Visible-Light-Driven Mentha spicata L.-Mediated Ag-Doped Bi2Zr2O7 Nanocomposite for Enhanced Degradation of Organic Pollutants, Electrochemical Sensing, and Antibacterial Applications.

Novel visible-light-driven Ag (X)-doped Bi2Zr2O7 (BZO) nanocomposites in pudina (P) extract (Mentha spicata L.), X-1, 3, 5, 7, and 9 mol %, were synthesized by the one-pot greener solution combustion method. The as-synthesized nanocomposite materials were characterized by using various spectral [X-ray diffraction (XRD), Fourier transform infrared, UV-visible, UV- diffuse reflectance spectra, X-ray photoelectron spectroscopy], electrochemical (cyclic voltammetry, electrochemical impedance spectroscopy), and analytical (scanning electron microscopy-energy-dispersive X-ray spectroscopy, transmission electron microscopy, Brunauer-Emmett-Teller) techniques. The average particle size of the nanocomposite material was found to be between 14.8 and 39.2 nm by XRD. The well-characterized Ag-doped BZOP nanocomposite materials exhibited enhanced photocatalytic degradation activity toward hazardous dyes such as methylene blue (MB) and rose bengal (RB) under visible light irradiation ranges between 400 and 800 nm due to their low energy band gap. As a result, 7 mol % of Ag-doped BZOP nanocomposite material exhibited excellent photodegradation activity against MB (D.E. = 98.7%) and RB (D.E. = 99.3%) as compared to other Ag-doped BZOP nanocomposite materials and pure BZOP nanocomposite, respectively, due to enhanced semiconducting and optical behaviors, high binding energy, and mechanical and thermal stabilities. The Ag-doped BZOP nanocomposite material-based electrochemical sensor showed good sensing ability toward the determination of lead nitrate and dextrose with the lowest limit of detection (LOD) of 18 µM and 12 µM, respectively. Furthermore, as a result of the initial antibacterial screening study, the Ag-doped BZOP nanocomposite material was found to be more effective against Gram-negative bacteria (Escherichia coli) as compared to Gram-positive (Staphylococcus aureus) bacteria. The scavenger study reveals that radicals such as O2•- and •OH are responsible for MB and RB mineralization. TOC removal percentages were found to be 96.8 and 98.5% for MB and RB dyes, and experimental data reveal that the Ag-doped BZOP enhances the radical (O2•- and •OH) formation and MB and RB degradation under visible-light irradiation.

Anti-GD2 CAR-NKT cells in relapsed or refractory neuroblastoma: updated phase 1 trial interim results.

Vα24-invariant natural killer T cells (NKTs) have anti-tumor properties that can be enhanced by chimeric antigen receptors (CARs). Here we report updated interim results from the first-in-human phase 1 evaluation of autologous NKTs co-expressing a GD2-specific CAR with interleukin 15 (IL15) (GD2-CAR.15) in 12 children with neuroblastoma (NB). The primary objectives were safety and determination of maximum tolerated dose (MTD). The anti-tumor activity of GD2-CAR.15 NKTs was assessed as a secondary objective. Immune response evaluation was an additional objective. No dose-limiting toxicities occurred; one patient experienced grade 2 cytokine release syndrome that was resolved by tocilizumab. The MTD was not reached. The objective response rate was 25% (3/12), including two partial responses and one complete response. The frequency of CD62L+NKTs in products correlated with CAR-NKT expansion in patients and was higher in responders (n = 5; objective response or stable disease with reduction in tumor burden) than non-responders (n = 7). BTG1 (BTG anti-proliferation factor 1) expression was upregulated in peripheral GD2-CAR.15 NKTs and is a key driver of hyporesponsiveness in exhausted NKT and T cells. GD2-CAR.15 NKTs with BTG1 knockdown eliminated metastatic NB in a mouse model. We conclude that GD2-CAR.15 NKTs are safe and can mediate objective responses in patients with NB. Additionally, their anti-tumor activity may be enhanced by targeting BTG1. ClinicalTrials.gov registration: NCT03294954 .

Green-engineered synthesis of Bi2Zr2O7 NPs: excellent performance on electrochemical sensor and sunlight-driven photocatalytic studies.

In this rapid growing eco-friendly research world, synthesis of non-toxic, highly effective photocatalyst for potential applications is necessary. Herein, a strong ability Bi2Zr2O7 nanoparticle (BZO NP) with pyrochlore structure was fabricated by solution combustion synthesis using green (Mentha spicata) and chemical (Glycine) fuels. The X-ray diffraction analysis confirms the formation of pure phase for synthesized BZO NP using pudina extract (BZOP NP) compared to BZO NP using Glycine fuel (BZOG NP). The lower energy band gap of synthesized BZOP NP was observed than BZOG NP and its values were found to be 2.26 and 2.49 eV measured by UV-visible absorbance spectral technique. The morphological analysis of pores and voids formation as examined by scanning electron microscopy (SEM) technique. The synthesized BZOP NP shows excellent photocatalytic activity for degradation of three different dyes under sunlight irradiation for about 150 min with 97.9% for Rose Bengal (RB) dye with lower charge transfer resistance (Rct) value. For the very first time, the synthesized NPs can be utilized as supercapacitor with good specific capacitance (SPCcv) value of 14.3 F/g and SPCGD (12.5 F/g) for BZOP compared to BZOG indicating pseudocapacitance nature. The synthesized nanoparticles (NPs) can sense lead nitrate and dextrose at concentration 1-5 mM in the potential range of - 1.0 to + 1.0 V. Accordingly, the reduction potential peak at - 0.25 V and oxidation potential peak found at - 0.82 V confirms the presence of lead content and presence of additional potential peaks at - 0.37 V and - 0.71 V for detection of dextrose biochemical. Recyclability experiment showed the retainment of photocatalytic activity up to five cycles indicating the photostability.

Single-cell RNA-seq reveals cell type-specific molecular and genetic associations to lupus.

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease. Knowledge of circulating immune cell types and states associated with SLE remains incomplete. We profiled more than 1.2 million peripheral blood mononuclear cells (162 cases, 99 controls) with multiplexed single-cell RNA sequencing (mux-seq). Cases exhibited elevated expression of type 1 interferon-stimulated genes (ISGs) in monocytes, reduction of naïve CD4+ T cells that correlated with monocyte ISG expression, and expansion of repertoire-restricted cytotoxic GZMH+ CD8+ T cells. Cell type-specific expression features predicted case-control status and stratified patients into two molecular subtypes. We integrated dense genotyping data to map cell type-specific cis-expression quantitative trait loci and to link SLE-associated variants to cell type-specific expression. These results demonstrate mux-seq as a systematic approach to characterize cellular composition, identify transcriptional signatures, and annotate genetic variants associated with SLE.

CloudPred: Predicting Patient Phenotypes From Single-cell RNA-seq.

Single-cell RNA sequencing (scRNA-seq) has the potential to provide powerful, high-resolution signatures to inform disease prognosis and precision medicine. This paper takes an important first step towards this goal by developing an interpretable machine learning algorithm, CloudPred, to predict individuals' disease phenotypes from their scRNA-seq data. Predicting phenotype from scRNA-seq is challenging for standard machine learning methods-the number of cells measured can vary by orders of magnitude across individuals and the cell populations are also highly heterogeneous. Typical analysis creates pseudo-bulk samples which are biased toward prior annotations and also lose the single cell resolution. CloudPred addresses these challenges via a novel end-to-end differentiable learning algorithm which is coupled with a biologically informed mixture of cell types model. CloudPred automatically infers the cell subpopulation that are salient for the phenotype without prior annotations. We developed a systematic simulation platform to evaluate the performance of CloudPred and several alternative methods we propose, and find that CloudPred outperforms the alternative methods across several settings. We further validated CloudPred on a real scRNA-seq dataset of 142 lupus patients and controls. CloudPred achieves AUROC of 0.98 while identifying a specific subpopulation of CD4 T cells whose presence is highly indicative of lupus. CloudPred is a powerful new framework to predict clinical phenotypes from scRNA-seq data and to identify relevant cells.

Optimized design of single-cell RNA sequencing experiments for cell-type-specific eQTL analysis.

Single-cell RNA-sequencing (scRNA-Seq) is a compelling approach to directly and simultaneously measure cellular composition and state, which can otherwise only be estimated by applying deconvolution methods to bulk RNA-Seq estimates. However, it has not yet become a widely used tool in population-scale analyses, due to its prohibitively high cost. Here we show that given the same budget, the statistical power of cell-type-specific expression quantitative trait loci (eQTL) mapping can be increased through low-coverage per-cell sequencing of more samples rather than high-coverage sequencing of fewer samples. We use simulations starting from one of the largest available real single-cell RNA-Seq data from 120 individuals to also show that multiple experimental designs with different numbers of samples, cells per sample and reads per cell could have similar statistical power, and choosing an appropriate design can yield large cost savings especially when multiplexed workflows are considered. Finally, we provide a practical approach on selecting cost-effective designs for maximizing cell-type-specific eQTL power which is available in the form of a web tool.

Author Correction: Multiplexed droplet single-cell RNA-sequencing using natural genetic variation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

GBAT: a gene-based association test for robust detection of trans-gene regulation.

The observation that disease-associated genetic variants typically reside outside of exons has inspired widespread investigation into the genetic basis of transcriptional regulation. While associations between the mRNA abundance of a gene and its proximal SNPs (cis-eQTLs) are now readily identified, identification of high-quality distal associations (trans-eQTLs) has been limited by a heavy multiple testing burden and the proneness to false-positive signals. To address these issues, we develop GBAT, a powerful gene-based pipeline that allows robust detection of high-quality trans-gene regulation signal.

Functional interpretation of single cell similarity maps.

We present Vision, a tool for annotating the sources of variation in single cell RNA-seq data in an automated and scalable manner. Vision operates directly on the manifold of cell-cell similarity and employs a flexible annotation approach that can operate either with or without preconceived stratification of the cells into groups or along a continuum. We demonstrate the utility of Vision in several case studies and show that it can derive important sources of cellular variation and link them to experimental meta-data even with relatively homogeneous sets of cells. Vision produces an interactive, low latency and feature rich web-based report that can be easily shared among researchers, thus facilitating data dissemination and collaboration.

Genetic and environmental perturbations lead to regulatory decoherence.

Correlation among traits is a fundamental feature of biological systems that remains difficult to study. To address this problem, we developed a flexible approach that allows us to identify factors associated with inter-individual variation in correlation. We use data from three human cohorts to study the effects of genetic and environmental variation on correlations among mRNA transcripts and among NMR metabolites. We first show that environmental exposures (infection and disease) lead to a systematic loss of correlation, which we define as 'decoherence'. Using longitudinal data, we show that decoherent metabolites are better predictors of whether someone will develop metabolic syndrome than metabolites commonly used as biomarkers of this disease. Finally, we demonstrate that correlation itself is under genetic control by mapping hundreds of 'correlation quantitative trait loci (QTLs)'. Together, this work furthers our understanding of how and why coordinated biological processes break down, and points to a potential role for decoherence in disease. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Genetic analysis of isoform usage in the human anti-viral response reveals influenza-specific regulation of ERAP2 transcripts under balancing selection.

While genetic variants are known to be associated with overall gene abundance in stimulated immune cells, less is known about their effects on alternative isoform usage. By analyzing RNA-seq profiles of monocyte-derived dendritic cells from 243 individuals, we uncovered thousands of unannotated isoforms synthesized in response to influenza infection and type 1 interferon stimulation. We identified more than a thousand quantitative trait loci (QTLs) associated with alternate isoform usage (isoQTLs), many of which are independent of expression QTLs (eQTLs) for the same gene. Compared with eQTLs, isoQTLs are enriched for splice sites and untranslated regions, but depleted of sequences upstream of annotated transcription start sites. Both eQTLs and isoQTLs explain a significant proportion of the disease heritability attributed to common genetic variants. At the ERAP2 locus, we shed light on the function of the gene and how two frequent, highly differentiated haplotypes with intermediate frequencies could be maintained by balancing selection. At baseline and following type 1 interferon stimulation, the major haplotype is associated with low ERAP2 expression caused by nonsense-mediated decay, while the minor haplotype, known to increase Crohn's disease risk, is associated with high ERAP2 expression. In response to influenza infection, we found two uncharacterized isoforms expressed from the major haplotype, likely the result of multiple perfectly linked variants affecting the transcription and splicing at the locus. Thus, genetic variants at a single locus could modulate independent gene regulatory processes in innate immune responses and, in the case of ERAP2, may confer a historical fitness advantage in response to virus.

Lineage dynamics of murine pancreatic development at single-cell resolution.

Organogenesis requires the complex interactions of multiple cell lineages that coordinate their expansion, differentiation, and maturation over time. Here, we profile the cell types within the epithelial and mesenchymal compartments of the murine pancreas across developmental time using a combination of single-cell RNA sequencing, immunofluorescence, in situ hybridization, and genetic lineage tracing. We identify previously underappreciated cellular heterogeneity of the developing mesenchyme and reconstruct potential lineage relationships among the pancreatic mesothelium and mesenchymal cell types. Within the epithelium, we find a previously undescribed endocrine progenitor population, as well as an analogous population in both human fetal tissue and human embryonic stem cells differentiating toward a pancreatic beta cell fate. Further, we identify candidate transcriptional regulators along the differentiation trajectory of this population toward the alpha or beta cell lineages. This work establishes a roadmap of pancreatic development and demonstrates the broad utility of this approach for understanding lineage dynamics in developing organs.

Genetic determinants of co-accessible chromatin regions in activated T cells across humans.

Over 90% of genetic variants associated with complex human traits map to non-coding regions, but little is understood about how they modulate gene regulation in health and disease. One possible mechanism is that genetic variants affect the activity of one or more cis-regulatory elements leading to gene expression variation in specific cell types. To identify such cases, we analyzed ATAC-seq and RNA-seq profiles from stimulated primary CD4+ T cells in up to 105 healthy donors. We found that regions of accessible chromatin (ATAC-peaks) are co-accessible at kilobase and megabase resolution, consistent with the three-dimensional chromatin organization measured by in situ Hi-C in T cells. Fifteen percent of genetic variants located within ATAC-peaks affected the accessibility of the corresponding peak (local-ATAC-QTLs). Local-ATAC-QTLs have the largest effects on co-accessible peaks, are associated with gene expression and are enriched for autoimmune disease variants. Our results provide insights into how natural genetic variants modulate cis-regulatory elements, in isolation or in concert, to influence gene expression.

Author Correction: Discovery of stimulation-responsive immune enhancers with CRISPR activation.

In this Letter, analysis of steady-state regulatory T (Treg) cell percentages from Il2ra enhancer deletion (EDEL) and wild-type (WT) mice revealed no differences between them (Extended Data Fig. 9d). This analysis included two mice whose genotypes were incorrectly assigned. Even after correction of the genotypes, no significant differences in Treg cell percentages were seen when data across experimental cohorts were averaged (as was done in Extended Data Fig. 9d). However, if we normalize the corrected data to account for variation among experimental cohorts, a subtle decrease in EDEL Treg cell percentages is revealed and, using the corrected and normalized data, we have redrawn Extended Data Fig. 9d in Supplementary Fig. 1. The Supplementary Information to this Amendment contains the corrected and reanalysed Extended Data Fig. 9d. The sentence "This enhancer deletion (EDEL) strain also had no obvious T cell phenotypes at steady state (Extended Data Fig. 9)." should read: "This enhancer deletion (EDEL) strain had a small decrease in the percentage of Treg cells (Extended Data Fig. 9).". This error does not affect any of the main figures in the Letter or the data from mice with the human autoimmune-associated single nucleotide polymorphism (SNP) knocked in or with a 12-base-pair deletion at the site (12DEL). In addition, we stated in the Methods that we observed consistent immunophenotypes of EDEL mice across three founders, but in fact, we observed consistent phenotypes in mice from two founders. This does not change any of our conclusions and the original Letter has not been corrected.

Multiplexed droplet single-cell RNA-sequencing using natural genetic variation.

Droplet single-cell RNA-sequencing (dscRNA-seq) has enabled rapid, massively parallel profiling of transcriptomes. However, assessing differential expression across multiple individuals has been hampered by inefficient sample processing and technical batch effects. Here we describe a computational tool, demuxlet, that harnesses natural genetic variation to determine the sample identity of each droplet containing a single cell (singlet) and detect droplets containing two cells (doublets). These capabilities enable multiplexed dscRNA-seq experiments in which cells from unrelated individuals are pooled and captured at higher throughput than in standard workflows. Using simulated data, we show that 50 single-nucleotide polymorphisms (SNPs) per cell are sufficient to assign 97% of singlets and identify 92% of doublets in pools of up to 64 individuals. Given genotyping data for each of eight pooled samples, demuxlet correctly recovers the sample identity of >99% of singlets and identifies doublets at rates consistent with previous estimates. We apply demuxlet to assess cell-type-specific changes in gene expression in 8 pooled lupus patient samples treated with interferon (IFN)-ß and perform eQTL analysis on 23 pooled samples.

Discovery of stimulation-responsive immune enhancers with CRISPR activation.

The majority of genetic variants associated with common human diseases map to enhancers, non-coding elements that shape cell-type-specific transcriptional programs and responses to extracellular cues. Systematic mapping of functional enhancers and their biological contexts is required to understand the mechanisms by which variation in non-coding genetic sequences contributes to disease. Functional enhancers can be mapped by genomic sequence disruption, but this approach is limited to the subset of enhancers that are necessary in the particular cellular context being studied. We hypothesized that recruitment of a strong transcriptional activator to an enhancer would be sufficient to drive target gene expression, even if that enhancer was not currently active in the assayed cells. Here we describe a discovery platform that can identify stimulus-responsive enhancers for a target gene independent of stimulus exposure. We used tiled CRISPR activation (CRISPRa) to synthetically recruit a transcriptional activator to sites across large genomic regions (more than 100 kilobases) surrounding two key autoimmunity risk loci, CD69 and IL2RA. We identified several CRISPRa-responsive elements with chromatin features of stimulus-responsive enhancers, including an IL2RA enhancer that harbours an autoimmunity risk variant. Using engineered mouse models, we found that sequence perturbation of the disease-associated Il2ra enhancer did not entirely block Il2ra expression, but rather delayed the timing of gene activation in response to specific extracellular signals. Enhancer deletion skewed polarization of naive T cells towards a pro-inflammatory T helper (TH17) cell state and away from a regulatory T cell state. This integrated approach identifies functional enhancers and reveals how non-coding variation associated with human immune dysfunction alters context-specific gene programs.

Determination of ubiquitin fitness landscapes under different chemical stresses in a classroom setting.

Ubiquitin is essential for eukaryotic life and varies in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies indicate that ubiquitin is highly tolerant to single mutations. We hypothesized that this tolerance would be reduced by chemically induced physiologic perturbations. To test this hypothesis, a class of first year UCSF graduate students employed deep mutational scanning to determine the fitness landscape of all possible single residue mutations in the presence of five different small molecule perturbations. These perturbations uncover 'shared sensitized positions' localized to areas around the hydrophobic patch and the C-terminus. In addition, we identified perturbation specific effects such as a sensitization of His68 in HU and a tolerance to mutation at Lys63 in DTT. Our data show how chemical stresses can reduce buffering effects in the ubiquitin proteasome system. Finally, this study demonstrates the potential of lab-based interdisciplinary graduate curriculum.

Generation of knock-in primary human T cells using Cas9 ribonucleoproteins.

T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently "knock out" genes and "knock in" targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4(+) T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs). Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ∼40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 (PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ∼20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.

The landscape of genomic imprinting across diverse adult human tissues.

Genomic imprinting is an important regulatory mechanism that silences one of the parental copies of a gene. To systematically characterize this phenomenon, we analyze tissue specificity of imprinting from allelic expression data in 1582 primary tissue samples from 178 individuals from the Genotype-Tissue Expression (GTEx) project. We characterize imprinting in 42 genes, including both novel and previously identified genes. Tissue specificity of imprinting is widespread, and gender-specific effects are revealed in a small number of genes in muscle with stronger imprinting in males. IGF2 shows maternal expression in the brain instead of the canonical paternal expression elsewhere. Imprinting appears to have only a subtle impact on tissue-specific expression levels, with genes lacking a systematic expression difference between tissues with imprinted and biallelic expression. In summary, our systematic characterization of imprinting in adult tissues highlights variation in imprinting between genes, individuals, and tissues.