The hypothalamus controls essential social behaviors and homeostatic functions. However, the cellular architecture of hypothalamic nuclei, including the molecular identity, spatial organization, and function of distinct cell types, is poorly understood. Here, we developed an imaging-based in situ cell type identification and mapping method and combined it with single-cell RNA-sequencing to create a molecularly annotated and spatially resolved cell atlas of the mouse hypothalamic preoptic region. We profiled ~1 million cells, identified ~70 neuronal populations characterized by distinct neuromodulatory signatures and spatial organizations, and defined specific neuronal populations activated during social behaviors in male and female mice, providing a high-resolution framework for mechanistic investigation of behavior circuits. The approach described opens a new avenue for the construction of cell atlases in diverse tissues and organisms.

Image-based approaches to single-cell transcriptomics, in which RNA species are identified and counted in situ via imaging, have emerged as a powerful complement to single-cell methods based on RNA sequencing of dissociated cells. These image-based approaches naturally preserve the native spatial context of RNAs within a cell and the organization of cells within tissue, which are important for addressing many biological questions. However, the throughput of these imagebased approaches is relatively low. Here we report advances that lead to a drastic increase in the measurement throughput of multiplexed error-robust fluorescence in situ hybridization (MERFISH), an imagebased approach to single-cell transcriptomics. In MERFISH, RNAs are identified via a combinatorial labeling approach that encodes RNA species with error-robust barcodes followed by sequential rounds of single-molecule fluorescence in situ hybridization (smFISH) to read out these barcodes. Here we increase the throughput of MERFISH by two orders of magnitude through a combination of improvements, including using chemical cleavage instead of photobleaching to remove fluorescent signals between consecutive rounds of smFISH imaging, increasing the imaging field of view, and using multicolor imaging. With these improvements, we performed RNA profiling in more than 100,000 human cells, with as many as 40,000 cells measured in a single 18-h measurement. This throughput should substantially extend the range of biological questions that can be addressed by MERFISH.single-cell analysis | fluorescence | in situ hybridization | transcriptomics | multiplexed imaging

The expression profiles and spatial distributions of RNAs regulate many cellular functions. Image-based transcriptomic approaches provide powerful means to measure both expression and spatial information of RNAs in individual cells within their native environment. Among these approaches, multiplexed error-robust fluorescence in situ hybridization (MERFISH) has achieved spatially resolved RNA quantification at transcriptome scale by massively multiplexing single-molecule FISH measurements. Here, we increased the gene throughput of MERFISH and demonstrated simultaneous measurements of RNA transcripts from ∼10,000 genes in individual cells with ∼80% detection efficiency and ∼4% misidentification rate. We combined MERFISH with cellular structure imaging to determine subcellular compartmentalization of RNAs. We validated this approach by showing enrichment of secretome transcripts at the endoplasmic reticulum, and further revealed enrichment of long noncoding RNAs, RNAs with retained introns, and a subgroup of protein-coding mRNAs in the cell nucleus. Leveraging spatially resolved RNA profiling, we developed an approach to determine RNA velocity in situ using the balance of nuclear versus cytoplasmic RNA counts. We applied this approach to infer pseudotime ordering of cells and identified cells at different cell-cycle states, revealing ∼1,600 genes with putative cell cycle-dependent expression and a gradual transcription profile change as cells progress through cell-cycle stages. Our analysis further revealed cell cycle-dependent and cell cycle-independent spatial heterogeneity of transcriptionally distinct cells. We envision that the ability to perform spatially resolved, genome-wide RNA profiling with high detection efficiency and accuracy by MERFISH could help address a wide array of questions ranging from the regulation of gene expression in cells to the development of cell fate and organization in tissues.

Highly multiplexed single-molecule FISH has emerged as a promising approach to spatially resolved single-cell transcriptomics because of its ability to directly image and profile numerous RNA species in their native cellular context. However, backgroundfrom off-target binding of FISH probes and cellular autofluorescence-can become limiting in a number of important applications, such as increasing the degree of multiplexing, imaging shorter RNAs, and imaging tissue samples. Here, we developed a sample clearing approach for FISH measurements. We identified off-target binding of FISH probes to cellular components other than RNA, such as proteins, as a major source of background. To remove this source of background, we embedded samples in polyacrylamide, anchored RNAs to this polyacrylamide matrix, and cleared cellular proteins and lipids, which are also sources of autofluorescence. To demonstrate the efficacy of this approach, we measured the copy number of 130 RNA species in cleared samples using multiplexed error-robust FISH (MERFISH). We observed a reduction both in the background because of off-target probe binding and in the cellular autofluorescence without detectable loss in RNA. This process led to an improved detection efficiency and detection limit of MERFISH, and an increased measurement throughput via extension of MERFISH into four color channels. We further demonstrated MERFISH measurements of complex tissue samples from the mouse brain using this matrix-imprinting and -clearing approach. We envision that this method will improve the performance of a wide range of in situ hybridization-based techniques in both cell culture and tissues.tissue clearing | fluorescence in situ hybridization | multiplexed imaging | single-cell transcriptomics | brain S ingle-molecule FISH (smFISH) is a powerful technique that allows the direct imaging of individual RNA molecules within single cells (1, 2). In this approach, RNAs are labeled via the hybridization of fluorescently labeled oligonucleotide probes, producing bright fluorescent spots for single RNA molecules, which reveal both the abundance and the spatial distribution of these RNAs inside cells (1, 2). The ability of smFISH to image gene expression at the single-cell level in both cell culture and tissue has led to exciting advances in our understanding of the natural noise in gene expression and its role in cellular response (3, 4), the intracellular spatial organization of RNAs and its role in posttranscriptional regulation (5, 6), and the spatial variation in gene expression within complex tissues and its role in the molecular definition of cell types and tissue functions (6, 7).To extend the benefits of this technique to systems-level questions and high-throughput gene-expression profiling, approaches to increase the multiplexing of smFISH (i.e., the number of different RNA species that can be simultaneously quantified within the same cell) have been developed (8-13). Most of these approaches take advantage of color multiplexing, which has allowed a few tens...

To improve the quality and durability of inorganic halide perovskite nanocrystals (NCs), ammonium halide and lead oxide (PbO) were separately employed for the synthesis of NCs with a tunable ratio of metal to halide. The halide-rich circumstance was therefore successfully set up and found to be beneficial for obtaining good quality NCs with high photoluminescence quantum yields and remarkable stability against purification compared to those qualities of previous regular methods with lead halide. The fabricated light-emitting diode (LED) devices with NCs made in a halide-rich circumstance demonstrated performance better than that of devices with NCs made in a halide-poor circumstance. A LED with CsPbBr 3 NCs with a 1:4 Pb:Br ratio showed an obvious improved maximal luminance of 12090 cd m −2 , a current efficiency of 3.1 cd A −1 , and an external quantum efficiency of 1.194%, which were much higher than those of devices with NCs synthesized by the regular method.

Background With evidence of sustained transmission in more than 190 countries, coronavirus disease 2019 (COVID-19) has been declared a global pandemic. Data are urgently needed about risk factors associated with clinical outcomes. Methods A retrospective review of 323 hospitalized patients with COVID-19 in Wuhan was conducted. Patients were classified into three disease severity groups (non-severe, severe, and critical), based on initial clinical presentation. Clinical outcomes were designated as favorable and unfavorable, based on disease progression and response to treatments. Logistic regression models were performed to identify risk factors associated with clinical outcomes, and log-rank test was conducted for the association with clinical progression. Results Current standard treatments did not show significant improvement in patient outcomes. By univariate logistic regression analysis, 27 risk factors were significantly associated with clinical outcomes. Multivariate regression indicated age over 65 years (p<0.001), smoking (p=0.001), critical disease status (p=0.002), diabetes (p=0.025), high hypersensitive troponin I (>0.04 pg/mL, p=0.02), leukocytosis (>10 x 109/L, p<0.001) and neutrophilia (>75 x 109/L, p<0.001) predicted unfavorable clinical outcomes. By contrast, the administration of hypnotics was significantly associated with favorable outcomes (p<0.001), which was confirmed by survival analysis. Conclusions Hypnotics may be an effective ancillary treatment for COVID-19. We also found novel risk factors, such as higher hypersensitive troponin I, predicted poor clinical outcomes. Overall, our study provides useful data to guide early clinical decision making to reduce mortality and improve clinical outcomes of COVID-19.

Background With evidence of sustained transmission in more than 190 countries, coronavirus disease 2019 (COVID-19) has been declared a global pandemic. As such, data are urgently needed about risk factors associated with clinical outcomes. Methods A retrospective chart review of 323 hospitalized patients with COVID-19 in Wuhan was conducted. Patients were classified into three disease severity groups (non-severe, severe, and critical), based on their initial clinical presentation. Clinical outcomes were designated as favorable and unfavorable, based on disease progression and response to treatments. Logistic regression models were performed to identify factors associated with clinical outcomes, and logrank test was conducted for the association with clinical progression. Results Current standard treatments did not show significant improvement on patient outcomes in the study. By univariate logistic regression model, 27 risk factors were significantly associated with clinical outcomes. Further, multivariate regression indicated that age over 65 years, smoking, critical disease status, diabetes, high hypersensitive troponin I (>0.04 pg/mL), leukocytosis (>10 x 109/L) and neutrophilia (>75 x 109/L) predicted unfavorable clinical outcomes. By contrast, the use of hypnotics was significantly associated with favorable outcomes. Survival analysis also confirmed that patients receiving hypnotics had significantly better survival. Conclusions To our knowledge, this is the first indication that hypnotics could be an effective ancillary treatment for COVID-19. We also found that novel risk factors, such as higher hypersensitive troponin I, predicted poor clinical outcomes. Overall, our study provides useful data to guide early clinical decision making to reduce mortality and improve clinical outcomes of COVID-19.

WRKY proteins are a large family of transcriptional regulators involved in a variety of biological processes in plants. Here we report functional characterization of a rice WRKY gene, OsWRKY89. RNA gel blot analysis indicated that OsWRKY89 was strongly induced by treatments of methyl jasmonate and UV-B radiation. The transient expression analysis of the OsWRKY89-eGFP reporter in onion epidermal cells revealed that OsWRKY89 was targeted to nuclei. Transcriptional activity assays of OsWRKY89 and its mutants fused with a GAL4 DNA binding domain indicated that the 67 C-terminal amino acids were required for the transcriptional activation and that the leucine zipper region at the N-terminus enhanced its transcriptional activity. Overexpression of OsWRKY89 led to growth retardation at the early stage and reduction of internode length. Scanning electron microscopy revealed an increase in wax deposition on leaf surfaces of the OsWRKY89 overexpression lines and a decrease in wax loading in the RNAi-mediated OsWRKY89 suppression lines. Moreover, extractable and cell-wall-bound phenolic compounds were decreased in the overexpressor lines, but its SA levels were increased. Lignin staining showed an increase in lignification in culms of the overexpressor lines. Interestingly, overexpression of the OsWRKY89 gene enhanced resistance to the rice blast fungus and white-backed planthopper as well as tolerance to UV-B irradiation. These results suggest that OsWRKY89 plays an important role in response to biotic and abiotic stresses.