Community-developed checklists for publishing images and image analyses.

Images document scientific discoveries and are prevalent in modern biomedical research. Microscopy imaging in particular is currently undergoing rapid technological advancements. However, for scientists wishing to publish obtained images and image-analysis results, there are currently no unified guidelines for best practices. Consequently, microscopy images and image data in publications may be unclear or difficult to interpret. Here, we present community-developed checklists for preparing light microscopy images and describing image analyses for publications. These checklists offer authors, readers and publishers key recommendations for image formatting and annotation, color selection, data availability and reporting image-analysis workflows. The goal of our guidelines is to increase the clarity and reproducibility of image figures and thereby to heighten the quality and explanatory power of microscopy data.

Single-cell and single-nucleus RNA-sequencing from paired normal-adenocarcinoma lung samples provide both common and discordant biological insights.

Whether single-cell RNA-sequencing (scRNA-seq) captures the same biological information as single-nucleus RNA-sequencing (snRNA-seq) remains uncertain and likely to be context-dependent. Herein, a head-to-head comparison was performed in matched normal-adenocarcinoma human lung samples to assess biological insights derived from scRNA-seq versus snRNA-seq and better understand the cellular transition that occurs from normal to tumoral tissue. Here, the transcriptome of 160,621 cells/nuclei was obtained. In non-tumor lung, cell type proportions varied widely between scRNA-seq and snRNA-seq with a predominance of immune cells in the former (81.5%) and epithelial cells (69.9%) in the later. Similar results were observed in adenocarcinomas, in addition to an overall increase in cell type heterogeneity and a greater prevalence of copy number variants in cells of epithelial origin, which suggests malignant assignment. The cell type transition that occurs from normal lung tissue to adenocarcinoma was not always concordant whether cells or nuclei were examined. As expected, large differential expression of the whole-cell and nuclear transcriptome was observed, but cell-type specific changes of paired normal and tumor lung samples revealed a set of common genes in the cells and nuclei involved in cancer-related pathways. In addition, we showed that the ligand-receptor interactome landscape of lung adenocarcinoma was largely different whether cells or nuclei were evaluated. Immune cell depletion in fresh specimens partly mitigated the difference in cell type composition observed between cells and nuclei. However, the extra manipulations affected cell viability and amplified the transcriptional signatures associated with stress responses. In conclusion, research applications focussing on mapping the immune landscape of lung adenocarcinoma benefit from scRNA-seq in fresh samples, whereas snRNA-seq of frozen samples provide a low-cost alternative to profile more epithelial and cancer cells, and yield cell type proportions that more closely match tissue content.

Hierarchical organization of cortical and thalamic connectivity.

The mammalian cortex is a laminar structure containing many areas and cell types that are densely interconnected in complex ways, and for which generalizable principles of organization remain mostly unknown. Here we describe a major expansion of the Allen Mouse Brain Connectivity Atlas resource1, involving around a thousand new tracer experiments in the cortex and its main satellite structure, the thalamus. We used Cre driver lines (mice expressing Cre recombinase) to comprehensively and selectively label brain-wide connections by layer and class of projection neuron. Through observations of axon termination patterns, we have derived a set of generalized anatomical rules to describe corticocortical, thalamocortical and corticothalamic projections. We have built a model to assign connection patterns between areas as either feedforward or feedback, and generated testable predictions of hierarchical positions for individual cortical and thalamic areas and for cortical network modules. Our results show that cell-class-specific connections are organized in a shallow hierarchy within the mouse corticothalamic network.

A Test to Comprehensively Capture the Known Genetic Component of Familial Pulmonary Fibrosis.

The recent European Respiratory Society statement on familial pulmonary fibrosis supports the need for genetic testing in the care of patients and their relatives. However, no solution (i.e., a concrete test) was provided to implement genetic testing in daily practice. Herein, we tabulated and standardized the nomenclature of 128 genetic variants in 20 genes implicated in adult-onset pulmonary fibrosis. The objective was to develop a laboratory-developed test (LDT) on the basis of standard Sanger sequencing to capture all known familial pulmonary fibrosis-associated variants. Targeted DNA fragments were amplified using harmonized PCR conditions to perform the LDT in a single 96-well plate. The new genetic test was evaluated in 62 sporadic cases of idiopathic pulmonary fibrosis. As expected in this population, we observed a low yield of disease-causing mutations. More important, 100% of targeted variants by the LDT were successfully evaluated. Furthermore, four variants of uncertain significance with in silico-predicted deleterious scores were identified in three patients, suggesting novel pathogenic variants in genes known to cause idiopathic pulmonary fibrosis. Finally, the MUC5B promoter variant rs35705950 was strongly enriched in these patients with a minor allele frequency of 41.1% compared with 10.6% in a matched population-based cohort (n = 29,060), leading to an estimation that this variant may explain up to 35% of the population-attributable risk. This LDT provides a solution for rapid clinical translation. Technical laboratory details are provided so that specialized pulmonary centers can implement the LDT in house to expedite the clinical recommendations of expert panels.

Novel filamin C (FLNC) variant causes a severe form of familial mixed hypertrophic-restrictive cardiomyopathy.

Variants of filamin C (FLNC) have been identified as rare genetic substrate for hypertrophic cardiomyopathy (HCM). Data on the clinical course of FLNC-related HCM are conflicting with some studies suggesting mild phenotypes whereas other studies have reported more severe outcomes. In this study, we present a novel FLNC variant (Ile1937Asn) that was identified in a large family of French-Canadian descent with excellent segregation data. FLNC-Ile1937Asn is a novel missense variant characterized by full penetrance and poor clinical outcomes. End stage heart failure requiring transplantation occurred in 43% and sudden cardiac death in 29% of affected family members. Other particular features of FLNC-Ile1937Asn include an early disease onset (mean age of 19 years) and the development of a marked atrial myopathy (severe biatrial dilatation with remodeling and multiple complex atrial arrhythmias) that was present in all gene carriers. The FLNC-Ile1937Asn variant is a novel, pathogenic mutation resulting in a severe form of HCM with full disease penetrance. The variant is associated with a high proportion of end-stage heart failure, heart transplantation, and disease-related mortality. Close follow-up and appropriate risk stratification of affected individuals at specialized heart centers is recommended.

A deep generative model of 3D single-cell organization.

We introduce a framework for end-to-end integrative modeling of 3D single-cell multi-channel fluorescent image data of diverse subcellular structures. We employ stacked conditional ß-variational autoencoders to first learn a latent representation of cell morphology, and then learn a latent representation of subcellular structure localization which is conditioned on the learned cell morphology. Our model is flexible and can be trained on images of arbitrary subcellular structures and at varying degrees of sparsity and reconstruction fidelity. We train our full model on 3D cell image data and explore design trade-offs in the 2D setting. Once trained, our model can be used to predict plausible locations of structures in cells where these structures were not imaged. The trained model can also be used to quantify the variation in the location of subcellular structures by generating plausible instantiations of each structure in arbitrary cell geometries. We apply our trained model to a small drug perturbation screen to demonstrate its applicability to new data. We show how the latent representations of drugged cells differ from unperturbed cells as expected by on-target effects of the drugs.

Leucocyte-derived micro-RNAs as candidate biomarkers in Brugada syndrome.

AIMS: Risk stratification for sudden cardiac death in patients with Brugada syndrome remains a major challenge. Contemporary risk prediction models have only modest predictive value. The aim of this study was to assess the role of micro-RNAs from peripheral blood as candidate biomarkers in Brugada syndrome. METHODS AND RESULTS: In this prospective study, Brugada patients and unaffected control individuals were enrolled for analysis of leucocyte-derived microRNAs (miRNAs) levels. Expression levels of 798 different circulating miRNAs were analysed on the NanoString® nCounter platform. All results were cross-validated by using a quantitative polymerase chain reaction. Micro-RNA expression levels of Brugada patients were compared with clinical data. A total of 21 definite Brugada patients (38% with a history of ventricular arrhythmia or cardiac arrest) and 30 unaffected control individuals were included in the study. Micro-RNA analysis showed a distinct expression profile in Brugada patients with 42 differentially expressed markers (38 up-regulated, 4 down-regulated miRNAs). The symptom status of Brugada patients was associated with a distinct miRNA signature. Micro-RNAs 145-5p and 585-3p were significantly up-regulated in symptomatic Brugada patients (P = 0.04). Incorporating miRNAs 145-5p and 585-3p into a multivariable model demonstrated significantly increased symptom prediction (area under the curve = 0.96; 95% confidence interval: 0.88-1.00). CONCLUSION: Brugada patients display a distinct miRNA expression profile compared with unaffected control individuals. There is also evidence that certain miRNAs (miR-145-5p and miR-585-3p) are associated with the symptom status of Brugada patients. The results suggest the principal utility of leucocyte-derived miRNAs as prognostic biomarkers for Brugada syndrome.

Intra- and inter-evaluator reliability of the MyotonPRO for the assessment of the viscoelastic properties of caesarean section scar and unscarred skin.

BACKGROUND: The clinical evaluation of viscoelastic properties of the Caesarean section (C-section) scar, such as stiffness and elasticity, is usually carried out using subjective scales and palpation techniques. There is currently no reliable and valid tool that objectively quantifies these properties. The MyotonPRO could fill this gap. MATERIALS AND METHODS: Nineteen healthy women aged between 21 and 40 years with C-section scars participated in this reliability study. Two points, one on the scar and one on unscarred skin, were measured four times successively with the MyotonPRO by three independent evaluators on the same day. The intra-class correlation (ICC) coefficients were estimated using a two-factor ANOVA to determine the inter- and intra-rater reliability. The capacity of the MyotonPRO to discriminate the viscoelastic properties of the C-Section scar against unscarred skin was assessed using the Wilcoxon signed rank test. RESULTS: The intra- and inter-rater reliability of the viscoelastic property measurements was good to excellent (ICC 0.99-1.00 and 0.87-0.98, respectively). There was no significant difference between C-section scar and unscarred skin in terms of elasticity (P = .737). Significant differences between C-section scars and unscarred skin tissue were observed for tone (P < .001), stiffness (P < .001), creep (P < .001), and mechanical stress relaxation time (P < .001). CONCLUSION: The MyotonPRO is a reliable tool for an objective measurement of the viscoelastic properties of the C-section scar and unscarred skin. The MyotonPRO can discriminate the viscoelastic properties of the C-section scar against the unscarred skin, for tone, stiffness, creep and relaxation times, but not for elasticity.

Transcriptome-wide association study reveals candidate causal genes for lung cancer.

We have recently completed the largest GWAS on lung cancer including 29,266 cases and 56,450 controls of European descent. The goal of our study has been to integrate the complete GWAS results with a large-scale expression quantitative trait loci (eQTL) mapping study in human lung tissues (n = 1,038) to identify candidate causal genes for lung cancer. We performed transcriptome-wide association study (TWAS) for lung cancer overall, by histology (adenocarcinoma, squamous cell carcinoma and small cell lung cancer) and smoking subgroups (never- and ever-smokers). We performed replication analysis using lung data from the Genotype-Tissue Expression (GTEx) project. DNA damage assays were performed in human lung fibroblasts for selected TWAS genes. As expected, the main TWAS signal for all histological subtypes and ever-smokers was on chromosome 15q25. The gene most strongly associated with lung cancer at this locus using the TWAS approach was IREB2 (pTWAS = 1.09E-99), where lower predicted expression increased lung cancer risk. A new lung adenocarcinoma susceptibility locus was revealed on 9p13.3 and associated with higher predicted expression of AQP3 (pTWAS = 3.72E-6). Among the 45 previously described lung cancer GWAS loci, we mapped candidate target gene for 17 of them. The association AQP3-adenocarcinoma on 9p13.3 was replicated using GTEx (pTWAS = 6.55E-5). Consistent with the effect of risk alleles on gene expression levels, IREB2 knockdown and AQP3 overproduction promote endogenous DNA damage. These findings indicate genes whose expression in lung tissue directly influences lung cancer risk.

Granularity of SERPINA1 alleles by DNA sequencing in CanCOLD.

DNA sequencing of the SERPINA1 gene to detect α1-antitrypsin (AAT) deficiency (AATD) may provide a better appreciation of the individual and cumulative impact of genetic variants on AAT serum levels and COPD phenotypes.AAT serum level and DNA sequencing of the coding regions of SERPINA1 were performed in 1359 participants of the Canadian Cohort Obstructive Lung Disease (CanCOLD) study. Clinical assessment for COPD included questionnaires, pulmonary function testing and computed tomography (CT) imaging. Phenotypes were tested for association with SERPINA1 genotypes collated into four groups: normal (MM), mild (MS and MI), intermediate (heterozygote MZ, non-S/non-Z/non-I, compound IS, and homozygote SS) and severe (ZZ and SZ) deficiency. Smoking strata and MZ-only analyses were also performed.34 genetic variants were identified including 25 missense mutations. Overall, 8.1% of alleles in this Canadian cohort were deficient and 15.5% of 1359 individuals were carriers of at least one deficient allele. Four AATD subjects were identified and had statistically lower diffusion capacity and greater CT-based emphysema. No COPD phenotypes were associated with mild and intermediate AATD in the overall cohort or stratified by smoking status. MZ heterozygotes had similar CT-based emphysema, but lowered diffusion capacity compared with normal and mild deficiency.In this Canadian population-based cohort, comprehensive genetic testing for AATD reveals a variety of deficient alleles affecting 15.5% of subjects. COPD phenotype was demonstrated in severe deficiency and MZ heterozygotes. This study shows the feasibility of implementing a diagnostic test for AATD using DNA sequencing in a large cohort.

Transcriptomic data helps refining classification of pulmonary carcinoid tumors with increased mitotic counts.

Pulmonary neuroendocrine neoplasms are classified by WHO as either typical or atypical carcinoids, large cell (LCNEC) or small cell (SCLC) neuroendocrine carcinoma based on mitotic count, morphology, and necrosis assessment. LCNEC with low mitotic count and sharing morphologic features with carcinoids are in a gray zone for classification and their rare prevalence and the paucity of studies precludes proper validation of the current grading system. In this study, we aim to investigate their clinicopathological and transcriptomic profiles. Lung resection specimens obtained from 18 patients diagnosed with carcinoids or LCNEC were selected. Four of them were characterized as borderline tumors based on a mitotic rate ranging between 10 and 30 mitoses per 2 mm2. Comprehensive morphological and immunohistochemical (IHC) evaluation was performed and tumor-based transcriptomic profiles were analyzed through unsupervised clustering. Clustering analysis revealed two distinct molecular groups characterized by low (C1) and high (C2) proliferation. C1 was comprised of seven carcinoids and three borderline tumors, while C2 was comprised of seven LCNEC and one borderline tumor. Furthermore, patients in cluster C1 had a better recurrence-free survival compared with patients in cluster C2 (20% vs 75%). Histological features, IHC profile, and molecular analysis showed that three out of four borderline tumors showed features consistent with carcinoids. Therefore, our findings convey that the current diagnostic guidelines are suboptimal for classification of pulmonary neuroendocrine tumors with increased proliferative index and carcinoid-like morphology. These results support the emerging concept that neuroendocrine tumors with carcinoid-like features and mitotic count of <20 mitoses per 2 mm2 should be regarded as pulmonary carcinoids instead of LCNEC.

Altered DNA Methylation of Long Noncoding RNA H19 in Calcific Aortic Valve Disease Promotes Mineralization by Silencing NOTCH1.

BACKGROUND: Calcific aortic valve disease is characterized by an abnormal mineralization of the aortic valve. Osteogenic activity in the aortic valve is under the control of NOTCH1, which regulates the expression of key pro-osteogenic genes such as RUNX2 and BMP2. Long noncoding RNAs (lncRNAs) may reprogram cells by altering the gene expression pattern. METHODS: Multidimensional genomic profiling was performed in human aortic valves to document the expression of lncRNAs and the DNA methylation pattern in calcific aortic valve disease. In-depth functional assays were carried out to document the impact of lncRNA on the mineralization of the aortic valve. RESULTS: We documented that lncRNA H19 (H19) was increased in calcific aortic valve disease. Hypomethylation of the promoter region was observed in mineralized aortic valves and was inversely associated with H19 expression. Knockdown and overexpression experiments showed that H19 induces a strong osteogenic phenotype by altering the NOTCH1 pathway. Gene promoter analyses showed that H19 silenced NOTCH1 by preventing the recruitment of p53 to its promoter. A knockdown of H19 in valve interstitial cells (VICs) increased the expression of NOTCH1 and decreased the level of RUNX2 and BMP2, 2 downstream targets repressed by NOTCH1. In rescue experiments, the transfection of a vector encoding for the active Notch intracellular domain prevented H19-induced mineralization of valve interstitial cells. CONCLUSIONS: These findings indicate that a dysregulation of DNA methylation in the promoter of H19 during calcific aortic valve disease is associated with a higher expression of this lncRNA, which promotes an osteogenic program by interfering with the expression of NOTCH1.

RNA expression profile of calcified bicuspid, tricuspid, and normal human aortic valves by RNA sequencing.

The molecular mechanisms leading to premature development of aortic valve stenosis (AS) in individuals with a bicuspid aortic valve are unknown. The objective of this study was to identify genes differentially expressed between calcified bicuspid aortic valves (BAVc) and tricuspid valves with (TAVc) and without (TAVn) AS using RNA sequencing (RNA-Seq). We collected 10 human BAVc and nine TAVc from men who underwent primary aortic valve replacement. Eight TAVn were obtained from men who underwent heart transplantation. mRNA levels were measured by RNA-Seq and compared between valve groups. Two genes were upregulated, and none were downregulated in BAVc compared with TAVc, suggesting a similar gene expression response to AS in individuals with bicuspid and tricuspid valves. There were 462 genes upregulated and 282 downregulated in BAVc compared with TAVn. In TAVc compared with TAVn, 329 genes were up- and 170 were downregulated. A total of 273 upregulated and 147 downregulated genes were concordantly altered between BAVc vs. TAVn and TAVc vs. TAVn, which represent 56 and 84% of significant genes in the first and second comparisons, respectively. This indicates that extra genes and pathways were altered in BAVc. Shared pathways between calcified (BAVc and TAVc) and normal (TAVn) aortic valves were also more extensively altered in BAVc. The top pathway enriched for genes differentially expressed in calcified compared with normal valves was fibrosis, which support the remodeling process as a therapeutic target. These findings are relevant to understand the molecular basis of AS in patients with bicuspid and tricuspid valves.

Hyperglycemia impairs atherosclerosis regression in mice.

Diabetic patients are known to be more susceptible to atherosclerosis and its associated cardiovascular complications. However, the effects of hyperglycemia on atherosclerosis regression remain unclear. We hypothesized that hyperglycemia impairs atherosclerosis regression by modulating the biological function of lesional macrophages. HypoE (Apoe(h/h)Mx1-Cre) mice express low levels of apolipoprotein E (apoE) and develop atherosclerosis when fed a high-fat diet. Atherosclerosis regression occurs in these mice upon plasma lipid lowering induced by a change in diet and the restoration of apoE expression. We examined the morphological characteristics of regressed lesions and assessed the biological function of lesional macrophages isolated with laser-capture microdissection in euglycemic and hyperglycemic HypoE mice. Hyperglycemia induced by streptozotocin treatment impaired lesion size reduction (36% versus 14%) and lipid loss (38% versus 26%) after the reversal of hyperlipidemia. However, decreases in lesional macrophage content and remodeling in both groups of mice were similar. Gene expression analysis revealed that hyperglycemia impaired cholesterol transport by modulating ATP-binding cassette A1, ATP-binding cassette G1, scavenger receptor class B family member (CD36), scavenger receptor class B1, and wound healing pathways in lesional macrophages during atherosclerosis regression. Hyperglycemia impairs both reduction in size and loss of lipids from atherosclerotic lesions upon plasma lipid lowering without significantly affecting the remodeling of the vascular wall.

Inducible ApoE gene repair in hypomorphic ApoE mice deficient in the low-density lipoprotein receptor promotes atheroma stabilization with a human-like lipoprotein profile.

OBJECTIVE: To study atherosclerosis regression in mice after plasma lipid reduction to moderately elevated apolipoprotein B (apoB)-lipoprotein levels. APPROACH AND RESULTS: Chow-fed hypomorphic Apoe mice deficient in low-density lipoprotein receptor expression (Apoe(h/h)Ldlr(-/-)Mx1-cre mice) develop hyperlipidemia and atherosclerosis. These mice were studied before and after inducible cre-mediated Apoe gene repair. By 1 week, induced mice displayed a 2-fold reduction in plasma cholesterol and triglyceride levels and a decrease in the non-high-density lipoprotein:high-density lipoprotein-cholesterol ratio from 87%:13% to 60%:40%. This halted atherosclerotic lesion growth and promoted macrophage loss and accumulation of thick collagen fibers for up to 8 weeks. Concomitantly, blood Ly-6C(high) monocytes were decreased by 2-fold but lesional macrophage apoptosis was unchanged. The expression of several genes involved in extracellular matrix remodeling and cell migration was changed in lesional macrophages 1 week after Apoe gene repair. However, mRNA levels of numerous genes involved in cholesterol efflux and inflammation were not significantly changed at this time point. CONCLUSIONS: Restoring apoE expression in Apoe(h/h)Ldlr(-/-)Mx1-cre mice resulted in lesion stabilization in the context of a human-like ratio of non-high-density lipoprotein:high-density lipoprotein-cholesterol. Our data suggest that macrophage loss derived in part from reduced blood Ly-6C(high) monocytes levels and genetic reprogramming of lesional macrophages.

Prognostic implication of methylation-based circulating tumor DNA detection prior to surgery in stage I non-small cell lung cancer.

BACKGROUND: Circulating tumor DNA (ctDNA) positivity at diagnosis, which is associated with worse outcomes in multiple solid tumors including stage I-III non-small cell lung cancer (NSCLC), may have utility to guide (neo)adjuvant therapy. METHODS: In this retrospective study, 260 patients with clinical stage I NSCLC (180 adenocarcinoma, 80 squamous cell carcinoma) were allocated (2:1) to high- and low-risk groups based on relapse versus disease-free status ≤5 years post-surgery. We evaluated the association of preoperative ctDNA detection by a plasma-only targeted methylation-based multi-cancer early detection (MCED) test with NSCLC relapse ≤5 years post-surgery in the overall population, followed by histology-specific subgroup analyses. RESULTS: Across clinical stage I patients, preoperative ctDNA detection did not associate with relapse within 5 years post-surgery. Sub-analyses confined to lung adenocarcinoma suggested a histology-specific association between ctDNA detection and outcome. In this group, ctDNA positivity tended to associate with relapse within 2 years, suggesting prognostic implications of MCED test positivity may be histology- and time-dependent in stage I NSCLC. Preoperative ctDNA detection was associated with upstaging of clinical stage I to pathological stage II-III NSCLC. CONCLUSIONS: Our findings suggest preoperative ctDNA detection in patients with resectable clinical stage I NSCLC using MCED, a pan-cancer screening test developed for use in an asymptomatic population, has no detectable prognostic value for relapse ≤5 years post-surgery. MCED detection may be associated with early adenocarcinoma relapse and increased pathological upstaging rates in stage I NSCLC. However, given the exploratory nature of these findings, independent validation is required.

Integrative genomic analyses identify candidate causal genes for calcific aortic valve stenosis involving tissue-specific regulation.

There is currently no medical therapy to prevent calcific aortic valve stenosis (CAVS). Multi-omics approaches could lead to the identification of novel molecular targets. Here, we perform a genome-wide association study (GWAS) meta-analysis including 14,819 cases among 941,863 participants of European ancestry. We report 32 genomic loci, among which 20 are novel. RNA sequencing of 500 human aortic valves highlights an enrichment in expression regulation at these loci and prioritizes candidate causal genes. Homozygous genotype for a risk variant near TWIST1, a gene involved in endothelial-mesenchymal transition, has a profound impact on aortic valve transcriptomics. We identify five genes outside of GWAS loci by combining a transcriptome-wide association study, colocalization, and Mendelian randomization analyses. Using cross-phenotype and phenome-wide approaches, we highlight the role of circulating lipoproteins, blood pressure and inflammation in the disease process. Our findings pave the way for the development of novel therapies for CAVS.