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 1645 single mitochondria 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 co-segregation 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. CONCLUSIONS: 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.

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.

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.

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.

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.

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.

Effects of an 8-Week Mindfulness Course on Affective Polarization.

Objectives: The European Union Brexit referendum has split the British electorate into two camps, with high levels of affective polarization between those who affiliate with the Remain side (Remainers) and the Leave side (Leavers) of the debate. Previous research has shown that a brief meditation intervention can reduce affective polarization, but no study has thus far investigated the effects of an 8-week mindfulness program on affective polarization. This is what will be examined in this study. Methods: The present study used a randomized waitlist control design (n = 177) with a 1-month post-intervention follow-up to investigate whether an 8-week mindfulness program delivered online would have an effect on affective polarization among Remainers and Leavers. Results: Results showed significantly greater reductions in affective polarization over time for participants in the mindfulness condition relative to participants in the waitlist control condition (time X group B = - 0.087, p = .024). Conclusions: Taken together, the findings highlight the potential of mindfulness training as a means to reduce intergroup biases in political contexts. Trial Registration: Preregistered on the Open Science Framework at https://osf.io/px8m2.

Effects of an eight-week, online mindfulness program on anxiety and depression in university students during COVID-19: A randomized controlled trial.

The COVID-19 pandemic has had adverse mental health effects for many groups in British society, especially young adults and university students. The present study reports secondary outcomes (i.e., symptoms of anxiety and depression) from a randomized waitlist controlled trial, with a one-month post-intervention follow-up, on the effects of a guided, eight-week mindfulness program delivered online during the COVID-19 pandemic among students at the University of Oxford. Longitudinal multilevel models showed greater reductions in anxiety but not depression symptoms for participants in the mindfulness condition relative to participants in the waitlist control condition (time X group B=-0.36, p=.025).

Water quality performance of wetlands receiving nonpoint-source nitrogen loads: Nitrate and total nitrogen removal efficiency and controlling factors.

Nonpoint-source nitrogen (N) loads in the U.S. Corn Belt are a major concern both for local impacts on receiving waters and for contributing to hypoxia in the Gulf of Mexico. Nonpoint-source nutrient loads can be ameliorated by a combination of in-field and offsite practices, and wetland restoration is a particularly promising approach for reducing N loads from agricultural drainage. However, there is considerable variability among wetlands, and adequate performance data are available for relatively few systems receiving unregulated nonpoint-source loads. We measured N mass balances of 26 restored wetlands receiving a wide range of unregulated, naturally varying hydraulic and nutrient loads to evaluate the N removal performance of these systems and the effects of major factors controlling their performance. Nitrogen loads were primarily in the form of nitrate, and all of the wetlands were effective in reducing both nitrate and total N loads. Nitrate N and total N removal rates averaged 1,500 and 1,440 kg N ha-1  yr-1 , respectively, with the slightly lower total N removal rates reflecting a small net export of reduced N (averaging 66 kg N ha-1  yr-1 ). Average nitrate and total N removal rates were substantially higher than typically reported for Corn Belt wetlands but comparable with highly loaded systems elsewhere. Nitrate removal efficiency ranged from 9 to 92% and was strongly related to hydraulic loading rate and temperature. Results demonstrate the substantial capacity of wetlands to reduce unregulated and highly variable nonpoint-source N loads over a broad range of weather and loading conditions and provide a reasonable basis for predicting average wetland performance based on hydraulic loading rate, temperature, and nitrate concentration.

Percutaneous CT guided bone biopsy for suspected osteomyelitis: Diagnostic yield and impact on patient's treatment change and recovery.

AIM: To evaluate the utility of percutaneous CT guided bone biopsy (PCBB) for suspected osteomyelitis (OM) and its eventual impact on patient management and recovery. MATERIAL AND METHODS: Patients who received a PCBB for suspected osteomyelitis from years 2012-2018. Patient demographics, lesion location, ulcer grade, signs of toxemia, serology, wound and blood cultures, bone biopsy and cross-sectional imaging results were recorded. Diagnostic yield of the bone biopsy and its role in influencing the final treatment plan and patient recovery were evaluated. Chi-square test was used. P-value less than 0.05 was considered statistically significant. RESULTS: 115 patients with mean age 50.86 ± 14.49 years were included. The common locations were sacrum/ischium (49/115, 43%) and spine (35/115, 30%). Clinically, 40/115 (35%) had toxemia and 67/115 (58%) had ulcers. Per serology, 17/111 (15%), 95/106 (90%), and 86/98 (88%) had an elevated WBC, CRP, and sedimentation rate, respectively. 22/91 (24%) had a positive blood culture and all 23/23 had a positive wound culture. On imaging, definitive and possible OM were reported in 84.1% and 14.2%, respectively, with 1.8% as no OM. Only 24/115 (21%) had a positive bone biopsy culture and only 10/24 (42%) total positive bone cultures impacted the treatment plan. There was no significant effect of antibiotics on the diagnostic yield of culture (p = 0.08). No statistical significance was found when comparing treatment change based on bone culture results versus all other factors combined (p = 0.33), or when comparing clinical improvement with and without positive bone cultures (p = 0.12). CONCLUSION: Despite positive cross-sectional imaging findings of OM, bone biopsy yield of positive culture is low, and it leads to a small impact in changing the treatment plan or altering the course of patient recovery.

miRNA Profiling of Magnetic Nanopore-Isolated Extracellular Vesicles for the Diagnosis of Pancreatic Cancer.

Improved diagnostics for pancreatic ductal adenocarcinoma (PDAC) to detect the disease at earlier, curative stages and to guide treatments is crucial to progress against this disease. The development of a liquid biopsy for PDAC has proven challenging due to the sparsity and variable phenotypic expression of circulating biomarkers. Here we report methods we developed for isolating specific subsets of extracellular vesicles (EV) from plasma using a novel magnetic nanopore capture technique. In addition, we present a workflow for identifying EV miRNA biomarkers using RNA sequencing and machine-learning algorithms, which we used in combination to classify distinct cancer states. Applying this approach to a mouse model of PDAC, we identified a biomarker panel of 11 EV miRNAs that could distinguish mice with PDAC from either healthy mice or those with precancerous lesions in a training set of n = 27 mice and a user-blinded validation set of n = 57 mice (88% accuracy in a three-way classification). These results provide strong proof-of-concept support for the feasibility of using EV miRNA profiling and machine learning for liquid biopsy.Significance: These findings present a panel of extracellular vesicle miRNA blood-based biomarkers that can detect pancreatic cancer at a precancerous stage in a transgenic mouse model. Cancer Res; 78(13); 3688-97. ©2018 AACR.

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.

Pathology-radiology correlation of common and uncommon computed tomographic patterns of organizing pneumonia.

Organizing pneumonia (OP) is a common pattern of lung injury that can be associated with a wide range of etiologies. Typical and not-so-typical imaging features of OP occur, as both common and rare lung pathologies can mimic the same imaging pattern as that of OP. This article will attempt to describe the difference between confusing terminologies that have been used in the past for OP and existence of primary versus secondary OP. The role of a multidisciplinary approach as an essential component to correctly diagnose and effectively manage challenging cases of OP will be highlighted. Additionally, we will discuss the limitation of transbronchial and importance of open lung biopsy to make the correct diagnosis. One example of an emerging diagnosis in the spectrum of OP and diffuse alveolar damage is acute fibrinous and organizing pneumonia. Ultimately, the reader should feel comfortable recognizing the many variable presentations of OP and be able to participate knowledgeably in a multidisciplinary team after reading this article. OP is a disease entity with variable radiographic and distinct histological characteristics that requires a multidisciplinary approach to correctly diagnose cryptogenic OP. Classic radiologic findings of OP occur in as low as 60% of cases. Secondary causes include infections, neoplasms, inflammatory disorders, and iatrogenic. Acute fibrinous and organizing pneumonia can appear similarly, but miliary nodules are a clue to diagnosis.

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.

Fate of triazoles in softwood upon environmental exposure.

Determining the fate of preservatives in commercial wood products is essential to minimize their losses and improve protective impregnation techniques. The fate of triazole fungicides in ponderosa pine wood was investigated in both outdoor and controlled-environment experiments using a representative triazole, tebuconazole (TAZ), which was accompanied by propiconazole (PAZ) in selected experiments. The study was designed to mimic industrial settings used in window frame manufacturing. To investigate the TAZ fate in detail, loosely and strongly bound fractions were differentiated using a multi-step extraction. The loosely bound TAZ fraction extracted through two sonications accounted for 85± 5% of the total TAZ, while the strongly bound TAZ was extracted only with an exhaustive Soxhlet extraction and corresponded to the remaining 15± 5%. A significant fraction (∼80%) of the original TAZ remained in the wood despite a six-month exposure to harsh environmental conditions, maintaining wood preservation and assuring minimal environmental impact. Depletion of loosely bound TAZ was observed from cross-sectional surfaces when exposed to rain, high humidity and sunlight. Water leaching was deemed to be the major route leading to triazole losses from wood. Leaching rate was found to be slightly higher for TAZ than for PAZ. The contribution of bio-, photo- and thermal degradation of triazoles was negligible as both PAZ and TAZ sorbed in wood remained intact. Triazole evaporation was also found to be minor at the moderate temperature (20-25 °C) recorded throughout the outdoor study.

Symptomatic Parietal Intradiploic Encephalocele-A Case Report and Literature Review.

Encephalocele is a rare condition that consists of herniation of cerebral matter through openings of dura and skull. A majority of encephaloceles are congenital and manifest in childhood. We present a case of a 45-year-old man presenting with contralateral hemiparesis and found to have an extremely rare phenomenon of a symptomatic posttraumatic parietal intradiploic encephalocele (IE) manifesting 36 years following pediatric traumatic head injury. Computed tomography and magnetic resonance imaging confirmed herniation of brain tissue into the intradiploic space. Surgical treatment with reduction of the encephalocele achieved near resolution of preoperative hemiparesis on follow-up. The pathogenesis and a literature review of IE are discussed.