Prevalence of glaucoma in Canada: results from the 2016-2019 Canadian Health Measures Survey.

OBJECTIVE: To estimate the prevalence of glaucoma in Canada based on self-reports and test data, including Frequency Doubling Technology Perimetry (FDT), optic nerve vertical cup-to-disc ratio (CDR), intraocular pressure (IOP), and use of glaucoma medications. DESIGN: Cross-sectional survey. PARTICIPANTS: 2,600-4,100 participants aged 40-79 in the Canadian Health Measures Survey 2016-2019 with available information from self-report, CDR, FDT, and IOP. METHODS: Glaucoma was defined by self-reports, CDR ≥ 0.7 only, or failed FDT only. Incorporating results of CDR, FDT, IOP, and use of glaucoma medications, participants were further classified as definite glaucoma (failed FDT and CDR ≥ 0.7) or glaucoma suspects (CDR ≥ 0.7 only, failed FDT only, or IOP > 21 mmHg only, or "normal" values of FDT, CDR, and IOP but used glaucoma medications). Survey weights were used in analyses. RESULTS: The glaucoma prevalence was 2.5% (95% confidence interval [CI] 1.7%-3.3%) utilizing self-reports, 3.0% (95% CI 2.1%-3.9%) by CDR ≥ 0.7 only and 10.3% (7.8%-12.8%) with failed FDT only. Merging test data, the prevalence of definite glaucoma was 0.7% (95% CI 0.3%-1.1%) and the prevalence of suspected glaucoma was 16.3% (95% CI 13.2%-19.4%). Among the patients suspected of having glaucoma, 44.4% had ocular hypertension (OHT, mean IOP 22.8 mmHg) and 6.8% used glaucoma medications. IOP ≥28 mmHg was found in 2.4% of OHT individuals, and none used glaucoma medications.37.5% of Canadians with definite glaucoma were unaware they had glaucoma. CONCLUSIONS: Glaucoma prevalence in Canadians aged 40-79 varied between 0.7% and 10.3% depending on definition used. 16.3% of Canadians were labeled "glaucoma suspects". Nearly 40% of Canadians with definite glaucoma were unaware of having glaucoma.

A methodology for gene level omics-WAS integration identifies genes influencing traits associated with cardiovascular risks: the Long Life Family Study.

The Long Life Family Study (LLFS) enrolled 4953 participants in 539 pedigrees displaying exceptional longevity. To identify genetic mechanisms that affect cardiovascular risks in the LLFS population, we developed a multi-omics integration pipeline and applied it to 11 traits associated with cardiovascular risks. Using our pipeline, we aggregated gene-level statistics from rare-variant analysis, GWAS, and gene expression-trait association by Correlated Meta-Analysis (CMA). Across all traits, CMA identified 64 significant genes after Bonferroni correction (p ≤ 2.8 × 10-7), 29 of which replicated in the Framingham Heart Study (FHS) cohort. Notably, 20 of the 29 replicated genes do not have a previously known trait-associated variant in the GWAS Catalog within 50 kb. Thirteen modules in Protein-Protein Interaction (PPI) networks are significantly enriched in genes with low meta-analysis p-values for at least one trait, three of which are replicated in the FHS cohort. The functional annotation of genes in these modules showed a significant over-representation of trait-related biological processes including sterol transport, protein-lipid complex remodeling, and immune response regulation. Among major findings, our results suggest a role of triglyceride-associated and mast-cell functional genes FCER1A, MS4A2, GATA2, HDC, and HRH4 in atherosclerosis risks. Our findings also suggest that lower expression of ATG2A, a gene we found to be associated with BMI, may be both a cause and consequence of obesity. Finally, our results suggest that ENPP3 may play an intermediary role in triglyceride-induced inflammation. Our pipeline is freely available and implemented in the Nextflow workflow language, making it easily runnable on any compute platform ( https://nf-co.re/omicsgenetraitassociation ).

MAPLES-DR: MESSIDOR Anatomical and Pathological Labels for Explainable Screening of Diabetic Retinopathy.

Reliable automatic diagnosis of Diabetic Retinopathy (DR) and Macular Edema (ME) is an invaluable asset in improving the rate of monitored patients among at-risk populations and in enabling earlier treatments before the pathology progresses and threatens vision. However, the explainability of screening models is still an open question, and specifically designed datasets are required to support the research. We present MAPLES-DR (MESSIDOR Anatomical and Pathological Labels for Explainable Screening of Diabetic Retinopathy), which contains, for 198 images of the MESSIDOR public fundus dataset, new diagnoses for DR and ME as well as new pixel-wise segmentation maps for 10 anatomical and pathological biomarkers related to DR. This paper documents the design choices and the annotation procedure that produced MAPLES-DR, discusses the interobserver variability and the overall quality of the annotations, and provides guidelines on using the dataset in a machine learning context.

Novel Loci (EIF4A2, ADIPOQ, TPRG1) for Triglyceride / High-density Lipoprotein Cholesterol Ratio Longitudinal Change (ΔTHR) among Subjects without Type 2 Diabetes: Evidence from the Long Life Family Study (LLFS) and the Framingham Heart Study (FHS) Offspring Cohort (OS).

Aims/hypothesis: Triglyceride (TG) /High density lipoprotein cholesterol (HDL-C) ratio (THR) represents a single surrogate predictor of hyperinsulinemia or insulin resistance that is associated with premature aging processes, risk of diabetes and increased mortality. To identify novel genetic loci for THR change over time (ΔTHR), we conducted genome-wide association study (GWAS) and genome-wide linkage scan (GWLS) among subjects of European ancestry who had complete data from two exams collected about seven years apart from the Long Life Family Study (LLFS, n=1384), a study with familial clustering of exceptional longevity in the US and Denmark. Methods: Subjects with diabetes or using medications for dyslipidemia were excluded from this analysis. ΔTHR was derived using growth curve modeling, and adjusted for age, sex, field centers, and principal components (PCs). GWAS was conducted using a linear mixed model accounted for familial relatedness. Our linkage scan was built on haplotype-based IBD estimation with 0.5 cM average spacing. Results: Heritability of ΔTHR was moderate (46%). Our GWAS identified a significant locus at the LPL (p=1.58e-9) for ΔTHR; this gene locus has been reported before influencing baseline THR levels. Our GWLS found evidence for a significant linkage with a logarithm of the odds (LODs) exceeding 3 on 3q28 (LODs=4.1). Using a subset of 25 linkage enriched families (pedigree-specific LODs>0.1), we assessed sequence elements under 3q28 and identified two novel variants (EIF4A2/ADIPOQ-rs114108468, p=5e-6, MAF=1.8%; TPRG1-rs16864075, p=3e-6, MAF=8%; accounted for ~28% and ~29% of the linkage, respectively, and 57% jointly). While the former variant was associated with EIF4A2 (p=7e-5) / ADIPOQ (p=3.49e-2) RNA transcriptional levels, the latter variant was not associated with TPRG1 (p=0.23) RNA transcriptional levels. Replication in FHS OS observed modest effect of these loci on ΔTHR. Of 188 metabolites from 13 compound classes assayed in LLFS, we observed multiple metabolites (e.g., DG.38.5, PE.36.4, TG.58.3) that were significantly associated with the variants (p<3e-4). Conclusions: our linkage-guided sequence analysis approach permitted our discovery of two novel gene variants EIF4A2/ADIPOQ-rs114108468 and TPRG1-rs16864075 on 3q28 for ΔTHR among subjects without diabetes selected for exceptional survival and healthy aging.

A Novel Gene ARHGAP44 for Longitudinal Changes in Glycated Hemoglobin (HbA1c) in Subjects without Type 2 Diabetes: Evidence from the Long Life Family Study (LLFS) and the Framingham Offspring Study (FOS).

Glycated hemoglobin (HbA1c) indicates average glucose levels over three months and is associated with insulin resistance and type 2 diabetes (T2D). Longitudinal changes in HbA1c (ΔHbA1c) are also associated with aging processes, cognitive performance, and mortality. We analyzed ΔHbA1c in 1,886 non-diabetic Europeans from the Long Life Family Study to uncover gene variants influencing ΔHbA1c. Using growth curve modeling adjusted for multiple covariates, we derived ΔHbA1c and conducted linkage-guided sequence analysis. Our genome-wide linkage scan identified a significant locus on 17p12. In-depth analysis of this locus revealed a variant rs56340929 (explaining 27% of the linkage peak) in the ARHGAP44 gene that was significantly associated with ΔHbA1c. RNA transcription of ARHGAP44 was associated with ΔHbA1c. The Framingham Offspring Study data further supported these findings on the gene level. Together, we found a novel gene ARHGAP44 for ΔHbA1c in family members without T2D. Follow-up studies using longitudinal omics data in large independent cohorts are warranted.

Discovery of genomic and transcriptomic pleiotropy between kidney function and soluble receptor for advanced glycation end products using correlated meta-analyses: The Long Life Family Study.

Patients with chronic kidney disease (CKD) have increased oxidative stress and chronic inflammation, which may escalate the production of advanced glycation end-products (AGEs). High soluble receptor for AGE (sRAGE) and low estimated glomerular filtration rate (eGFR) levels are associated with CKD and aging. We evaluated whether eGFR calculated from creatinine and cystatin C share pleiotropic genetic factors with sRAGE. We employed whole-genome sequencing and correlated meta-analyses on combined genome-wide association study (GWAS) p-values in 4182 individuals (age range: 24-110) from the Long Life Family Study (LLFS). We also conducted transcriptome-wide association studies (TWAS) on whole blood in a subset of 1209 individuals. We identified 59 pleiotropic GWAS loci (p < 5 × 10-8) and 17 TWAS genes (Bonferroni-p < 2.73 × 10-6) for eGFR traits and sRAGE. TWAS genes, LSP1 and MIR23AHG, were associated with eGFR and sRAGE located within GWAS loci, lncRNA-KCNQ1OT1 and CACNA1A/CCDC130, respectively. GWAS variants were eQTLs in the kidney glomeruli and tubules, and GWAS genes predicted kidney carcinoma. TWAS genes harbored eQTLs in the kidney, predicted kidney carcinoma, and connected enhancer-promoter variants with kidney function-related phenotypes at p < 5 × 10-8. Additionally, higher allele frequencies of protective variants for eGFR traits were detected in LLFS than in ALFA-Europeans and TOPMed, suggesting better kidney function in healthy-aging LLFS than in general populations. Integrating genomic annotation and transcriptional gene activity revealed the enrichment of genetic elements in kidney function and aging-related processes. The identified pleiotropic loci and gene expressions for eGFR and sRAGE suggest their underlying shared genetic effects and highlight their roles in kidney- and aging-related signaling pathways.

Whole Genome Linkage and Association Analyses Identify DLG Associated Protein-1 as a Novel Positional and Biological Candidate Gene for Muscle Strength: The Long Life Family Study.

BACKGROUND: Grip strength is a robust indicator of overall health, is moderately heritable, and predicts longevity in older adults. METHODS: Using genome-wide linkage analysis, we identified a novel locus on chromosome 18p (mega-basepair region: 3.4-4.0) linked to grip strength in 3 755 individuals from 582 families aged 64 ±â€…12 years (range 30-110 years; 55% women). There were 26 families that contributed to the linkage peak (cumulative logarithm of the odds [LOD] score = 10.94), with 6 families (119 individuals) accounting for most of the linkage signal (LOD = 6.4). In these 6 families, using whole genome sequencing data, we performed association analyses between the 7 312 single nucleotide (SNVs) and insertion deletion (INDELs) variants in the linkage region and grip strength. Models were adjusted for age, age2, sex, height, field center, and population substructure. RESULTS: We found significant associations between genetic variants (8 SNVs and 4 INDELs, p < 5 × 10-5) in the Disks Large-associated Protein 1 (DLGAP1) gene and grip strength. Haplotypes constructed using these variants explained up to 98.1% of the LOD score. Finally, RNAseq data showed that these variants were significantly associated with the expression of nearby Myosin Light Chain 12A (MYL12A), Structural Maintenance of Chromosomes Flexible Hinge Domain Containing 1 (SMCHD1), Erythrocyte Membrane Protein Band 4.1 Like 3 (EPB41L3) genes (p < .0004). CONCLUSIONS: The DLGAP1 gene plays an important role in the postsynaptic density of neurons; thus, it is both a novel positional and biological candidate gene for follow-up studies aimed at uncovering genetic determinants of muscle strength.

The response of Dual-leucine zipper kinase (DLK) to nocodazole: Evidence for a homeostatic cytoskeletal repair mechanism.

Genetic and pharmacological perturbation of the cytoskeleton enhances the regenerative potential of neurons. This response requires Dual-leucine Zipper Kinase (DLK), a neuronal stress sensor that is a central regulator of axon regeneration and degeneration. The damage and repair aspects of this response are reminiscent of other cellular homeostatic systems, suggesting that a cytoskeletal homeostatic response exists. In this study, we propose a framework for understanding DLK mediated neuronal cytoskeletal homeostasis. We demonstrate that low dose nocodazole treatment activates DLK signaling. Activation of DLK signaling results in a DLK-dependent transcriptional signature, which we identify through RNA-seq. This signature includes genes likely to attenuate DLK signaling while simultaneously inducing actin regulating genes. We identify alterations to the cytoskeleton including actin-based morphological changes to the axon. These results are consistent with the model that cytoskeletal disruption in the neuron induces a DLK-dependent homeostatic mechanism, which we term the Cytoskeletal Stress Response (CSR) pathway.

Diabetic Disease of the Eye in Canada: Consensus Statements from a Retina Specialist Working Group.

Despite advances in systemic care, diabetic disease of the eye (DDE) remains the leading cause of blindness worldwide. There is a critical gap of up-to-date, evidence-based guidance for ophthalmologists in Canada that includes evidence from recent randomized controlled trials. Previous guidance has not always given special consideration to applying treatments and managing DDE in the context of the healthcare system. This consensus statement aims to assist practitioners in the field by providing a spectrum of acceptable opinions on DDE treatment and management from recognized experts in the field. In compiling evidence and generating consensus, a working group of retinal specialists in Canada addressed clinical questions surrounding the four themes of disease, patient, management, and collaboration. The working group reviewed literature representing the highest level of evidence on DDE and shared their opinions on topics surrounding the epidemiology and pathophysiology of diabetic retinopathy and diabetic macular edema; diagnosis and monitoring; considerations around diabetes medication use; strategic considerations for management given systemic comorbidities, ocular comorbidities, and pregnancy; treatment goals and modalities for diabetic macular edema, non-proliferative and proliferative diabetic retinopathy, and retinal detachment; and interdisciplinary collaboration. Ultimately, this work highlighted that the retinal examination in DDE not only informs the treating ophthalmologist but can serve as a global index for disease progression across many tissues of the body. It highlighted further that DDE can be treated regardless of diabetic control, that a systemic approach to patient care will result in the best health outcomes, and prevention of visual complications requires a multidisciplinary management approach. Ophthalmologists must tailor their clinical approach to the needs and circumstances of individual patients and work within the realities of their healthcare setting.

Multi-omics Integration Identifies Genes Influencing Traits Associated with Cardiovascular Risks: The Long Life Family Study.

The Long Life Family Study (LLFS) enrolled 4,953 participants in 539 pedigrees displaying exceptional longevity. To identify genetic mechanisms that affect cardiovascular risks in the LLFS population, we developed a multi-omics integration pipeline and applied it to 11 traits associated with cardiovascular risks. Using our pipeline, we aggregated gene-level statistics from rare-variant analysis, GWAS, and gene expression-trait association by Correlated Meta-Analysis (CMA). Across all traits, CMA identified 64 significant genes after Bonferroni correction (p ≤ 2.8×10-7), 29 of which replicated in the Framingham Heart Study (FHS) cohort. Notably, 20 of the 29 replicated genes do not have a previously known trait-associated variant in the GWAS Catalog within 50 kb. Thirteen modules in Protein-Protein Interaction (PPI) networks are significantly enriched in genes with low meta-analysis p-values for at least one trait, three of which are replicated in the FHS cohort. The functional annotation of genes in these modules showed a significant over-representation of trait-related biological processes including sterol transport, protein-lipid complex remodeling, and immune response regulation. Among major findings, our results suggest a role of triglyceride-associated and mast-cell functional genes FCER1A, MS4A2, GATA2, HDC, and HRH4 in atherosclerosis risks. Our findings also suggest that lower expression of ATG2A, a gene we found to be associated with BMI, may be both a cause and consequence of obesity. Finally, our results suggest that ENPP3 may play an intermediary role in triglyceride-induced inflammation. Our pipeline is freely available and implemented in the Nextflow workflow language, making it easily runnable on any compute platform (https://nf-co.re/omicsgenetraitassociation).

Fasted C-Peptide Distribution and Associated Clinical Factors in Adults With Longstanding Type 1 Diabetes: Analysis of the Canadian Study of Longevity in Type 1 Diabetes.

OBJECTIVE: Although insulin production is reportedly retained in many people with longstanding type 1 diabetes (T1D), the magnitude and relevance of connecting peptide (C-peptide) production are uncertain. In this study, we aimed to define fasted C-peptide distributions and associated clinical factors. METHODS: In a cross-sectional analysis of the Canadian Study of Longevity, fasted serum and urinary C-peptide was measured in 74 patients with longstanding T1D (duration ≥50 years) and 75 age- and sex-matched controls. Extensive phenotyping for complications was performed and patient-reported variables were included. C-peptide distributions were analyzed, and multivariable logistic regression was used to assess the variable association in participants with T1D. RESULTS: The 74 participants with T1D had a mean age of 66±8 years, a disease duration of 54 (interquartile range 52 to 58) years, and a glycated hemoglobin (A1C) of 7.4%±0.8% (56.8±9.15 mmol/mol). The 75 controls had a mean age of 65±8 years and an A1C of 5.7%±0.4% (38.4±4.05 mmol/mol). Participants with T1D had lower fasted serum C-peptide than controls (0.013±0.022 vs 1.595±1.099 nmol/L, p<0.001). Of the participants with T1D, C-peptide was detectable in 30 of 73 (41%) serum samples, 32 of 74 (43%) urine samples, and 48 of 74 (65%) for either serum or urine. The variables independently associated with detectable serum or urinary C-peptide were lower total daily insulin requirement (odds ratio 2.351 [for 1 lower unit/kg], p=0.013) and lower hypoglycemia worry score (odds ratio 1.059 [for 1 point lower on the worry subscore of the Hypoglycemia Fear Survey], p=0.030). CONCLUSIONS: Although detectable C-peptide in longstanding diabetes was common, the magnitude of concentration was extremely low when compared with age- and sex-matched controls. Despite minimal detectability, its presence is validated by lower insulin requirements and strongly associated with lower hypoglycemia worry.

Prevalence of self-reported visual impairment among people in Canada with and without diabetes: findings from population-based surveys from 1994 to 2014.

BACKGROUND: Diabetes, a leading cause of visual impairment, is on the rise in Canada. We assessed trends in the prevalence of visual impairment among people in Canada with and without diabetes to inform the development of strategies and policies for the management of visual impairment. METHODS: We analyzed self-reported data from respondents aged 45 years and older in 7 cycles of nationwide surveys (National Population Health Survey and Canadian Community Health Survey) from 1994/95 to 2013/14. The age- and sex-standardized prevalence of visual impairment was calculated. We assessed comparisons by levels of education and income, using sex-standardized prevalence owing to sparse data. RESULTS: Among people in Canada with diabetes, the age- and sex-standardized prevalence of visual impairment was 7.37% (95% confidence interval [CI] 5.31%-9.43%) in 1994/95 and 1996/97 combined, decreasing to 3.03% (95% CI 2.48%-3.57%) in 2013/14, giving a standardized prevalence ratio of 0.41 (95% CI 0.30-0.56) comparing 2013/14 with 1994/95 and 1996/97 combined. Among people in Canada without diabetes, visual impairment prevalence decreased from 3.72% (95% CI 3.31%-4.14%) in 1994/95 and 1996/97 combined to 1.69% (95% CI 1.52%-1.87%) in 2013/14, with a standardized prevalence ratio of 0.45 (95% CI 0.40-0.52). Decreased sex-standardized prevalence of visual impairment was observed among people with high and low education levels and incomes among those with and without diabetes. INTERPRETATION: Visual impairment prevalence was roughly 2 times higher among those with versus without diabetes in all survey years; from 1994 to 2014, visual impairment prevalence decreased among those with and without diabetes irrespective of education and income levels. These results suggest effective collective efforts by clinicians, researchers, the public and government.

Unbiased discovery of natural sequence variants that influence fungal virulence.

Isolates of Cryptococcus neoformans, a fungal pathogen that kills over 112,000 people each year, differ from a 19-Mb reference genome at a few thousand up to almost a million DNA sequence positions. We used bulked segregant analysis and association analysis, genetic methods that require no prior knowledge of sequence function, to address the key question of which naturally occurring sequence variants influence fungal virulence. We identified a region containing such variants, prioritized them, and engineered strains to test our findings in a mouse model of infection. At one locus, we identified a 4-nt variant in the PDE2 gene that occurs in common laboratory strains and severely truncates the encoded phosphodiesterase. The resulting loss of phosphodiesterase activity significantly impacts virulence. Our studies demonstrate a powerful and unbiased strategy for identifying key genomic regions in the absence of prior information and provide significant sequence and strain resources to the community.

The response of Dual-Leucine Zipper Kinase (DLK) to nocodazole: evidence for a homeostatic cytoskeletal repair mechanism.

Genetic and pharmacological perturbation of the cytoskeleton enhances the regenerative potential of neurons. This response requires Dual-leucine Zipper Kinase (DLK), a neuronal stress sensor that is a central regulator of axon regeneration and degeneration. The damage and repair aspects of this response are reminiscent of other cellular homeostatic systems, suggesting that a cytoskeletal homeostatic response exists. In this study, we propose a framework for understanding DLK mediated neuronal cytoskeletal homeostasis. We demonstrate that a) low dose nocodazole treatment activates DLK signaling and b) DLK signaling mitigates the microtubule damage caused by the cytoskeletal perturbation. We also perform RNA-seq to discover a DLK-dependent transcriptional signature. This signature includes genes likely to attenuate DLK signaling while simultaneously inducing actin regulating genes and promoting actin-based morphological changes to the axon. These results are consistent with the model that cytoskeletal disruption in the neuron induces a DLK-dependent homeostatic mechanism, which we term the Cytoskeletal Stress Response (CSR) pathway.

Assessing the feasibility, acceptability, and fidelity of a tele-retinopathy-based intervention to encourage greater attendance to diabetic retinopathy screening in immigrants living with diabetes from China and African-Caribbean countries in Ottawa, Canada: a protocol.

BACKGROUND: Diabetic retinopathy is a leading cause of preventable blindness in Canada. Clinical guidelines recommend annual diabetic retinopathy screening for people living with diabetes to reduce the risk and progression of vision loss. However, many Canadians with diabetes do not attend screening. Screening rates are even lower in immigrants to Canada including people from China, Africa, and the Caribbean, and these groups are also at higher risk of developing diabetes complications. We aim to assess the feasibility, acceptability, and fidelity of a co-developed, linguistically and culturally tailored tele-retinopathy screening intervention for Mandarin-speaking immigrants from China and French-speaking immigrants from African-Caribbean countries living with diabetes in Ottawa, Canada, and identify how many from each population group attend screening during the pilot period. METHODS: We will work with our health system and patient partners to conduct a 6-month feasibility pilot of a tele-retinopathy screening intervention in a Community Health Centre in Ottawa. We anticipate recruiting 50-150 patients and 5-10 health care providers involved in delivering the intervention for the pilot. Acceptability will be assessed via a Theoretical Framework of Acceptability-informed survey with patients and health care providers. To assess feasibility, we will use a Theoretical Domains Framework-informed interview guide and to assess fidelity, and we will use a survey informed by the National Institutes of Health framework from the perspective of health care providers. We will also collect patient demographics (i.e., age, gender, ethnicity, health insurance status, and immigration information), screening outcomes (i.e., patients with retinopathy identified, patients requiring specialist care), patient costs, and other intervention-related variables such as preferred language. Survey data will be descriptively analyzed and qualitative data will undergo content analysis. DISCUSSION: This feasibility pilot study will capture how many people living with diabetes from each group attend the diabetic retinopathy screening, costs, and implementation processes for the tele-retinopathy screening intervention. The study will indicate the practicability and suitability of the intervention in increasing screening attendance in the target population groups. The study results will inform a patient-randomized trial, provide evidence to conduct an economic evaluation of the intervention, and optimize the community-based intervention.

Calling Cards: A Customizable Platform to Longitudinally Record Protein-DNA Interactions Over Time in Cells and Tissues.

Calling Cards is a platform technology to record a cumulative history of transient protein-DNA interactions in the genome of genetically targeted cell types. The record of these interactions is recovered by next-generation sequencing. Compared with other genomic assays, readouts of which provide a snapshot at the time of harvest, Calling Cards enables correlation of historical molecular states to eventual outcomes or phenotypes. To achieve this, Calling Cards uses the piggyBac transposase to insert self-reporting transposon "Calling Cards" into the genome, leaving permanent marks at interaction sites. Calling Cards can be deployed in a variety of in vitro and in vivo biological systems to study gene regulatory networks involved in development, aging, and disease. Out of the box, it assesses enhancer usage but can be adapted to profile-specific transcription factor (TF) binding with custom TF-piggyBac fusion proteins. The Calling Cards workflow has five main stages: delivery of Calling Cards reagents, sample preparation, library preparation, sequencing, and data analysis. Here, we first present a comprehensive guide for experimental design, reagent selection, and optional customization of the platform to study additional TFs. Then, we provide an updated protocol for the five steps, using reagents that improve throughput and decrease costs, including an overview of a newly deployed computational pipeline. This protocol is designed for users with basic molecular biology experience to process samples into sequencing libraries in 2 days. Familiarity with bioinformatic analysis and command line tools is required to set up the pipeline in a high-performance computing environment and to conduct downstream analyses. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Preparation and delivery of Calling Cards reagents Support Protocol 1: Next-generation sequencing quantification of barcode distribution within self-reporting transposon plasmid pool and adeno-associated virus genome Basic Protocol 2: Sample collection and RNA purification Support Protocol 2: Library density quantitative PCR Basic Protocol 3: Sequencing library preparation Basic Protocol 4: Library pooling and sequencing Basic Protocol 5: Data analysis.

Calling Cards: a customizable platform to longitudinally record protein-DNA interactions over time in cells and tissues.

Calling Cards is a platform technology to record a cumulative history of transient protein-DNA interactions in the genome of genetically targeted cell types. The record of these interactions is recovered by next generation sequencing. Compared to other genomic assays, whose readout provides a snapshot at the time of harvest, Calling Cards enables correlation of historical molecular states to eventual outcomes or phenotypes. To achieve this, Calling Cards uses the piggyBac transposase to insert self-reporting transposon (SRT) "Calling Cards" into the genome, leaving permanent marks at interaction sites. Calling Cards can be deployed in a variety of in vitro and in vivo biological systems to study gene regulatory networks involved in development, aging, and disease. Out of the box, it assesses enhancer usage but can be adapted to profile specific transcription factor binding with custom transcription factor (TF)-piggyBac fusion proteins. The Calling Cards workflow has five main stages: delivery of Calling Card reagents, sample preparation, library preparation, sequencing, and data analysis. Here, we first present a comprehensive guide for experimental design, reagent selection, and optional customization of the platform to study additional TFs. Then, we provide an updated protocol for the five steps, using reagents that improve throughput and decrease costs, including an overview of a newly deployed computational pipeline. This protocol is designed for users with basic molecular biology experience to process samples into sequencing libraries in 1-2 days. Familiarity with bioinformatic analysis and command line tools is required to set up the pipeline in a high-performance computing environment and to conduct downstream analyses. Basic Protocol 1: Preparation and delivery of Calling Cards reagentsBasic Protocol 2: Sample preparationBasic Protocol 3: Sequencing library preparationBasic Protocol 4: Library pooling and sequencingBasic Protocol 5: Data analysis.

How host-like signals drive gene expression and gene expression drives capsule expansion in Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen with a polysaccharide capsule that becomes greatly enlarged in the mammalian host and during in vitro growth in response to host-like conditions. To understand how individual host-like signals affect capsule size and gene expression, we grew cells with and without all combinations of 5 signals suspected of affecting capsule size and systematically measured cell and capsule sizes of 47,458 cells. We also collected samples for RNA-Seq at 30, 90, 180, and 1440 minutes and carried out RNA-Seq in quadruplicate, yielding 881 RNA-Seq samples. This massive, uniformly collected dataset will be a significant resource for the research community. Analysis revealed that capsule induction requires both tissue culture medium and either CO2 or exogenous cyclic AMP, a second messenger. Rich medium (YPD) blocks capsule growth completely, DMEM permits it, and RPMI yields the largest capsules. Medium has the biggest impact on overall gene expression, followed by CO2, mammalian body temperature (37° compared to 30°), and then cAMP. Surprisingly, adding CO2 or cAMP pushes overall gene expression in the opposite direction from tissue culture media, even though both tissue culture medium and CO2 or cAMP are required for capsule development. By modeling the relationship between gene expression and capsule size, we identified novel genes whose deletion affects capsule size.