Coordination of olfactory receptor choice with guidance receptor expression and function in olfactory sensory neurons.

Olfactory sensory neurons choose to express a single odorant receptor (OR) from a large gene repertoire and extend axons to reproducible, OR-specific locations within the olfactory bulb. This developmental process produces a topographically organized map of odorant experience in the brain. The axon guidance mechanisms that generate this pattern of connectivity, as well as those that coordinate OR choice and axonal guidance receptor expression, are incompletely understood. We applied the powerful approach of single-cell RNA-seq on newly born olfactory sensory neurons (OSNs) in young zebrafish larvae to address these issues. Expression profiles were generated for 56 individual Olfactory Marker Protein (OMP) positive sensory neurons by single-cell (SC) RNA-seq. We show that just as in mouse OSNs, mature zebrafish OSNs typically express a single predominant OR transcript. Our previous work suggests that OSN targeting is related to the OR clade from which a sensory neuron chooses to express its odorant receptor. We categorized each of the mature cells based on the clade of their predominantly expressed OR. Transcripts expressed at higher levels in each of three clade-related categories were identified using Penalized Linear Discriminant Analysis (PLDA). A genome-wide approach was used to identify membrane-associated proteins that are most likely to have guidance-related activity. We found that OSNs that choose to express an OR from a particular clade also express specific subsets of potential axon guidance genes and transcription factors. We validated our identification of candidate axon guidance genes for one clade of OSNs using bulk RNA-seq from a subset of transgene-labeled neurons that project to a single protoglomerulus. The differential expression patterns of selected candidate guidance genes were confirmed using fluorescent in situ hybridization. Most importantly, we observed axonal mistargeting in knockouts of three candidate axonal guidance genes identified in this analysis: nrp1a, nrp1b, and robo2. In each case, targeting errors were detected in the subset of axons that normally express these transcripts at high levels, and not in the axons that express them at low levels. Our findings demonstrate that specific, functional, axonal guidance related genes are expressed in subsets of OSNs that that can be categorized by their patterns of OR expression.

PIVOT: platform for interactive analysis and visualization of transcriptomics data.

BACKGROUND: Many R packages have been developed for transcriptome analysis but their use often requires familiarity with R and integrating results of different packages requires scripts to wrangle the datatypes. Furthermore, exploratory data analyses often generate multiple derived datasets such as data subsets or data transformations, which can be difficult to track. RESULTS: Here we present PIVOT, an R-based platform that wraps open source transcriptome analysis packages with a uniform user interface and graphical data management that allows non-programmers to interactively explore transcriptomics data. PIVOT supports more than 40 popular open source packages for transcriptome analysis and provides an extensive set of tools for statistical data manipulations. A graph-based visual interface is used to represent the links between derived datasets, allowing easy tracking of data versions. PIVOT further supports automatic report generation, publication-quality plots, and program/data state saving, such that all analysis can be saved, shared and reproduced. CONCLUSIONS: PIVOT will allow researchers with broad background to easily access sophisticated transcriptome analysis tools and interactively explore transcriptome datasets.

Enucleation or exenteration in two dogs with previous parotid duct transposition: parotid duct ligation versus reverse parotid duct transposition.

Two dogs with previous parotid duct transpositions presented with unrelated ocular disease. In both cases, ophthalmic examination revealed the need for enucleation or exenteration. In case 1, systemic coccidioidomycosis was diagnosed with panuveitis and secondary glaucoma of the left eye. In this case, the parotid duct was ligated at the time of enucleation to stop salivary secretions. This dog encountered morbidity in the form of a sialocele that did not resolve for 11 months. In case 2, ultrasound and computed tomography revealed a discrete mass within the left medial orbit that was suspected to arise from the nictitating membrane. A combination of exenteration and parotid duct transposition reversal was performed to avoid morbidity associated with ligation of the parotid duct. The dog encountered no complications from this novel procedure. This case report represents the first report of re-routing a transposed parotid duct from the ventral conjunctival sac back to the mouth at the time of enucleation or exenteration in the dog.

Transcriptome in vivo analysis (TIVA) of spatially defined single cells in live tissue.

Transcriptome profiling of single cells resident in their natural microenvironment depends upon RNA capture methods that are both noninvasive and spatially precise. We engineered a transcriptome in vivo analysis (TIVA) tag, which upon photoactivation enables mRNA capture from single cells in live tissue. Using the TIVA tag in combination with RNA sequencing (RNA-seq), we analyzed transcriptome variance among single neurons in culture and in mouse and human tissue in vivo. Our data showed that the tissue microenvironment shapes the transcriptomic landscape of individual cells. The TIVA methodology is, to our knowledge, the first noninvasive approach for capturing mRNA from live single cells in their natural microenvironment.

Single-cell transcriptomics and functional target validation of brown adipocytes show their complex roles in metabolic homeostasis.

Brown adipocytes (BAs) are specialized for adaptive thermogenesis and, upon sympathetic stimulation, activate mitochondrial uncoupling protein (UCP)-1 and oxidize fatty acids to generate heat. The capacity for brown adipose tissue (BAT) to protect against obesity and metabolic disease is recognized, yet information about which signals activate BA, besides ß3-adrenergic receptor stimulation, is limited. Using single-cell transcriptomics, we confirmed the presence of mRNAs encoding traditional BAT markers (i.e., UCP1, expressed in 100% of BAs Adrb3, expressed in <50% of BAs) in mouse and have shown single-cell variability (>1000-fold) in their expression at both the mRNA and protein levels. We further identified mRNAs encoding novel markers, orphan GPCRs, and many receptors that bind the classic neurotransmitters, neuropeptides, chemokines, cytokines, and hormones. The transcriptome variability between BAs suggests a much larger range of responsiveness of BAT than previously recognized and that not all BAs function identically. We examined the in vivo functional expression of 12 selected receptors by microinjecting agonists into live mouse BAT and analyzing the metabolic response. In this manner, we expanded the number of known receptors on BAs at least 25-fold, while showing that the expression of classic BA markers is more complex and variable than previously thought.

Tumor Enhancement and Heterogeneity Are Associated With Treatment Response to Drug-Eluting Bead Chemoembolization for Hepatocellular Carcinoma.

PURPOSE: Treatment response to drug-eluting bead chemoembolization (DEB-TACE) is well established for patients with hepatocellular carcinoma (HCC); however, few studies have evaluated tumor imaging characteristics associated with treatment responses. The aim of our study was to identify imaging characteristics associated with treatment responses and overall survival after DEB-TACE of HCC. METHODS: This is a retrospective cohort study of 33 tumors in 32 patients who underwent DEB-TACE for inoperable HCC in a single, large academic medical center. Arterial phase computed tomography data were reviewed to assess tumor size, edge characteristics, tumor enhancement on pixel density histogram, and heterogeneity using coefficient of variation. We assessed correlation between these markers of tumor morphology and response to DEB-TACE using mRECIST criteria, progression-free survival, and overall survival. RESULTS: Tumor heterogeneity (P = 0.01) and tumor enhancement greater than 50% (P = 0.05) were significantly associated with complete response to DEB-TACE in patients with HCC; however, neither was associated with overall or progression-free survival. Tumor size and edge characteristics were not associated with complete response to DEB-TACE, although tumor size greater than 6 cm was associated with worse overall survival (hazard ratio, 3.349; P = 0.02). CONCLUSIONS: Tumor heterogeneity and enhancement on arterial phase imaging may be predictive markers of treatment response to DEB-TACE among patients with HCC.

Assessing characteristics of RNA amplification methods for single cell RNA sequencing.

BACKGROUND: Recently, measurement of RNA at single cell resolution has yielded surprising insights. Methods for single-cell RNA sequencing (scRNA-seq) have received considerable attention, but the broad reliability of single cell methods and the factors governing their performance are still poorly known. RESULTS: Here, we conducted a large-scale control experiment to assess the transfer function of three scRNA-seq methods and factors modulating the function. All three methods detected greater than 70% of the expected number of genes and had a 50% probability of detecting genes with abundance greater than 2 to 4 molecules. Despite the small number of molecules, sequencing depth significantly affected gene detection. While biases in detection and quantification were qualitatively similar across methods, the degree of bias differed, consistent with differences in molecular protocol. Measurement reliability increased with expression level for all methods and we conservatively estimate measurements to be quantitative at an expression level greater than ~5-10 molecules. CONCLUSIONS: Based on these extensive control studies, we propose that RNA-seq of single cells has come of age, yielding quantitative biological information.

Serotonergic neuron regulation informed by in vivo single-cell transcriptomics.

Despite the recognized importance of the dorsal raphe (DR) serotonergic (5-HT) nuclei in the pathophysiology of depression and anxiety, the molecular components/putative drug targets expressed by these neurons are poorly characterized. Utilizing the promoter of an ETS domain transcription factor that is a stable marker of 5-HT neurons (Pet-1) to drive 5-HT neuronal expression of YFP, we identified 5-HT neurons in live acute slices. We isolated RNA from single 5-HT neurons in the ventromedial and lateral wings of the DR and performed single-cell RNA-Seq analysis identifying >500 G-protein coupled receptors (GPCRs) including receptors for classical transmitters, lipid signals, and peptides as well as dozens of orphan-GPCRs. Using these data to inform our selection of receptors to assess, we found that oxytocin and lysophosphatidic acid 1 receptors are translated and active in costimulating, with the α1-adrenergic receptor, the firing of DR 5-HT neurons, while the effects of histamine are inhibitory and exerted at H3 histamine receptors. The inhibitory histamine response provides evidence for tonic in vivo histamine inhibition of 5-HT neurons. This study illustrates that unbiased single-cell transcriptomics coupled with functional analyses provides novel insights into how neurons and neuronal systems are regulated.

T-cell chronic lymphocytic leukemia or small-cell variant of T-cell prolymphocytic leukemia: a historical perspective and search for consensus.

There is a rich history behind the extinct entity 'T-cell chronic lymphocytic leukemia (T-CLL)' and the now-established replacement, small-cell variant of T-cell prolymphocytic leukemia (T-PLL-sv). Herein, we review the history of the events, observations, and discussions that led to this replacement. We also provide a systematic analysis of all previously reported cases of T-PLL-sv as well as our four new additional cases. Despite the higher frequency of a normal karyotype and perhaps an overrepresented CD4(-) CD8(-) immunophenotype among these patients (compared to T-PLL in general) as well as bland morphology (that makes them superficially appear more similar to B-CLL), we argue that the current World Health Organization (WHO)-based classification as T-PLL-sv is adequate and should continue for the time being. Morphologically, T-PLL-sv represents approximately one-fifth of all T-PLL cases. However, morphology alone does not determine the clinical course and should not be the basis for clinical decision making and prognostication. We propose a clonal evolution model in which mature T-cell leukemias classified in the past as T-CLL are perhaps T-PLL diagnosed early in the course of the disease. Future research using next-generation sequencing, comparative genomic hybridization, and molecular array studies, including serial analyses of individual cases over time, is needed to better identify this rarely diagnosed, inherently controversial form of T-cell leukemia.

Advanced MR imaging of peripheral nerve sheath tumors including diffusion imaging.

Peripheral nerve sheath tumors (PNSTs) are neoplasms derived from neoplastic Schwann cells or their precursors. Whereas benign PNSTs are relatively common and considered curable lesions, their malignant counterparts are rare but highly aggressive and require early diagnosis and treatment. MR imaging has been the modality of choice for noninvasive evaluation of PNSTs. This article discusses the features of PNSTs in conventional and advanced MR imaging, and it emphasizes the features that help differentiate benign and malignant variants.

Acute myeloid leukemia following solid organ transplantation: entity or novelty?

Due to the rarity of the disease, the characteristics of acute myeloid leukemia following solid organ transplantation (post-transplant AML; PT-AML) are unclear; furthermore, it is not known for certain whether PT-AML is a separate entity or not. We provide a systematic review of all previously reported cases of PT-AML in the English literature (n = 51). 45% of cases occurred after renal transplantation, and 72% were males. The median age at diagnosis of AML was 50 yr, with a median transplant-to-AML interval of 3.8 yr and a rapid decline in incidence after 5 yr. 26% of patients were asymptomatic at the time of presentation, and 42% were pancytopenic. M0/M1/M2, M3, M4/M5, and M6/M7 subtypes comprised 17%, 25%, 39%, and 19% of all cases, respectively. 36% of patients had unfavorable cytogenetic risk disease. The median overall survival was only 3 months. We observed several transplant-specific features: (i) The transplant-to-AML interval follows two very different patterns between renal vs. liver transplant patients. (ii) All 4 cases of donor cell leukemia occurred after liver transplant. (iii) Unfavorable risk disease was marginally significantly more common among renal compared with liver transplant patients (P = 0.057). Our results suggest that PT-AML is a separate entity with distinct characteristics, which need to be investigated further in future research. Heavy post-transplant immunosuppression likely plays a key role in the pathogenesis of PT-AML.

Mindfulness meditation use in Britain during the COVID-19 pandemic.

OBJECTIVES: The objectives of this study were to examine the prevalence and associations of mindfulness meditation use and also its perceived mental health effects during the COVID-19 pandemic. METHODS: Using repeated cross-sectional data from broad online samples weighted to be representative of the adult population in Britain, we estimated the prevalence of mindfulness meditation use and employed logistic regression models to investigate sociodemographic and political associations of mindfulness meditation use and also its perceived mental health effects during the COVID-19 pandemic. RESULTS: The findings suggest that 16 percent of adults in Britain had learnt to practice mindfulness in 2021. In covariate-adjusted regression models, having learnt to practice mindfulness was more common among young and middle-aged adults, residents in London, and respondents who voted for the Liberal Democrats. Among mindfulness meditation users who reported having practiced mindfulness during the COVID-19 pandemic, 60 percent reported that it positively affected their mental health and 24 percent reported that it negatively affected their mental health. Notably, 41 percent of respondents with children under 18 (versus 13 percent of those without minors) reported negative mental health effects. In covariate-adjusted regression models, negative mental health effects from mindfulness practice during the COVID-19 pandemic were not concentrated in any particular groups, except for respondents with children under 18. CONCLUSIONS: Mindfulness meditation has become widespread in Britain, but the results in this study suggest that mindfulness meditation use may be concentrated in certain sociodemographic and political groups. The results also suggest that practicing mindfulness during the COVID-19 pandemic had positive mental health effects for a majority of users, but approximately one-quarter of users reported negative mental health effects. It is therefore important for future research to continue monitoring the prevalence of mindfulness meditation use in society and to investigate under what circumstances, for whom, and in what ways mindfulness-based practices may have negative effects on mental health.

Single-Mitochondrion Sequencing Uncovers Distinct Mutational Patterns and Heteroplasmy Landscape in Mouse Astrocytes and Neurons.

Background: Mitochondrial (mt) heteroplasmy can cause adverse biological consequences when deleterious mtDNA mutations accumulate disrupting 'normal' mt-driven processes and cellular functions. To investigate the heteroplasmy of such mtDNA changes we developed a moderate throughput mt isolation procedure to quantify the mt single-nucleotide variant (SNV) landscape in individual mouse neurons and astrocytes In this study we amplified mt-genomes from 1,645 single mitochondria (mts) isolated from mouse single astrocytes and neurons to 1. determine the distribution and proportion of mt-SNVs as well as mutation pattern in specific target regions across the mt-genome, 2. assess differences in mtDNA SNVs between neurons and astrocytes, and 3. Study cosegregation of variants in the mouse mtDNA. Results: 1. The data show that specific sites of the mt-genome are permissive to SNV presentation while others appear to be under stringent purifying selection. Nested hierarchical analysis at the levels of mitochondrion, cell, and mouse reveals distinct patterns of inter- and intra-cellular variation for mt-SNVs at different sites. 2. Further, differences in the SNV incidence were observed between mouse neurons and astrocytes for two mt-SNV 9027:G>A and 9419:C>T showing variation in the mutational propensity between these cell types. Purifying selection was observed in neurons as shown by the Ka/Ks statistic, suggesting that neurons are under stronger evolutionary constraint as compared to astrocytes. 3. Intriguingly, these data show strong linkage between the SNV sites at nucleotide positions 9027 and 9461. Conclusion: This study suggests that segregation as well as clonal expansion of mt-SNVs is specific to individual genomic loci, which is important foundational data in understanding of heteroplasmy and disease thresholds for mutation of pathogenic variants.

Primary adrenal lymphoma: a systematic review.

Fewer than 200 cases of primary adrenal lymphoma (PAL) have been reported. We have systematically reviewed all 187 cases of PAL reported in the English literature until June 2013, from which we drew the following conclusions: PAL is typically a highly symptomatic and aggressive, metabolically hyperactive, hypovascular, hypoechoic (and heterogeneous on ultrasound), hypodense (with slight to moderate enhancement on computed tomography), high-grade lymphoma, primarily affecting elderly males and presenting with large bilateral adrenal masses. Most cases have adrenal insufficiency, B-symptoms, and elevated lactate dehydrogenase. Hepatosplenomegaly, lymphadenopathy, concurrent or prior immune dysregulation, and bone marrow involvement are uncommon. Epstein-Barr virus positivity is observed in more than half of cases and the disease is disseminated at presentation in 18 % of cases. The two most common WHO 2008-defined PAL subtypes are diffuse large B cell lymphoma (78 %) and peripheral T cell lymphoma (7 %). The prognosis of PAL has improved with the advent of rituximab-containing chemotherapeutic regimens. According to our results, administration of chemotherapy and adrenal insufficiency are significant independent predictors of prognosis.

Mindfulness in Politics: A Qualitative Study on Mindfulness Training in the UK Parliament.

Objectives: While mindfulness in the workplace has received substantial scientific attention in the past decades, it is not yet well-understood if, under what circumstances, and in what ways mindfulness training may be helpful for individuals working in political environments. The aim of this study was to explore the experience of mindfulness training among British politicians, as well as mindfulness facilitators who had taught mindfulness to politicians in the UK Parliament. Method: Between September and November 2021, semi-structured in-depth interviews were conducted with British politicians (n = 18) who had experience of mindfulness training and mindfulness facilitators (n = 4) who had taught mindfulness to politicians in the UK Parliament. The interview material was analyzed using reflexive thematic analysis. Results: Two main themes related to the experience of mindfulness training in politics were developed during the analytic process: (1) mindfulness approaches addressing particular challenges in political work, and (2) mindfulness sessions and group dynamics. Taken together, mindfulness training helped the politicians to better deal with the demands and stresses of political work, to reconnect with themselves and be more grounded, and - especially when taught in a group setting - to relate to other politicians and their viewpoints in a more humane and constructive way. Conclusions: The results suggest that mindfulness training can be beneficial at both the personal and professional level for individuals working in political contexts, which can provide a rationale for governments to introduce mindfulness-based programs to politicians. Supplementary Information: The online version contains supplementary material available at 10.1007/s12671-023-02156-x.

Systematic Sampling of the Female Reproductive System for Molecular Characterization.

As part of the National Institutes of Health Human BioMolecular Atlas Program to develop a global platform to map the 37 trillion cells in the adult human body, we are generating a comprehensive molecular characterization of the female reproductive system. Data gathered from multiple single-cell/single-nucleus and spatial molecular assays will be used to build a 3D molecular atlas. Herein, we describe our multistep protocol, beginning with an optimized organ procurement workflow that maintains functional characteristics of the uterus, ovaries, and fallopian tubes by perfusing these organs with preservation solution. We have also developed a structured tissue sampling procedure that retains information on individual-level anatomic, physiologic, and individual diversity of the female reproductive system, toward full exploration of the function and structure of female reproductive cells. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Preparation and preservation of the female reproductive system (ovaries, fallopian tubes, and uterus) prior to procurement Basic Protocol 2: Removal of the female reproductive system en bloc Basic Protocol 3: Postsurgical dissection of ovaries Basic Protocol 4: Postsurgical dissection of fallopian tubes Basic Protocol 5: Postsurgical dissection of cervix Basic Protocol 6: Postsurgical dissection of uterine body Support Protocol 1: OCT-embedded tissue protocol Support Protocol 2: Tissue fixation protocol Support Protocol 3: Snap-frozen tissue protocol Basic Protocol 7: Tissue slice preparation for Visium analysis Support Protocol 4: Hematoxylin and eosin staining for 10X Visium imaging Basic Protocol 8: Manual tissue dissociation for Multiome analysis Basic Protocol 9: Tissue dissociation for Multiome analysis using S2 Singulator.

CHEX-seq detects single-cell genomic single-stranded DNA with catalytical potential.

Genomic DNA (gDNA) undergoes structural interconversion between single- and double-stranded states during transcription, DNA repair and replication, which is critical for cellular homeostasis. We describe "CHEX-seq" which identifies the single-stranded DNA (ssDNA) in situ in individual cells. CHEX-seq uses 3'-terminal blocked, light-activatable probes to prime the copying of ssDNA into complementary DNA that is sequenced, thereby reporting the genome-wide single-stranded chromatin landscape. CHEX-seq is benchmarked in human K562 cells, and its utilities are demonstrated in cultures of mouse and human brain cells as well as immunostained spatially localized neurons in brain sections. The amount of ssDNA is dynamically regulated in response to perturbation. CHEX-seq also identifies single-stranded regions of mitochondrial DNA in single cells. Surprisingly, CHEX-seq identifies single-stranded loci in mouse and human gDNA that catalyze porphyrin metalation in vitro, suggesting a catalytic activity for genomic ssDNA. We posit that endogenous DNA enzymatic activity is a function of genomic ssDNA.