to encourage more research to address the immunological issues associated with current and next-generation HSC-based gene therapies to help realize the full potential of this field.

Stochastic models for single-cell data: Current challenges and the way forward.

Although the quantity and quality of single-cell data have progressed rapidly, making quantitative predictions with single-cell stochastic models remains challenging. The stochastic nature of cellular processes leads to at least three challenges in building models with single-cell data: (a) because variability in single-cell data can be attributed to multiple different sources, it is difficult to rule out conflicting mechanistic models that explain the same data equally well; (b) the distinction between interesting biological variability and experimental variability is sometimes ambiguous; (c) the nonstandard distributions of single-cell data can lead to violations of the assumption of symmetric errors in least-squares fitting. In this review, we first discuss recent studies that overcome some of the challenges or set up a promising direction and then introduce some powerful statistical approaches utilized in these studies. We conclude that applying and developing statistical approaches could lead to further progress in building stochastic models for single-cell data.

A functionally divergent intrinsically disordered region underlying the conservation of stochastic signaling.

Stochastic signaling dynamics expand living cells' information processing capabilities. An increasing number of studies report that regulators encode information in their pulsatile dynamics. The evolutionary mechanisms that lead to complex signaling dynamics remain uncharacterized, perhaps because key interactions of signaling proteins are encoded in intrinsically disordered regions (IDRs), whose evolution is difficult to analyze. Here we focused on the IDR that controls the stochastic pulsing dynamics of Crz1, a transcription factor in fungi downstream of the widely conserved calcium signaling pathway. We find that Crz1 IDRs from anciently diverged fungi can all respond transiently to calcium stress; however, only Crz1 IDRs from the Saccharomyces clade support pulsatility, encode extra information, and rescue fitness in competition assays, while the Crz1 IDRs from distantly related fungi do none of the three. On the other hand, we find that Crz1 pulsing is conserved in the distantly related fungi, consistent with the evolutionary model of stabilizing selection on the signaling phenotype. Further, we show that a calcineurin docking site in a specific part of the IDRs appears to be sufficient for pulsing and show evidence for a beneficial increase in the relative calcineurin affinity of this docking site. We propose that evolutionary flexibility of functionally divergent IDRs underlies the conservation of stochastic signaling by stabilizing selection.

Metric-Based Analysis of Convergence in Complex Molecule Synthesis.

Convergent syntheses are characterized by the coupling of two or more synthetic intermediates of similar complexity, often late in a pathway. At its limit, a fully convergent synthesis is achieved when commercial or otherwise readily available intermediates are coupled to form the final target in a single step. Of course, in all but exceptional circumstances this level of convergence is purely hypothetical; in practice, additional steps are typically required to progress from fragment coupling to the target. Additionally, the length of the sequence required to access each target is a primary consideration in synthetic design.In this Account, we provide an overview of alkaloid, polyketide, and diterpene metabolites synthesized in our laboratory and present parameters that may be used to put the degree of convergence of each synthesis on quantitative footing. We begin with our syntheses of the antiproliferative, antimicrobial bacterial metabolite (-)-kinamycin F (1) and related dimeric structure (-)-lomaiviticin aglycon (2). These synthetic routes featured a three-step sequence to construct a complex diazocyclopentadiene found in both targets and an oxidative dimerization to unite the two halves of (-)-lomaiviticin aglycon (2). We then follow with our synthesis of the antineurodegenerative alkaloid (-)-huperzine A (3). Our route to (-)-huperzine A (3) employed a diastereoselective three-component coupling reaction, followed by the intramolecular α-arylation of a ß-ketonitrile intermediate, to form the carbon skeleton of the target. We then present our syntheses of the hasubanan alkaloids (-)-hasubanonine (4), (-)-delavayine (5), (-)-runanine (6), (+)-periglaucine B (7), and (-)-acutumine (8). These alkaloids bear a 7-azatricyclo[4.3.3.01,6]dodecane (propellane) core and a highly oxidized cyclohexenone ring. The propellane structure was assembled by the addition of an aryl acetylide to a complex iminium ion, followed by intramolecular 1,4-addition. We then present our synthesis of the guanidinium alkaloid (+)-batzelladine B (9), which contains two complex polycyclic guanidine residues united by an ester linkage. This target was logically disconnected by an esterification to allow for the independent synthesis of each guanidine residue. A carefully orchestrated cascade reaction provided (+)-batzelladine B (9) in a single step following fragment coupling by esterification. We then discuss our synthesis of the diterpene fungal metabolite (+)-pleuromutilin (10). The synthesis of (+)-pleuromutilin (10) proceeded via a fragment coupling involving two neopentylic reagents and employed a nickel-catalyzed reductive cyclization reaction to close the eight-membered ring, ultimately providing access to (+)-pleuromutilin (10), (+)-12-epi-pleuromutilin (11), and (+)-12-epi-mutilin (12). Finally, we discuss our synthesis of (-)-myrocin G (13), a tricyclic pimarane diterpene that was assembled by a convergent annulation.In the final section of this Account, we present several paramaters to analyze and quantitatively assess the degree of convergence of each synthesis. These parameters include: (1) the number of steps required following the point of convergence, (2) the difference in the number of steps required to prepare each coupling partner, (3) the percentage of carbons (or, more broadly, atoms) present at the point of convergence, and (4) the complexity generated in the fragment coupling step. While not an exhaustive list, these parameters bring the strengths and weaknesses each synthetic strategy to light, emphasizing the key contributors to the degree of convergence of each route while also highlighting the nuances of these analyses.

Cas9-AAV6 gene correction of beta-globin in autologous HSCs improves sickle cell disease erythropoiesis in mice.

CRISPR/Cas9-mediated beta-globin (HBB) gene correction of sickle cell disease (SCD) patient-derived hematopoietic stem cells (HSCs) in combination with autologous transplantation represents a recent paradigm in gene therapy. Although several Cas9-based HBB-correction approaches have been proposed, functional correction of in vivo erythropoiesis has not been investigated previously. Here, we use a humanized globin-cluster SCD mouse model to study Cas9-AAV6-mediated HBB-correction in functional HSCs within the context of autologous transplantation. We discover that long-term multipotent HSCs can be gene corrected ex vivo and stable hemoglobin-A production can be achieved in vivo from HBB-corrected HSCs following autologous transplantation. We observe a direct correlation between increased HBB-corrected myeloid chimerism and normalized in vivo red blood cell (RBC) features, but even low levels of chimerism resulted in robust hemoglobin-A levels. Moreover, this study offers a platform for gene editing of mouse HSCs for both basic and translational research.

Fragment Coupling Reactions in Total Synthesis That Form Carbon-Carbon Bonds via Carbanionic or Free Radical Intermediates.

Fragment coupling reactions that form carbon-carbon bonds are valuable transformations in synthetic design. Advances in metal-catalyzed cross-coupling reactions in the early 2000s brought a high level of predictability and reliability to carbon-carbon bond constructions involving the union of unsaturated fragments. By comparison, recent years have witnessed an increase in fragment couplings proceeding via carbanionic and open-shell (free radical) intermediates. The latter has been driven by advances in methods to generate and utilize carbon-centered radicals under mild conditions. In this Review, we survey a selection of recent syntheses that have implemented carbanion- or radical-based fragment couplings to form carbon-carbon bonds. We aim to highlight the strategic value of these disconnections in their respective settings and to identify extensible lessons from each example that might be instructive to students.

Stickiness of extracellular polymeric substances on different surfaces via magnetic tweezers.

Organic particle dynamics in the surface ocean plays a critical part in the marine carbon cycle. Aggregation of marine organic particles drives their downward transport to support various marine organisms on their transit to the sediments. Extracellular polymeric substances (EPS) from various microbes are a major contributor to the oceanic organic particle pool. The stickiness of EPS is expected to play a determining role in the aggregation process of particles; however, stickiness parameters are usually indirectly estimated through data fitting without direct assessment. Here a magnetic tweezer method was developed to quantitatively assess the stickiness of three model EPS produced by: Amphora sp., (diatom), Emiliania huxleyi (coccolithophore), and Sagittula stellata (bacteria), under different in vitro environmental conditions (salinity or EDTA complexed cations) and surface matrices (EPS-EPS and bare glass). Our results showed the stickiness of three microbial EPS decreasing for S. stellata > E. huxleyi > Amphora sp., in line with their decreasing protein-to-carbohydrate (P/C) ratios (related to their relative hydrophobicity). The data not only emphasize the importance of hydrophobicity on EPS stickiness, but also demonstrates that salinity and the nature of the substrate surface can influence the stickiness. Furthermore, we investigated stickiness between various types of EPS, and the observed selective stickiness of EPS between species may shed light on the interactions among heterogeneous marine microorganisms. Overall, this newly developed system provides a platform to assess the EPS stickiness to advance our understanding of the aggregation and sedimentation process of organic particles that are critical for the fate of organic carbon as well as for biofilm formation and microbial colonization of surfaces in the ocean.

Depression, Anxiety, and Acute Stress Disorder Among Patients Hospitalized With COVID-19: A Prospective Cohort Study.

BACKGROUND: The epidemiology of psychiatric symptoms among patients with coronavirus disease 2019 is poorly characterized. OBJECTIVE: This article sought to identify the prevalence of anxiety, depression, and acute stress disorder among hospitalized patients with coronavirus disease 2019. METHODS: Adult patients recently admitted to nonintensive care unit medical ward settings with coronavirus disease 2019 were eligible for enrollment. Enrolled patients were screened for depression, anxiety, and delirium. Subsequently, patients were followed up by phone after 2 weeks and rescreened for depression, anxiety, and acute stress disorder symptoms. Subjects' medical records were abstracted for clinical data. RESULTS: A total of 58 subjects were enrolled; of whom, 44 completed the study. Initially, 36% of subjects had elevated anxiety symptoms and 29% had elevated depression symptoms. At 2-week follow-up, 9% had elevated anxiety symptoms, 20% had elevated depression symptoms, and 25% had mild-to-moderate acute stress disorder symptoms. Discharge to home was not associated with improvement in psychiatric symptoms. CONCLUSIONS: A significant number of patients hospitalized with coronavirus disease 2019 experienced symptoms of depression and anxiety. While anxiety improved after index admission, depression remained fairly stable. Furthermore, a significant minority of patients experienced acute stress disorder symptoms, though these were largely mild to moderate.

Evaluation of plant seed DNA and botanical evidence for potential forensic applications.

Seeds, the reproductive organs of plants, are common as trace evidence from crime scenes. Seed evidence could be grouped into several categories based on the types of crimes they are associated with, including child abuse, homicides and drugs. Most commonly, seeds are examined microscopically and identified to the plant species level to show a linkage between persons and places. More recently, forensic researchers have evaluated the potential for extracting and typing DNA from seeds to further individualize the samples. As a model system, tomato seeds were examined microscopically after different cooking treatments and assessed for the potential to DNA type seeds for variety identification. A sufficient quantity and quality of DNA were recovered from uncooked, digested and undigested tomato seeds for amplified fragment length polymorphism (AFLP) analysis; however, any form of cooking destroyed the seed DNA. A simple microscopic analysis was able to distinguish between a cooked tomato seed versus an uncooked seed. This article is intended to provide an overview of case examples and current techniques for the forensic examination of seeds as plant-derived evidence.

Use of polyvinyl alcohol for chimeric antigen receptor T-cell expansion.

Serum albumin has long been an essential supplement for ex vivo hematopoietic and immune cell cultures. However, serum albumin medium supplements represent a major source of biological contamination in cell cultures and often cause loss of cellular function. As serum albumin exhibits significant batch-to-batch variability, it has also been blamed for causing major issues in experimental reproducibility. We recently discovered the synthetic polymer polyvinyl alcohol (PVA) as an inexpensive, Good Manufacturing Practice-compatible, and biologically inert serum albumin replacement for ex vivo hematopoietic stem cell cultures. Importantly, PVA is free of the biological contaminants that have plagued serum albumin-based media. Here, we describe that PVA can replace serum albumin in a range of blood and immune cell cultures including cell lines, primary leukemia samples, and human T lymphocytes. PVA can even replace human serum in the generation and expansion of functional chimeric antigen receptor (CAR) T cells, offering a potentially safer and more cost-efficient approach for this clinical cell therapy. In summary, PVA represents a chemically defined, biologically inert, and inexpensive alternative to serum albumin for a range of cell cultures in hematology and immunology.

YeastSpotter: accurate and parameter-free web segmentation for microscopy images of yeast cells.

SUMMARY: We introduce YeastSpotter, a web application for the segmentation of yeast microscopy images into single cells. YeastSpotter is user-friendly and generalizable, reducing the computational expertise required for this critical preprocessing step in many image analysis pipelines. AVAILABILITY AND IMPLEMENTATION: YeastSpotter is available at http://yeastspotter.csb.utoronto.ca/. Code is available at https://github.com/alexxijielu/yeast_segmentation. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.