Abstract
Background
Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold-standard nucleic acid tests are hampered by supply shortages in critical ...reagents including nasal swabs, RNA extraction kits, personal protective equipment, instrumentation, and labor.
Methods
To overcome these challenges, we developed a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe the optimization of saliva pretreatment protocols to enable analytically sensitive viral detection by RT-LAMP. We optimized the RT-LAMP reaction conditions and implemented high-throughput unbiased methods for assay interpretation. We tested whether saliva pretreatment could also enable viral detection by conventional reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Finally, we validated these assays on clinical samples.
Results
The optimized saliva pretreatment protocol enabled analytically sensitive extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP or RT-qPCR. In simulated samples, the optimized RT-LAMP assay had a limit of detection of 59 (95% confidence interval: 44–104) particle copies per reaction. We highlighted the flexibility of LAMP assay implementation using 3 readouts: naked-eye colorimetry, spectrophotometry, and real-time fluorescence. In a set of 30 clinical saliva samples, colorimetric RT-LAMP and RT-qPCR assays performed directly on pretreated saliva samples without RNA extraction had accuracies greater than 90%.
Conclusions
Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP is a simple, sensitive, and cost-effective approach with broad potential to expand diagnostic testing for the virus causing COVID-19.
Molecular and behavioral responses to opioids are thought to be primarily mediated by neurons, although there is accumulating evidence that other cell types play a prominent role in drug addiction. ...To investigate cell-type-specific opioid responses, we performed single-cell RNA sequencing (scRNA-seq) of the nucleus accumbens of mice following acute morphine treatment. Differential expression analysis uncovered unique morphine-dependent transcriptional responses by oligodendrocytes and astrocytes. We examined the expression of selected genes, including Cdkn1a and Sgk1, by FISH, confirming their induction by morphine in oligodendrocytes. Further analysis using RNA-seq of FACS-purified oligodendrocytes revealed a large cohort of morphine-regulated genes. The affected genes are enriched for roles in cellular pathways intimately linked to oligodendrocyte maturation and myelination, including the unfolded protein response. Altogether, our data illuminate the morphine-dependent transcriptional response by oligodendrocytes and offer mechanistic insights into myelination defects associated with opioid abuse.
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•Single-cell RNA-seq uncovers cell-type-specific responses to morphine in mice•Among glia, the most robust transcriptional response are in oligodendrocytes•Oligodendrocyte- and neuron-specific responses to morphine are validated by FISH•Bulk RNA-seq of purified oligodendrocytes reveals ∼1,000 morphine-regulated genes
Avey et al. use single-cell RNA-seq to uncover cell-type-specific responses to morphine in the brains of mice, revealing a unique and robust response by oligodendrocytes, which they further validate by multiple approaches. These analyses offer insights into the molecular mechanisms underlying oligodendrocyte dysfunction in the context of opioid abuse.
In order to facilitate understanding of pigment cell biology, we developed a method to concomitantly purify melanocytes, iridophores, and retinal pigmented epithelium from zebrafish, and analyzed ...their transcriptomes. Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co-enrichment in melanocytes and retinal pigmented epithelium, as well as in melanocytes and iridophores. We found 214 genes co-enriched in melanocytes and retinal pigmented epithelium, indicating the shared functions of melanin-producing cells. We found 62 genes significantly co-enriched in melanocytes and iridophores, illustrative of their shared developmental origins from the neural crest. This is also the first analysis of the iridophore transcriptome. Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine-based reflective pigment. We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate-limiting substrate for purine synthesis, phosphoribosyl pyrophosphate, thus constituting a guanine cycle. The purification procedure and expression analysis described here, along with the accompanying transcriptome-wide expression data, provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types, and will be a useful resource for further studies of pigment cell biology.
The overwhelming success of exome- and genome-wide association studies in discovering thousands of disease-associated genes necessitates developing novel high-throughput functional genomics ...approaches to elucidate the molecular mechanisms of these genes. Here, we have coupled multiplexed repression of neurodevelopmental disease-associated genes to single-cell transcriptional profiling in differentiating human neurons to rapidly assay the functions of multiple genes in a disease-relevant context, assess potentially convergent mechanisms, and prioritize genes for specific functional assays. For a set of 13 autism spectrum disorder (ASD)-associated genes, we show that this approach generated important mechanistic insights, revealing two functionally convergent modules of ASD genes: one that delays neuron differentiation and one that accelerates it. Five genes that delay neuron differentiation (
,
,
,
, and
) mechanistically converge, as they all dysregulate genes involved in cell-cycle control and progenitor cell proliferation. Live-cell imaging after individual ASD-gene repression validated this functional module, confirming that these genes reduce neural progenitor cell proliferation and neurite growth. Finally, these functionally convergent ASD gene modules predicted shared clinical phenotypes among individuals with mutations in these genes. Altogether, these results show the utility of a novel and simple approach for the rapid functional elucidation of neurodevelopmental disease-associated genes.
Objective
To define the genetic landscape of amyotrophic lateral sclerosis (ALS) and assess the contribution of possible oligogenic inheritance, we aimed to comprehensively sequence 17 known ALS ...genes in 391 ALS patients from the United States.
Methods
Targeted pooled‐sample sequencing was used to identify variants in 17 ALS genes. Fragment size analysis was used to define ATXN2 and C9ORF72 expansion sizes. Genotype–phenotype correlations were made with individual variants and total burden of variants. Rare variant associations for risk of ALS were investigated at both the single variant and gene level.
Results
A total of 64.3% of familial and 27.8% of sporadic subjects carried potentially pathogenic novel or rare coding variants identified by sequencing or an expanded repeat in C9ORF72 or ATXN2; 3.8% of subjects had variants in >1 ALS gene, and these individuals had disease onset 10 years earlier (p = 0.0046) than subjects with variants in a single gene. The number of potentially pathogenic coding variants did not influence disease duration or site of onset.
Interpretation
Rare and potentially pathogenic variants in known ALS genes are present in >25% of apparently sporadic and 64% of familial patients, significantly higher than previous reports using less comprehensive sequencing approaches. A significant number of subjects carried variants in >1 gene, which influenced the age of symptom onset and supports oligogenic inheritance as relevant to disease pathogenesis. ANN NEUROL 2015;77:100–113
Abstract
Gene expression changes are orchestrated by transcription factors (TFs), which bind to DNA to regulate gene expression. It remains surprisingly difficult to predict basic features of the ...transcriptional process, including in vivo TF occupancy. Existing thermodynamic models of TF function are often not concordant with experimental measurements, suggesting undiscovered biology. Here, we analyzed one of the most well-studied TFs, the yeast zinc cluster Gal4, constructed a Shea–Ackers thermodynamic model to describe its binding, and compared the results of this model to experimentally measured Gal4p binding in vivo. We found that at many promoters, the model predicted no Gal4p binding, yet substantial binding was observed. These outlier promoters lacked canonical binding motifs, and subsequent investigation revealed Gal4p binds unexpectedly to DNA sequences with high densities of its half site (CGG). We confirmed this novel mode of binding through multiple experimental and computational paradigms; we also found most other zinc cluster TFs we tested frequently utilize this binding mode, at 27% of their targets on average. Together, these results demonstrate a novel mode of binding where zinc clusters, the largest class of TFs in yeast, bind DNA sequences with high densities of half sites.
Graphical Abstract
Graphical Abstract
A novel mode of non-canonical binding in budding yeast.
Pathogenic mutations in APP, PSEN1, PSEN2, MAPT and GRN have previously been linked to familial early onset forms of dementia. Mutation screening in these genes has been performed in either very ...small series or in single families with late onset AD (LOAD). Similarly, studies in single families have reported mutations in MAPT and GRN associated with clinical AD but no systematic screen of a large dataset has been performed to determine how frequently this occurs. We report sequence data for 439 probands from late-onset AD families with a history of four or more affected individuals. Sixty sequenced individuals (13.7%) carried a novel or pathogenic mutation. Eight pathogenic variants, (one each in APP and MAPT, two in PSEN1 and four in GRN) three of which are novel, were found in 14 samples. Thirteen additional variants, present in 23 families, did not segregate with disease, but the frequency of these variants is higher in AD cases than controls, indicating that these variants may also modify risk for disease. The frequency of rare variants in these genes in this series is significantly higher than in the 1,000 genome project (p = 5.09 × 10⁻⁵; OR = 2.21; 95%CI = 1.49-3.28) or an unselected population of 12,481 samples (p = 6.82 × 10⁻⁵; OR = 2.19; 95%CI = 1.347-3.26). Rare coding variants in APP, PSEN1 and PSEN2, increase risk for or cause late onset AD. The presence of variants in these genes in LOAD and early-onset AD demonstrates that factors other than the mutation can impact the age at onset and penetrance of at least some variants associated with AD. MAPT and GRN mutations can be found in clinical series of AD most likely due to misdiagnosis. This study clearly demonstrates that rare variants in these genes could explain an important proportion of genetic heritability of AD, which is not detected by GWAS.
Abstract
Summary
Transposon calling cards is a genomic assay for identifying transcription factor binding sites in both bulk and single cell experiments. Here, we describe the qBED format, an open, ...text-based standard for encoding and analyzing calling card data. In parallel, we introduce the qBED track on the WashU Epigenome Browser, a novel visualization that enables researchers to inspect calling card data in their genomic context. Finally, through examples, we demonstrate that qBED files can be used to visualize non-calling card datasets, such as Combined Annotation-Dependent Depletion scores and GWAS/eQTL hits, and thus may have broad utility to the genomics community.
Availability and implementation
The qBED track is available on the WashU Epigenome Browser (http://epigenomegateway.wustl.edu/browser), beginning with version 46. Source code for the WashU Epigenome Browser with qBED support is available on GitHub (http://github.com/arnavm/eg-react and http://github.com/lidaof/eg-react). A complete definition of the qBED format is available as part of the WashU Epigenome Browser documentation (https://eg.readthedocs.io/en/latest/tracks.html#qbed-track). We have also released a tutorial on how to upload qBED data to the browser (http://dx.doi.org/10.17504/protocols.io.bca8ishw).
Epithelial-to-mesenchymal transition (EMT) is a process by which cells lose their epithelial characteristics and gain mesenchymal phenotypes. In cancer, EMT is thought to drive tumor invasion and ...metastasis. Recent efforts to understand EMT biology have uncovered that cells undergoing EMT attain a spectrum of intermediate "hybrid E/M" states, which exist along an epithelial-mesenchymal continuum. Here, we summarize recent studies characterizing the epigenetic drivers of hybrid E/M states. We focus on the histone-modification writers, erasers, and readers that assist or oppose the canonical hybrid E/M transcription factors that modulate hybrid E/M state transitions. We also examine the role of chromatin remodelers and DNA methylation in hybrid E/M states. Finally, we highlight the challenges of targeting hybrid E/M pharmacologically, and we propose future directions that might reveal the specific and targetable mechanisms by which hybrid E/M drives metastasis in patients.
Human leukocyte antigen (HLA) typing at the allelic level can in theory be achieved using whole exome sequencing (exome-seq) data with no added cost but has been hindered by its computational ...challenge. We developed ATHLATES, a program that applies assembly, allele identification and allelic pair inference to short read sequences, and applied it to data from Illumina platforms. In 15 data sets with adequate coverage for HLA-A, -B, -C, -DRB1 and -DQB1 genes, ATHLATES correctly reported 74 out of 75 allelic pairs with an overall concordance rate of 99% compared with conventional typing. This novel approach should be broadly applicable to research and clinical laboratories.
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