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Detailed characterization of the cell types in the human brain requires scalable experimental approaches to examine multiple aspects of the molecular state of individual cells, and computational integration of the data to produce unified cell-state annotations. Here we report improved high-throughput methods for single-nucleus Droplet-based sequencing (snDrop-seq) and single-cell transposome hypersensitive-site sequencing (scTHS-seq). We used each method to acquire nuclear transcriptomic and DNA accessibility maps for >60,000 single cells from the human adult visual cortex, frontal cortex, and cerebellum. Integration of these data revealed regulatory elements and transcription factors that underlie cell-type distinctions, providing a basis for studying complex processes in the brain, such as genetic programs coordinating adult remyelination. We also mapped disease-associated risk variants to specific cellular populations, providing insights into normal and pathogenic cellular processes in the human brain. This integrative multi-omics approach permits more detailed single-cell interrogation of complex organs and tissues.

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Abstract:Detailed characterization of the cell types comprising the highly complex human brain is essential to understanding its function. Such tasks require highly scalable experimental approaches to examine different aspects of the molecular state of individual cells, as well as the computational integration to produce unified cell state annotations. Here we report the development of two highly scalable methods (snDrop-Seq and scTHS-Seq), that we have used to acquire nuclear transcriptome and DNA accessibility maps for thousands of single cells from the human adult visual and frontal cortex. This has led to the best-resolved human neuronal subtypes to date, identification of a majority of the non-neuronal cell types, as well as the celltype specific nuclear transcriptome and DNA accessibility maps. Integrative analysis allowed us to identify transcription factors and regulatory elements shaping the state of different brain cell types, and to map genetic risk factors of human brain common diseases to specific pathogenic cell types and subtypes. Main TextIntroduction. Our brain is an enormously complex network consisting of 100 billion spatially organized and functionally connected neurons embedded in an even larger population of glia and non-neural cells. Producing a complete cell atlas of the human brain would require highly scalable and unbiased approaches. Recent advances in droplet-based technologies has greatly enhanced the throughput of single-cell RNA-sequencing (RNA-seq) 1-3 , enabling simultaneous

Transcriptional profiling, which is directly or indirectly associated with expressed protein levels, has been used in various applications including clinical prognosis and pharmaceutical investigation of drug activities. Although the widely used reverse transcription polymerase chain reaction (RT-PCR) allows for the quantification of absolute amounts of mRNA (mRNA) from inputs as small as a single cell, it is an indirect detection method that requires the amplification of cDNA copies of target mRNAs. Here, we report the quantification of unmodified full-length transcripts, using poly(ethylene) glycol diacrylate (PEGDA) hydrogel microparticles synthesized via stop flow lithography (SFL). We show that PEG600 serves as an effective porogen to allow for the capture of large (∼1000-3700 nt long) mRNAs. Our relatively simple hydrogel-based mRNA detection scheme uses a multibiotinylated universal label probe and provides assay performance (limit of detection of ∼6 amol of an in-vitro-transcribed model target) comparable to an existing commercial bead-based technology that uses branched DNA (bDNA) signal amplification. We also demonstrate a 3-plex mRNA detection, without cross-reactivity, using shape-encoded "intraplex" hydrogel microparticles. Our ability to tune the porosity of encoded hydrogel microparticles expands the utility of this platform to now quantify biomacromolecules ranging in size from large mRNAs to small miRNAs.

Escherichia coli SdiA is a quorum-sensing (QS) receptor that responds to autoinducers produced by other bacterial species to control cell division and virulence. Crystal structures reveal that E. coli SdiA, which is composed of an N-terminal ligand-binding domain and a C-terminal DNA-binding domain (DBD), forms a symmetrical dimer. Although each domain shows structural similarity to other QS receptors, SdiA differs from them in the relative orientation of the two domains, suggesting that its ligand-binding and DNA-binding functions are independent. Consistently, in DNA gel-shift assays the binding affinity of SdiA for the ftsQP2 promoter appeared to be insensitive to the presence of autoinducers. These results suggest that autoinducers increase the functionality of SdiA by enhancing the protein stability rather than by directly affecting the DNA-binding affinity. Structural analyses of the ligand-binding pocket showed that SdiA cannot accommodate ligands with long acyl chains, which was corroborated by isothermal titration calorimetry and thermal stability analyses. The formation of an intersubunit disulfide bond that might be relevant to modulation of the DNA-binding activity was predicted from the proximal position of two Cys residues in the DBDs of dimeric SdiA. It was confirmed that the binding affinity of SdiA for the uvrY promoter was reduced under oxidizing conditions, which suggested the possibility of regulation of SdiA by multiple independent signals such as quorum-sensing inducers and the oxidation state of the cell.

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A 35-yr-old man sustained an anoxic brain injury resulting from cardiac arrest, with subsequent extreme lethargy and lack of response to stimuli. The patient's lethargy was unresponsive to trials of several medications in attempts to increase arousal. Administration of twice-daily zolpidem 8 mos after injury resulted in a dramatic increase in the level of alertness, including improved speech and gait. When the patient was not able to receive zolpidem for a brief period, the patient's lethargy returned, and he became bedbound until the medication was resumed.