Transcriptomic profiling of complex tissues by single-nucleus RNA-sequencing (snRNA-seq) affords some advantages over single-cell RNA-sequencing (scRNA-seq). snRNA-seq provides less biased cellular ...coverage, does not appear to suffer cell isolation-based transcriptional artifacts, and can be applied to archived frozen specimens. We used well-matched snRNA-seq and scRNA-seq datasets from mouse visual cortex to compare cell type detection. Although more transcripts are detected in individual whole cells (~11,000 genes) than nuclei (~7,000 genes), we demonstrate that closely related neuronal cell types can be similarly discriminated with both methods if intronic sequences are included in snRNA-seq analysis. We estimate that the nuclear proportion of total cellular mRNA varies from 20% to over 50% for large and small pyramidal neurons, respectively. Together, these results illustrate the high information content of nuclear RNA for characterization of cellular diversity in brain tissues.
Multiple biological processes are driven by oscillatory gene expression at different time scales. Pulsatile dynamics are thought to be widespread, and single-cell live imaging of gene expression has ...lead to a surge of dynamic, possibly oscillatory, data for different gene networks. However, the regulation of gene expression at the level of an individual cell involves reactions between finite numbers of molecules, and this can result in inherent randomness in expression dynamics, which blurs the boundaries between aperiodic fluctuations and noisy oscillators. This underlies a new challenge to the experimentalist because neither intuition nor pre-existing methods work well for identifying oscillatory activity in noisy biological time series. Thus, there is an acute need for an objective statistical method for classifying whether an experimentally derived noisy time series is periodic. Here, we present a new data analysis method that combines mechanistic stochastic modelling with the powerful methods of non-parametric regression with Gaussian processes. Our method can distinguish oscillatory gene expression from random fluctuations of non-oscillatory expression in single-cell time series, despite peak-to-peak variability in period and amplitude of single-cell oscillations. We show that our method outperforms the Lomb-Scargle periodogram in successfully classifying cells as oscillatory or non-oscillatory in data simulated from a simple genetic oscillator model and in experimental data. Analysis of bioluminescent live-cell imaging shows a significantly greater number of oscillatory cells when luciferase is driven by a Hes1 promoter (10/19), which has previously been reported to oscillate, than the constitutive MoMuLV 5' LTR (MMLV) promoter (0/25). The method can be applied to data from any gene network to both quantify the proportion of oscillating cells within a population and to measure the period and quality of oscillations. It is publicly available as a MATLAB package.
OBJECTIVE: The safety of dendritic cells to selectively suppress autoimmunity, especially in type 1 diabetes, has never been ascertained. We investigated the safety of autologous dendritic cells, ...stabilized into an immunosuppressive state, in established adult type 1 diabetic patients. RESEARCH DESIGN AND METHODS: A randomized, double-blind, phase I study was conducted. A total of 10, otherwise generally healthy, insulin-requiring type 1 diabetic patients between 18 and 60 years of age, without any other known or suspected health conditions, received autologous dendritic cells, unmanipulated or engineered ex vivo toward an immunosuppressive state. Ten million cells were administered intradermally in the abdomen once every 2 weeks for a total of four administrations. The primary end point determined the proportion of patients with adverse events on the basis of the physician’s global assessment, hematology, biochemistry, and immune monitoring for a period of 12 months. RESULTS: The dendritic cells were safely tolerated. There were no discernible adverse events in any patient throughout the study. Other than a significant increase in the frequency of peripheral B220+ CD11c– B cells, mainly seen in the recipients of engineered dendritic cells during the dendritic cell administration period, there were no statistically relevant differences in other immune populations or biochemical, hematological, and immune biomarkers compared with baseline. CONCLUSIONS: Treatment with autologous dendritic cells, in a native state or directed ex vivo toward a tolerogenic immunosuppressive state, is safe and well tolerated. Dendritic cells upregulated the frequency of a potentially beneficial B220+ CD11c– B-cell population, at least in type 1 diabetes autoimmunity.
This essay examines the state of Digital Humanities work in the subfield of literature from the Spanish speaking world. It provides a case study of a multiyear digital mapping project conducted with ...undergraduate students at a highly selective liberal arts college. This project serves as a means of examining the digital state of the field and advocates for further integration of these digital projects as part of the standard pedagogical and research practices in Spanish programs across higher education. Especially given the pandemic conditions of the 2020–21 academic year, Digital Humanities work in Spanish has the opportunity to foster new types of cultural and linguistic study that will help keep students engaged with second language learning while also expanding their capability to undertake literary analysis.
Rats emit ultrasonic vocalizations (USVs) that are thought to serve as situation-dependent affective signals and accomplish important communicative functions. In appetitive situations, rats produce ...50 kHz USVs, whereas 22 kHz USVs occur in aversive situations. Reception of 50 kHz USVs induces social approach behavior, while 22 kHz USVs lead to freezing behavior. These opposite behavioral responses are paralleled by distinct brain activation patterns, with 50 kHz USVs, but not 22 kHz USVs, activating neurons in the nucleus accumbens (NAcc). The NAcc mediates appetitive behavior and is critically modulated by dopaminergic afferents that are known to encode the value of reward. Therefore, we hypothesized that 50 kHz USVs, but not 22 kHz USVs, elicit NAcc dopamine release. While recording dopamine signaling with fast-scan cyclic voltammetry, freely moving rats were exposed to playback of four acoustic stimuli via an ultrasonic speaker in random order: (1) 50 kHz USVs, (2) 22 kHz USVs, (3) time- and amplitude-matched white noise, and (4) background noise. Only presentation of 50 kHz USVs induced phasic dopamine release and elicited approach behavior toward the speaker. Both of these effects, neurochemical and behavioral, were most pronounced during initial playback, but then declined rapidly with subsequent presentations, indicating a close temporal relationship between the two measures. Moreover, the magnitudes of these effects during initial playback were significantly correlated. Collectively, our findings show that NAcc dopamine release encodes pro-social 50 kHz USVs, but not alarming 22 kHz USVs. Thus, our results support the hypothesis that these call types are processed in distinct neuroanatomical regions and establish a functional link between pro-social communicative signals and reward-related neurotransmission.
Highlights ► We apply a multiple systems framework to Pavlovian valuation. ► Recent evidence supports a selective role for dopamine in model-free Pavlovian valuation. ► Recent evidence supports a ...preferential role for the orbitofrontal cortex in model-based Pavlovian valuation. ► We discuss the implications of interaction between valuation systems for learning and decision making.
The neocortex contains a multitude of cell types that are segregated into layers and functionally distinct areas. To investigate the diversity of cell types across the mouse neocortex, here we ...analysed 23,822 cells from two areas at distant poles of the mouse neocortex: the primary visual cortex and the anterior lateral motor cortex. We define 133 transcriptomic cell types by deep, single-cell RNA sequencing. Nearly all types of GABA (γ-aminobutyric acid)-containing neurons are shared across both areas, whereas most types of glutamatergic neurons were found in one of the two areas. By combining single-cell RNA sequencing and retrograde labelling, we match transcriptomic types of glutamatergic neurons to their long-range projection specificity. Our study establishes a combined transcriptomic and projectional taxonomy of cortical cell types from functionally distinct areas of the adult mouse cortex.
High-dispersion observations of the Na I D lambdalambda5890, 5896 and K I lambdalambda7665, 7699 interstellar lines, and the diffuse interstellar band at 5780 A in the spectra of 32 Type Ia ...supernovae are used as an independent means of probing dust extinction. We show that the dust extinction of the objects where the diffuse interstellar band at 5780 A is detected is consistent with the visual extinction derived from the supernova colors. This strongly suggests that the dust producing the extinction is predominantly located in the interstellar medium of the host galaxies and not in circumstellar material associated with the progenitor system. One quarter of the supernovae display anomalously large Nai column densities in comparison to the amount of dust extinction derived from their colors. Remarkably, all of the cases of unusually strong Na I D absorption correspond to "Blueshifted" profiles in the classification scheme of Sternberg et al. This coincidence suggests that outflowing circumstellar gas is responsible for at least some of the cases of anomalously large Na i column densities. Two supernovae with unusually strong Nai D absorption showed essentially normal K I column densities for the dust extinction implied by their colors, but this does not appear to be a universal characteristic. Overall, we find the most accurate predictor of individual supernova extinction to be the equivalent width of the diffuse interstellar band at 5780 A, and provide an empirical relation for its use. Finally, we identify ways of producing significant enhancements of the Na abundance of circumstellar material in both the single-degenerate and double-degenerate scenarios for the progenitor system.
The human pathogenic parasite Trypanosoma brucei possess both de novo and salvage routes for the biosynthesis of pyrimidine nucleotides. Consequently, they do not require salvageable pyrimidines for ...growth. Thymidine kinase (TK) catalyzes the formation of dTMP and dUMP and is one of several salvage enzymes that appear redundant to the de novo pathway. Surprisingly, we show through analysis of TK conditional null and RNAi cells that TK is essential for growth and for infectivity in a mouse model, and that a catalytically active enzyme is required for its function. Unlike humans, T. brucei and all other kinetoplastids lack dCMP deaminase (DCTD), which provides an alternative route to dUMP formation. Ectopic expression of human DCTD resulted in full rescue of the RNAi growth phenotype and allowed for selection of viable TK null cells. Metabolite profiling by LC-MS/MS revealed a buildup of deoxypyrimidine nucleosides in TK depleted cells. Knockout of cytidine deaminase (CDA), which converts deoxycytidine to deoxyuridine led to thymidine/deoxyuridine auxotrophy. These unexpected results suggested that T. brucei encodes an unidentified 5'-nucleotidase that converts deoxypyrimidine nucleotides to their corresponding nucleosides, leading to their dead-end buildup in TK depleted cells at the expense of dTTP pools. Bioinformatics analysis identified several potential candidate genes that could encode 5'-nucleotidase activity including an HD-domain protein that we show catalyzes dephosphorylation of deoxyribonucleotide 5'-monophosphates. We conclude that TK is essential for synthesis of thymine nucleotides regardless of whether the nucleoside precursors originate from the de novo pathway or through salvage. Reliance on TK in the absence of DCTD may be a shared vulnerability among trypanosomatids and may provide a unique opportunity to selectively target a diverse group of pathogenic single-celled eukaryotes with a single drug.
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