Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq.

A central goal of genetics is to define the relationships between genotypes and phenotypes. High-content phenotypic screens such as Perturb-seq (CRISPR-based screens with single-cell RNA-sequencing readouts) enable massively parallel functional genomic mapping but, to date, have been used at limited scales. Here, we perform genome-scale Perturb-seq targeting all expressed genes with CRISPR interference (CRISPRi) across >2.5 million human cells. We use transcriptional phenotypes to predict the function of poorly characterized genes, uncovering new regulators of ribosome biogenesis (including CCDC86, ZNF236, and SPATA5L1), transcription (C7orf26), and mitochondrial respiration (TMEM242). In addition to assigning gene function, single-cell transcriptional phenotypes allow for in-depth dissection of complex cellular phenomena-from RNA processing to differentiation. We leverage this ability to systematically identify genetic drivers and consequences of aneuploidy and to discover an unanticipated layer of stress-specific regulation of the mitochondrial genome. Our information-rich genotype-phenotype map reveals a multidimensional portrait of gene and cellular function.

Drivers of heterogeneity in synovial fibroblasts in rheumatoid arthritis.

Inflammation of non-barrier immunologically quiescent tissues is associated with a massive influx of blood-borne innate and adaptive immune cells. Cues from the latter are likely to alter and expand activated states of the resident cells. However, local communications between immigrant and resident cell types in human inflammatory disease remain poorly understood. Here, we explored drivers of fibroblast-like synoviocyte (FLS) heterogeneity in inflamed joints of patients with rheumatoid arthritis using paired single-cell RNA and ATAC sequencing, multiplexed imaging and spatial transcriptomics along with in vitro modeling of cell-extrinsic factor signaling. These analyses suggest that local exposures to myeloid and T cell-derived cytokines, TNF, IFN-γ, IL-1ß or lack thereof, drive four distinct FLS states some of which closely resemble fibroblast states in other disease-affected tissues including skin and colon. Our results highlight a role for concurrent, spatially distributed cytokine signaling within the inflamed synovium.

Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens.

Genetic screens help infer gene function in mammalian cells, but it has remained difficult to assay complex phenotypes-such as transcriptional profiles-at scale. Here, we develop Perturb-seq, combining single-cell RNA sequencing (RNA-seq) and clustered regularly interspaced short palindromic repeats (CRISPR)-based perturbations to perform many such assays in a pool. We demonstrate Perturb-seq by analyzing 200,000 cells in immune cells and cell lines, focusing on transcription factors regulating the response of dendritic cells to lipopolysaccharide (LPS). Perturb-seq accurately identifies individual gene targets, gene signatures, and cell states affected by individual perturbations and their genetic interactions. We posit new functions for regulators of differentiation, the anti-viral response, and mitochondrial function during immune activation. By decomposing many high content measurements into the effects of perturbations, their interactions, and diverse cell metadata, Perturb-seq dramatically increases the scope of pooled genomic assays.

A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response.

Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based single-cell RNA-seq with a strategy for barcoding CRISPR-mediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis. Subjecting ∼100 hits to Perturb-seq enabled high-precision functional clustering of genes. Single-cell analyses decoupled the three UPR branches, revealed bifurcated UPR branch activation among cells subject to the same perturbation, and uncovered differential activation of the branches across hits, including an isolated feedback loop between the translocon and IRE1α. These studies provide insight into how the three sensors of ER homeostasis monitor distinct types of stress and highlight the ability of Perturb-seq to dissect complex cellular responses.

Molecular recording of mammalian embryogenesis.

Ontogeny describes the emergence of complex multicellular organisms from single totipotent cells. This field is particularly challenging in mammals, owing to the indeterminate relationship between self-renewal and differentiation, variation in progenitor field sizes, and internal gestation in these animals. Here we present a flexible, high-information, multi-channel molecular recorder with a single-cell readout and apply it as an evolving lineage tracer to assemble mouse cell-fate maps from fertilization through gastrulation. By combining lineage information with single-cell RNA sequencing profiles, we recapitulate canonical developmental relationships between different tissue types and reveal the nearly complete transcriptional convergence of endodermal cells of extra-embryonic and embryonic origins. Finally, we apply our cell-fate maps to estimate the number of embryonic progenitor cells and their degree of asymmetric partitioning during specification. Our approach enables massively parallel, high-resolution recording of lineage and other information in mammalian systems, which will facilitate the construction of a quantitative framework for understanding developmental processes.

Antiretroviral Therapy Use, Viral Detectability and Fear of Onward Transmission Among People Living with HIV in Australia: Changes Between 1997 and 2018.

This paper examines how antiretroviral therapy (ART) use and fears towards the onward transmission of HIV have changed among people living with HIV (PLHIV) in Australia between 1997 and 2018. Participants were recruited as part of the HIV Futures study, a large cross-sectional survey of PLHIV in Australia, in 1997, 2003, 2012 and 2018 (total n = 3889). ART use, viral load detectability, and fear of onward HIV transmission were compared between years. Predictors of onward transmission fear were assessed among the 2018 subsample. While ART use within our sample decreased between 1997 and 2003, it subsequently increased to 97% in 2018. Self-reported viral load undetectability steadily increased over time, up to 88% in 2018. Notably, fewer PLHIV reported being fearful of transmitting HIV in 2018 compared to all other years. Being unfamiliar with the undetectable = untransmissible health movement, and having a detectable or uncertain viral load at last test, were significant predictors of being fearful of onward HIV transmission. Beyond the immediate medical considerations of HIV treatment, these results suggest that the undetectable = untransmissible movement may play a critical role in attenuating burdens experienced by PLHIV in Australia and that such messaging, in tandem with early and consistent ART use, should remain a salient feature of heath messaging among this population.

Development and validation of the Alcoholic Beverage Identification Deep Learning Algorithm version 2 for quantifying alcohol exposure in electronic images.

BACKGROUND: Seeing alcohol in media has been demonstrated to increase alcohol craving, impulsive decision-making, and hazardous drinking. Due to the exponential growth of (social) media use it is important to develop algorithms to quantify alcohol exposure efficiently in electronic images. In this article, we describe the development of an improved version of the Alcoholic Beverage Identification Deep Learning Algorithm (ABIDLA), called ABIDLA2. METHODS: ABIDLA2 was trained on 191,286 images downloaded from Google Image Search results (based on search terms) and Bing Image Search results. In Task-1, ABIDLA2 identified images as containing one of eight beverage categories (beer/cider cup, beer/cider bottle, beer/cider can, wine, champagne, cocktails, whiskey/cognac/brandy, other images). In Task-2, ABIDLA2 made a binary classification between images containing an "alcoholic beverage" or "other". An ablation study was performed to determine which techniques improved algorithm performance. RESULTS: ABIDLA2 was most accurate in identifying Whiskey/Cognac/Brandy (88.1%) followed by Beer/Cider Can (80.5%), Beer/Cider Bottle (78.3%), and Wine (77.8%). Its overall accuracy was 77.0% (Task-1) and 87.7% (Task-2). Even the identification of the least accurate beverage category (Champagne, 64.5%) was more than five times higher than random chance (12.5% = 1/8 categories). The implementation of balanced data sampler to address class skewness and the use of self-training to make use of a large, secondary, weakly labeled dataset particularly improved overall algorithm performance. CONCLUSION: With extended capabilities and a higher accuracy, ABIDLA2 outperforms its predecessor and enables the screening of any kind of electronic media rapidly to estimate the quantity of alcohol exposure. Quantifying alcohol exposure automatically through algorithms like ABIDLA2 is important because viewing images of alcoholic beverages in media tends to increase alcohol consumption and related harms.

Assessing driving-relevant attentional impairment after a multiday drinking session: A two-phase pilot study.

BACKGROUND: The possibility of residual impairment of cognitive performance after multiday drinking sessions is particularly important given the potential for the deleterious effects of fatigue and hangover. This pilot study aimed to devise a methodology to compare sober performance on driving-relevant attentional tasks at the end of a 4-day music festival with performance at varying levels of the breath-alcohol curve. METHODS: Fifty-two participants completed selective and sustained attention tasks at a breath alcohol concentration (BrAC) of 0.00%, 0.05%, and 0.08% following acute dosing in a controlled laboratory setting. A subset of participants (n = 13) were then tested at the conclusion of a 4-day music festival at 0.00% BrAC, with task performance compared with laboratory results. RESULTS: During the laboratory phase, sustained attention was poorer at the 0.05% ascending timepoint only (compared to 0.00% BrAC). During the festival phase, participants made a greater number of errors on the selective attention task predeparture than at 0.00% and 0.05% BrAC in the laboratory. Sustained attention performance was poorer while intoxicated in the laboratory. CONCLUSIONS: Our findings suggest that the absence of blood alcohol acutely may not be indicative of unimpaired cognitive performance and that other factors related to multiday drinking may produce driving-related attentional deficits. The findings reinforce the need to measure attentional performance in real-world drinking contexts despite the methodological complexities of doing so. A larger study is warranted to replicate the findings and should include attentional measures that either are more sensitive to the effects of acute alcohol intoxication than those in our study or are based on a driving simulator.

Health literacy, financial insecurity and health outcomes among people living with HIV in Australia.

It is well documented that lower socio-economic status is associated with poorer health outcomes, while health literacy is considered important for improving health. What is less clear, is the extent to which greater health literacy can improve health outcomes among people for whom poverty or financial insecurity are important barriers to health. The paper presents findings from an Australian survey of people living with HIV (PLHIV) (N = 835) in which we explored the relationship between financial insecurity and health outcomes, looking at the extent to which health literacy mediates this relationship. The study drew on a comprehensive definition of health literacy, measuring participant's confidence to communicate with healthcare providers, navigate the health system and take an active stance in relation to their health. Findings showed that financial insecurity was associated with lower health literacy and poorer self-reported physical and mental health. Health literacy mediated 16.2% of the effect of financial insecurity on physical health scores and 16.6% of the effect of financial insecurity on mental health scores. This suggests that programmes which seek to build health literacy among PLHIV may improve health outcomes among PLHIV who are struggling financially. Health literacy programmes are likely to be effective if they build confidence and resourcefulness among people to engage with health information, decision-making and care.

Lucio phenomenon with concomitant necrotizing fasciitis and acute kidney injury.

Lucio phenomenon is a rare vasculopathy that can occur in patients with Hansen disease, particularly diffuse lepromatous leprosy. It is characterized by retiform purpura and necrotic ulcerations, most commonly affecting the extremities. Diagnosing Lucio phenomenon can be challenging, especially when secondary bacterial infections occur. We report a patient with Lucio phenomenon who presented with acute necrotizing fasciitis of his left upper extremity and a 10-year history of chronic ulcerations. Shortly following admission, he also developed acute kidney injury. The necrotizing fasciitis was treated with prompt surgical debridement and intravenous antibiotics. Biopsy and PCR of a right upper extremity ulcer confirmed the presence of Mycobacterium lepromatosis. Multidrug therapy and prednisone were used to treat the Lucio phenomenon. After initiating treatment, no new lesions developed, kidney function improved, and the patient underwent successful skin graft of his left upper extremity. Although corticosteroid use is controversial, our patient's marked response to multidrug therapy with prednisone highlights the importance of this regimen in severe presentations of Lucio phenomenon. To the best of our knowledge, only two other cases of Lucio phenomenon confirmed to be caused by M. lepromatosis have been reported in living patients (rather than retrospectively identified post-mortem), underscoring the importance of the presented clinical course and treatment regimen.

Stochastic Switching of Cell Fate in Microbes.

Microbes transiently differentiate into distinct, specialized cell types to generate functional diversity and cope with changing environmental conditions. Though alternate programs often entail radically different physiological and morphological states, recent single-cell studies have revealed that these crucial decisions are often left to chance. In these cases, the underlying genetic circuits leverage the intrinsic stochasticity of intracellular chemistry to drive transition between states. Understanding how these circuits transform transient gene expression fluctuations into lasting phenotypic programs will require a combination of quantitative modeling and extensive, time-resolved observation of switching events in single cells. In this article, we survey microbial cell fate decisions demonstrated to involve a random element, describe theoretical frameworks for understanding stochastic switching between states, and highlight recent advances in microfluidics that will enable characterization of key dynamic features of these circuits.

Memory and modularity in cell-fate decision making.

Genetically identical cells sharing an environment can display markedly different phenotypes. It is often unclear how much of this variation derives from chance, external signals, or attempts by individual cells to exert autonomous phenotypic programs. By observing thousands of cells for hundreds of consecutive generations under constant conditions, we dissect the stochastic decision between a solitary, motile state and a chained, sessile state in Bacillus subtilis. We show that the motile state is 'memoryless', exhibiting no autonomous control over the time spent in the state. In contrast, the time spent as connected chains of cells is tightly controlled, enforcing coordination among related cells in the multicellular state. We show that the three-protein regulatory circuit governing the decision is modular, as initiation and maintenance of chaining are genetically separable functions. As stimulation of the same initiating pathway triggers biofilm formation, we argue that autonomous timing allows a trial commitment to multicellularity that external signals could extend.

An epigenetic switch governing daughter cell separation in Bacillus subtilis.

Growing cells of Bacillus subtilis are a bistable mixture of individual motile cells in which genes for daughter cell separation and motility are ON, and chains of sessile cells in which these genes are OFF. How this ON/OFF switch is controlled has been mysterious. Here we report that a complex of the SinR and SlrR proteins binds to and represses genes involved in cell separation and motility. We also report that SinR and SlrR constitute a double-negative feedback loop in which SinR represses the gene for SlrR (slrR), and, by binding to (titrating) SinR, SlrR prevents SinR from repressing slrR. Thus, SlrR indirectly derepresses its own gene, creating a self-reinforcing loop. Finally, we show that, once activated, the loop remains locked in a high SlrR state in which cell separation and motility genes are OFF for extended periods of time. SinR and SlrR constitute an epigenetic switch for controlling genes involved in cell separation and motility.

Constraints on Fluctuations in Sparsely Characterized Biological Systems.

Biochemical processes are inherently stochastic, creating molecular fluctuations in otherwise identical cells. Such "noise" is widespread but has proven difficult to analyze because most systems are sparsely characterized at the single cell level and because nonlinear stochastic models are analytically intractable. Here, we exactly relate average abundances, lifetimes, step sizes, and covariances for any pair of components in complex stochastic reaction systems even when the dynamics of other components are left unspecified. Using basic mathematical inequalities, we then establish bounds for whole classes of systems. These bounds highlight fundamental trade-offs that show how efficient assembly processes must invariably exhibit large fluctuations in subunit levels and how eliminating fluctuations in one cellular component requires creating heterogeneity in another.

Evidence that metabolism and chromosome copy number control mutually exclusive cell fates in Bacillus subtilis.

Bacillus subtilis chooses between matrix production and spore formation, which are both controlled by the regulator Spo0A~P. We report that metabolism and chromosome copy number dictate which fate is adopted. Conditions that favour low Spo0A~P levels promote matrix production, whereas conditions favouring high levels trigger sporulation. Spo0A~P directs the synthesis of SinI, an antirepressor for the SinR repressor of matrix genes. The regulatory region of sinI contains an activator site that Spo0A~P binds strongly and operators that bind Spo0A~P weakly. Evidence shows that low Spo0A~P levels turn sinI ON and high levels turn sinI OFF and instead switch sporulation ON. Cells in which sinI and sinR were transplanted from their normal position near the chromosome replication terminus to positions near the origin and cells that harboured an extra copy of the genes were blocked in matrix production. Thus, matrix gene expression is sensitive to the number of copies of sinI and sinR. Because cells at the start of sporulation have two chromosomes and matrix-producing cells one, chromosome copy number could contribute to cell-fate determination.

Analysis of Contact Allergens in Polyvinyl Chloride Examination Gloves in the United States.

Background: Polyvinyl chloride (PVC) gloves are recommended as a safe alternative for patients with rubber accelerator allergy. However, allergic contact dermatitis to other chemicals in PVC gloves has been reported. Objective: To analyze single-use PVC medical examination gloves in the United States for the presence of potential contact allergens. Methods: Using liquid chromatography-mass spectrometry, 20 unique PVC gloves were analyzed in triplicate for 6 chemicals: benzisothiazolinone, bisphenol A, mono(2-ethylhexyl) maleate, tricresyl phosphate, triphenyl phosphate, and triphenyl phosphite. Results: All 20 PVC gloves contained detectable quantities of benzisothiazolinone (range, 0.001-1.48 parts per million [ppm]), bisphenol A (0.01-0.11 ppm), triphenyl phosphate (0.01-2.11 ppm), and triphenyl phosphite (0.001-0.22 ppm). Eighteen (90%) gloves contained mono(2-ethylhexyl) maleate (0.001-0.14 ppm) and 3 (15%) contained tricresyl phosphate (0.001-0.002 ppm). Conclusions: Known allergens were present in all 20 PVC gloves. However, the detected levels were mostly low and their relationship with sensitization and elicitation thresholds requires further study.

Multiome Perturb-seq unlocks scalable discovery of integrated perturbation effects on the transcriptome and epigenome.

Single-cell CRISPR screens link genetic perturbations to transcriptional states, but high-throughput methods connecting these induced changes to their regulatory foundations are limited. Here we introduce Multiome Perturb-seq, extending single-cell CRISPR screens to simultaneously measure perturbation-induced changes in gene expression and chromatin accessibility. We apply Multiome Perturb-seq in a CRISPRi screen of 13 chromatin remodelers in human RPE-1 cells, achieving efficient assignment of sgRNA identities to single nuclei via an improved method for capturing barcode transcripts from nuclear RNA. We organize expression and accessibility measurements into coherent programs describing the integrated effects of perturbations on cell state, finding that ARID1A and SUZ12 knockdowns induce programs enriched for developmental features. Pseudotime analysis of perturbations connects accessibility changes to changes in gene expression, highlighting the value of multimodal profiling. Overall, our method provides a scalable and simply implemented system to dissect the regulatory logic underpinning cell state.

A Survey of Skin Reactions Associated With Acrylic Nail Cosmetics, With a Focus on Home Kits: Is There a Need for Regulation?

Background: Sensitization to (meth)acrylates, the most common nail cosmetic allergens, is rising. In recent years, home acrylic nail kits have become easily available. Objective: To investigate the characteristics of individuals reporting skin reactions associated with acrylic nail cosmetics, particularly home kits. Methods: Cross-sectional survey of Facebook nail allergy support groups. Inclusion criteria were self-reported skin reactions associated with acrylic nails and age ≥18 years. Results: There were 199 respondents, nearly all female (99%), mostly white (83%), and 25-54 years old (83%). Seventy-eight percent reported using home acrylic kits, more than half for the first time during COVID-19. They predominantly learned about kits through social media (68%) and received training through websites/online videos (74%). Most home users (83%) first developed skin reactions after starting to use home kits. Compared with nonhome users, significantly more home users reported skin reaction onset within 1 year of use, as well as nail damage (P < 0.05). Conclusions: Among online nail allergy support group members, home acrylic nail kit use was common and associated with earlier development of skin reactions and more frequent nail damage than professional acrylic manicures. These findings raise important questions about the need to regulate home acrylic nail kits.

Vesiculopustular drug reaction with eosinophilia and systemic symptoms induced by levetiracetam.

Drug reaction with eosinophilia and systemic symptoms (DRESS) is a severe cutaneous adverse reaction characterised by fever, lymphadenopathy, morbilliform rash, haematologic abnormalities, and multiorgan involvement. Herein, we describe a 32-year-old female presenting with a 9-day history of facial oedema, cervical and inguinal lymphadenopathy, and a pruritic rash comprised of vesicles and pustules on her face, trunk, and extremities. Her only medications were valproate, which she had been taking for several years, and levetiracetam, which was initiated 41 days prior to rash onset. On the 16th day of her rash, she was diagnosed with DRESS induced by levetiracetam (Registry of Severe Cutaneous Adverse Reactions: 5). At this point, her absolute eosinophil count was 0.9 × 109 cells/L and aspartate and alanine transaminase levels were 357 and 339 U/L, respectively. Pustules with a morbilliform rash may occur in up to 30% of DRESS cases. In rarer instances, as in our patient, DRESS can present with isolated pustules and vesicles. Similarly, although rare, DRESS can be induced by levetiracetam.