Sphingolipids in Childhood Asthma and Obesity (SOAP Study): A Protocol of a Cross-Sectional Study.

Asthma and obesity are two of the most common chronic conditions in children and adolescents. There is increasing evidence that sphingolipid metabolism is altered in childhood asthma and is linked to airway hyperreactivity. Dysregulated sphingolipid metabolism is also reported in obesity. However, the functional link between sphingolipid metabolism, asthma, and obesity is not completely understood. This paper describes the protocol of an ongoing study on sphingolipids that aims to examine the pathophysiology of sphingolipids in childhood asthma and obesity. In addition, this study aims to explore the novel biomarkers through a comprehensive multi-omics approach including genomics, genome-wide DNA methylation, RNA-Seq, microRNA (miRNA) profiling, lipidomics, metabolomics, and cytokine profiling. This is a cross-sectional study aiming to recruit 440 children from different groups: children with asthma and normal weight (n = 100), asthma with overweight or obesity (n = 100), overweight or obesity (n = 100), normal weight (n = 70), and siblings of asthmatic children with normal weight, overweight, or obesity (n = 70). These participants will be recruited from the pediatric pulmonology, pediatric endocrinology, and general pediatric outpatient clinics at Sidra Medicine, Doha, Qatar. Information will be obtained from self-reported questionnaires on asthma, quality of life, food frequency (FFQ), and a 3-day food diary that are completed by the children and their parents. Clinical measurements will include anthropometry, blood pressure, biochemistry, bioelectrical impedance, and pulmonary function tests. Blood samples will be obtained for sphingolipid analysis, serine palmitoyltransferase (SPT) assay, whole-genome sequencing (WGS), genome-wide DNA methylation study, RNA-Seq, miRNA profiling, metabolomics, lipidomics, and cytokine analysis. Group comparisons of continuous outcome variables will be carried out by a one-way analysis of variance or the Kruskal-Wallis test using an appropriate pairwise multiple comparison test. The chi-squared test or a Fisher's exact test will be used to test the associations between categorical variables. Finally, multivariate analysis will be carried out to integrate the clinical data with multi-omics data. This study will help us to understand the role of dysregulated sphingolipid metabolism in obesity and asthma. In addition, the multi-omics data from the study will help to identify novel genetic and epigenetic signatures, inflammatory markers, and mechanistic pathways that link asthma and obesity in children. Furthermore, the integration of clinical and multi-omics data will help us to uncover the potential interactions between these diseases and to offer a new paradigm for the treatment of pediatric obesity-associated asthma.

Genome-Wide RNA Tomography in the Mouse Whole Olfactory Mucosa.

Spatial transcriptomics allows for the genome-wide profiling of topographic gene expression patterns within a tissue of interest. Here, we describe our methodology to generate high-quality RNA-seq libraries from cryosections from fresh frozen mouse whole olfactory mucosae. This methodology can be extended to virtually any vertebrate organ or tissue sample.

Genome-wide association of dry (Tamar) date palm fruit color.

Date palm (Phoenix dactylifera) fruit (dates) are an economically and culturally significant crop in the Middle East and North Africa. There are hundreds of different commercial cultivars producing dates with distinctive shapes, colors, and sizes. Genetic studies of some date palm traits have been performed, including sex determination, sugar content, and fresh fruit color. In this study, we used genome sequences and image data of 199 dry dates (Tamar) collected from 14 countries to identify genetic loci associated with the color of this fruit stage. Here, we find loci across multiple linkage groups (LG) associated with dry fruit color phenotype. We recover both the previously identified VIRESCENS (VIR) genotype associated with fresh fruit yellow or red color and new associations with the lightness and darkness of dry fruit. This study will add resolution to our understanding of date color phenotype, especially at the most commercially important Tamar stage.

An integrated tumor, immune and microbiome atlas of colon cancer.

The lack of multi-omics cancer datasets with extensive follow-up information hinders the identification of accurate biomarkers of clinical outcome. In this cohort study, we performed comprehensive genomic analyses on fresh-frozen samples from 348 patients affected by primary colon cancer, encompassing RNA, whole-exome, deep T cell receptor and 16S bacterial rRNA gene sequencing on tumor and matched healthy colon tissue, complemented with tumor whole-genome sequencing for further microbiome characterization. A type 1 helper T cell, cytotoxic, gene expression signature, called Immunologic Constant of Rejection, captured the presence of clonally expanded, tumor-enriched T cell clones and outperformed conventional prognostic molecular biomarkers, such as the consensus molecular subtype and the microsatellite instability classifications. Quantification of genetic immunoediting, defined as a lower number of neoantigens than expected, further refined its prognostic value. We identified a microbiome signature, driven by Ruminococcus bromii, associated with a favorable outcome. By combining microbiome signature and Immunologic Constant of Rejection, we developed and validated a composite score (mICRoScore), which identifies a group of patients with excellent survival probability. The publicly available multi-omics dataset provides a resource for better understanding colon cancer biology that could facilitate the discovery of personalized therapeutic approaches.

Technical assessment of different extraction methods and transcriptome profiling of RNA isolated from small volumes of blood.

Transcriptome profiling of human whole blood is used to discover biomarkers of diseases and to assess phenotypic traits. Recently, finger-stick blood collection systems have allowed a less invasive and quicker collection of peripheral blood. Such non-invasive sampling of small volumes of blood offers practical advantages. The quality of gene expression data is strictly dependent on the steps used for the sample collection, extraction, preparation and sequencing. Here we have: (i) compared the manual and automated RNA extraction of small volumes of blood using the Tempus Spin RNA isolation kit and the MagMAX for Stabilized Blood RNA Isolation kit , respectively; and (ii) assessed the effect of TURBO DNA Free treatment on the transcriptomic data of RNA isolated from small volumes of blood. We have used the QuantSeq 3' FWD mRNA-Seq Library Prep kit to prepare RNA-seq libraries, which were sequenced on the Illumina NextSeq 500 system. The samples isolated manually displayed a higher variability in the transcriptomic data as compared to the other samples. The TURBO DNA Free treatment affected the RNA samples negatively, decreasing the RNA yield and reducing the quality and reproducibility of the transcriptomic data. We conclude that automated extraction systems should be preferred over manual extraction systems for data consistency, and that the TURBO DNA Free treatment should be avoided when working on RNA samples isolated manually from small volumes of blood.

High-temporal resolution profiling reveals distinct immune trajectories following the first and second doses of COVID-19 mRNA vaccines.

Knowledge of the mechanisms underpinning the development of protective immunity conferred by mRNA vaccines is fragmentary. Here, we investigated responses to coronavirus disease 2019 (COVID-19) mRNA vaccination via high-temporal resolution blood transcriptome profiling. The first vaccine dose elicited modest interferon and adaptive immune responses, which peaked on days 2 and 5, respectively. The second vaccine dose, in contrast, elicited sharp day 1 interferon, inflammation, and erythroid cell responses, followed by a day 5 plasmablast response. Both post-first and post-second dose interferon signatures were associated with the subsequent development of antibody responses. Yet, we observed distinct interferon response patterns after each of the doses that may reflect quantitative or qualitative differences in interferon induction. Distinct interferon response phenotypes were also observed in patients with COVID-19 and were associated with severity and differences in duration of intensive care. Together, this study also highlights the benefits of adopting high-frequency sampling protocols in profiling vaccine-elicited immune responses.

Transcriptome Profile Identifies Actin as an Essential Regulator of Cardiac Myosin Binding Protein C3 Hypertrophic Cardiomyopathy in a Zebrafish Model.

Variants in cardiac myosin-binding protein C (cMyBP-C) are the leading cause of inherited hypertrophic cardiomyopathy (HCM), demonstrating the key role that cMyBP-C plays in the heart's contractile machinery. To investigate the c-MYBPC3 HCM-related cardiac impairment, we generated a zebrafish mypbc3-knockout model. These knockout zebrafish displayed significant morphological heart alterations related to a significant decrease in ventricular and atrial diameters at systolic and diastolic states at the larval stages. Immunofluorescence staining revealed significant hyperplasia in the mutant's total cardiac and ventricular cardiomyocytes. Although cardiac contractility was similar to the wild-type control, the ejection fraction was significantly increased in the mypbc3 mutants. At later stages of larval development, the mutants demonstrated an early cardiac phenotype of myocardium remodeling, concurrent cardiomyocyte hyperplasia, and increased ejection fraction as critical processes in HCM initiation to counteract the increased ventricular myocardial wall stress. The examination of zebrafish adults showed a thickened ventricular cardiac wall with reduced heart rate, swimming speed, and endurance ability in both the mypbc3 heterozygous and homozygous groups. Furthermore, heart transcriptome profiling showed a significant downregulation of the actin-filament-based process, indicating an impaired actin cytoskeleton organization as the main dysregulating factor associated with the early ventricular cardiac hypertrophy in the zebrafish mypbc3 HCM model.

Micro-dissection and integration of long and short reads to create a robust catalog of kidney compartment-specific isoforms.

Studying isoform expression at the microscopic level has always been a challenging task. A classical example is kidney, where glomerular and tubulo-interstitial compartments carry out drastically different physiological functions and thus presumably their isoform expression also differs. We aim at developing an experimental and computational pipeline for identifying isoforms at microscopic structure-level. We microdissected glomerular and tubulo-interstitial compartments from healthy human kidney tissues from two cohorts. The two compartments were separately sequenced with the PacBio RS II platform. These transcripts were then validated using transcripts of the same samples by the traditional Illumina RNA-Seq protocol, distinct Illumina RNA-Seq short reads from European Renal cDNA Bank (ERCB) samples, and annotated GENCODE transcript list, thus identifying novel transcripts. We identified 14,739 and 14,259 annotated transcripts, and 17,268 and 13,118 potentially novel transcripts in the glomerular and tubulo-interstitial compartments, respectively. Of note, relying solely on either short or long reads would have resulted in many erroneous identifications. We identified distinct pathways involved in glomerular and tubulo-interstitial compartments at the isoform level, creating an important experimental and computational resource for the kidney research community.

A 3D transcriptomics atlas of the mouse nose sheds light on the anatomical logic of smell.

The sense of smell helps us navigate the environment, but its molecular architecture and underlying logic remain understudied. The spatial location of odorant receptor genes (Olfrs) in the nose is thought to be independent of the structural diversity of the odorants they detect. Using spatial transcriptomics, we create a genome-wide 3D atlas of the mouse olfactory mucosa (OM). Topographic maps of genes differentially expressed in space reveal that both Olfrs and non-Olfrs are distributed in a continuous and overlapping fashion over at least five broad zones in the OM. The spatial locations of Olfrs correlate with the mucus solubility of the odorants they recognize, providing direct evidence for the chromatographic theory of olfaction. This resource resolves the molecular architecture of the mouse OM and will inform future studies on mechanisms underlying Olfr gene choice, axonal pathfinding, patterning of the nervous system, and basic logic for the peripheral representation of smell.

Deletion of beta-fructofuranosidase (invertase) genes is associated with sucrose content in Date Palm fruit.

The fruit of date palm trees are an important part of the diet for a large portion of the Middle East and North Africa. The fruit is consumed both fresh and dry and can be stored dry for extended periods of time. Date fruits vary significantly across hundreds of cultivars identified in the main regions of cultivation. Most dried date fruit are low in sucrose but high in glucose and fructose. However, high sucrose content is a distinctive feature of some date fruit and affects flavor as well as texture and water retention. To identify the genes controlling high sucrose content, we analyzed date fruit metabolomics for association with genotype data from 120 date fruits. We found significant association of dried date sucrose content and a genomic region that contains 3 tandem copies of the beta-fructofuranosidase (invertase) gene in the reference Khalas genome, a low-sucrose fruit. High-sucrose cultivars including the popular Deglet Noor had a homozygous deletion of two of the 3 copies of the invertase gene. We show the deletion allele is derived when compared to the ancestral allele that retains all copies of the gene in 3 other species of Phoenix. The fact that 2 of the 3 tandem invertase copies are associated with dry fruit sucrose content will assist in better understanding the distinct roles of multiple date palm invertases in plant physiology. Identification of the recessive alleles associated with end-point sucrose content in date fruit may be used in selective breeding in the future.

Differential regulation of the immune system in a brain-liver-fats organ network during short-term fasting.

OBJECTIVE: Fasting regimens can promote health, mitigate chronic immunological disorders, and improve age-related pathophysiological parameters in animals and humans. Several ongoing clinical trials are using fasting as a potential therapy for various conditions. Fasting alters metabolism by acting as a reset for energy homeostasis, but the molecular mechanisms underlying the beneficial effects of short-term fasting (STF) are not well understood, particularly at the systems or multiorgan level. METHODS: We performed RNA-sequencing in nine organs from mice fed ad libitum (0 h) or subjected to fasting five times (2-22 h). We applied a combination of multivariate analysis, differential expression analysis, gene ontology, and network analysis for an in-depth understanding of the multiorgan transcriptome. We used literature mining solutions, LitLab™ and Gene Retriever™, to identify the biological and biochemical terms significantly associated with our experimental gene set, which provided additional support and meaning to the experimentally derived gene and inferred protein data. RESULTS: We cataloged the transcriptional dynamics within and between organs during STF and discovered differential temporal effects of STF among organs. Using gene ontology enrichment analysis, we identified an organ network sharing 37 common biological pathways perturbed by STF. This network incorporates the brain, liver, interscapular brown adipose tissue, and posterior-subcutaneous white adipose tissue; hence, we named it the brain-liver-fats organ network. Using Reactome pathways analysis, we identified the immune system, dominated by T cell regulation processes, as a central and prominent target of systemic modulations during STF in this organ network. The changes we identified in specific immune components point to the priming of adaptive immunity and parallel the fine-tuning of innate immune signaling. CONCLUSIONS: Our study provides a comprehensive multiorgan transcriptomic profiling of mice subjected to multiple periods of STF and provides new insights into the molecular modulators involved in the systemic immunotranscriptomic changes that occur during short-term energy loss.

Novel subpopulations in date palm (Phoenix dactylifera) identified by population-wide organellar genome sequencing.

BACKGROUND: The date palm is one of the oldest cultivated fruit trees. The tree can withstand high temperatures and low water and the fruit can be stored dry offering nutrition across the year. The first region of cultivation is believed to be near modern day Iraq, however, where and if the date palm was domesticated is still a topic of debate. Recent studies of chloroplast and genomic DNA revealed two major subpopulations of cultivars centered in both the Eastern range of date palm cultivation including Arabian Peninsula, Iraq and parts of South Asia, and the Western range, including North Africa. RESULTS: To better understand the origins of date palm cultivation we sequenced and analyzed over 200 mitochondrial and chloroplast genomes from a geographically diverse set of date palms. Here we show that, based on mitochondrial and chloroplast genome-wide genotyping data, the most common cultivated date palms contain 4 haplotypes that appear associated with geographical region of cultivar origin. CONCLUSIONS: These data suggest at least 3 and possibly 4 original maternal contributions to the current date palm population and doubles the original number. One new haplotype was found mainly in Tunisia, Algeria and Egypt and the second in Iraq, Iran and Oman. We propose that earliest date palm cultivation occurred independently in at least 3 distinct locations. This discovery will further inform understanding of the history and origins of cultivated date palm.

Author Correction: Genus-wide sequencing support a two-locus model for sex-determination in Phoenix.

In the original version of this Article, the affiliation of the first author, Maria F. Torres, 'Department of Biological Sciences, University of Cincinnati, Cincinnati, 45221, OH, USA' was incorrectly assigned as a present address and should have been listed as a full affiliation. This error has been corrected in both the PDF and HTML versions of the Article.

Genotyping-by-sequencing identifies date palm clone preference in agronomics of the State of Qatar.

Understanding the genetic diversity in a crop population is key to its targeted breeding for desired traits, such as higher yields, better fruit quality and resistance to disease and changing climates. Date fruits represent a major crop in the Middle East and are key to achieving future food independence in arid countries like Qatar. We previously determined the genome of the date palm Phoenix dactylifera and showed that date palm trees world-wide divide into two distinct subpopulations of Eastern and Western origins. Here we applied a resource of SNPs from 179 commercially available date fruits to assess the genetic diversity of date palm trees grown in the State of Qatar. We found that palm trees in Qatar are mainly of Eastern origin, and that their genetic diversity doesn't associate with regions of the State. Together with targeted genetic assays, our resource can be used in the future for date palm cultivar identification, to aid selecting suitable cultivars for targeted breeding, to improve a country's date palm genetic diversity, and to certify the origin of date fruits and trees.

Genus-wide sequencing supports a two-locus model for sex-determination in Phoenix.

The date palm tree is a commercially important member of the genus Phoenix whose 14 species are dioecious with separate male and female individuals. To identify sex determining genes we sequenced the genomes of 15 female and 13 male Phoenix trees representing all 14 species. We identified male-specific sequences and extended them using phased single-molecule sequencing or BAC clones. We observed that only four genes contained sequences conserved in all analyzed Phoenix males. Most of these sequences showed similarity to a single genomic locus in the closely related monoecious oil palm. CYP703 and GPAT3, two single copy genes present in males and critical for male flower development in other monocots, were absent in females. A LOG-like gene appears translocated into the Y-linked region and is suggested to play a role in suppressing female flowers. Our data are consistent with a two-mutation model for the evolution of dioecy in Phoenix.

Life-threatening influenza pneumonitis in a child with inherited IRF9 deficiency.

Life-threatening pulmonary influenza can be caused by inborn errors of type I and III IFN immunity. We report a 5-yr-old child with severe pulmonary influenza at 2 yr. She is homozygous for a loss-of-function IRF9 allele. Her cells activate gamma-activated factor (GAF) STAT1 homodimers but not IFN-stimulated gene factor 3 (ISGF3) trimers (STAT1/STAT2/IRF9) in response to IFN-α2b. The transcriptome induced by IFN-α2b in the patient's cells is much narrower than that of control cells; however, induction of a subset of IFN-stimulated gene transcripts remains detectable. In vitro, the patient's cells do not control three respiratory viruses, influenza A virus (IAV), parainfluenza virus (PIV), and respiratory syncytial virus (RSV). These phenotypes are rescued by wild-type IRF9, whereas silencing IRF9 expression in control cells increases viral replication. However, the child has controlled various common viruses in vivo, including respiratory viruses other than IAV. Our findings show that human IRF9- and ISGF3-dependent type I and III IFN responsive pathways are essential for controlling IAV.

A comprehensive metabolomic data set of date palm fruit.

This article provides detailed information on the phenotypes and the metabolic profiles of 196 date fruits from 123 unique date fruit varieties. These date fruits are extensively diverse in their country of origin, variety and post harvesting conditions. We used a non-targeted mass-spectrometry based metabolomics approach to metabolically characterize date fruits, and measured 427 metabolites from a wide range of metabolic pathways. The metabolomics data for all the date fruit samples are available at the NIH Common Fund's Data Repository and Coordinating Center (supported by NIH grant, U01-DK097430) website, http://www.metabolomicsworkbench.org), under Metabolomics Workbench StudyID: ST000867. The data are directly accessible at http://www.metabolomicsworkbench.org/data/DRCCMetadata.php?Mode=Study&StudyID=ST000867&StudyType=MS&ResultType=1.

A Genome-Wide Survey of Date Palm Cultivars Supports Two Major Subpopulations in Phoenix dactylifera.

The date palm (Phoenix dactylifera L.) is one of the oldest cultivated trees and is intimately tied to the history of human civilization. There are hundreds of commercial cultivars with distinct fruit shapes, colors, and sizes growing mainly in arid lands from the west of North Africa to India. The origin of date palm domestication is still uncertain, and few studies have attempted to document genetic diversity across multiple regions. We conducted genotyping-by-sequencing on 70 female cultivar samples from across the date palm-growing regions, including four Phoenix species as the outgroup. Here, for the first time, we generate genome-wide genotyping data for 13,000-65,000 SNPs in a diverse set of date palm fruit and leaf samples. Our analysis provides the first genome-wide evidence confirming recent findings that the date palm cultivars segregate into two main regions of shared genetic background from North Africa and the Arabian Gulf. We identify genomic regions with high densities of geographically segregating SNPs and also observe higher levels of allele fixation on the recently described X-chromosome than on the autosomes. Our results fit a model with two centers of earliest cultivation including date palms autochthonous to North Africa. These results adjust our understanding of human agriculture history and will provide the foundation for more directed functional studies and a better understanding of genetic diversity in date palm.

Dietary fat and protein intake are not associated with incident biliary sludge and stones during pregnancy.

BACKGROUND: Dietary composition can cause insulin resistance, elevated serum lipid levels, and obesity, all of which predispose to gallstone formation. The effects of dietary fat (including individual fatty acids) and protein on gallstone formation are controversial. The aim of this study was to examine the effects of dietary fat and protein intake on incident gallstone disease during pregnancy, a high-risk time for stone formation. METHODS: We prospectively studied 3070 pregnant women who underwent serial gallbladder ultrasound examinations during pregnancy and at 4-6 weeks postpartum. All women had at least 2 study ultrasounds for comparison. A semi-quantitative food frequency questionnaire was completed by subjects in the early third trimester. Multivariate logistic regression was performed to assess the risk of incident gallbladder disease across quartiles of intake of total fat, individual fatty acids (polyunsaturated, monounsaturated, saturated, and total trans-fatty acids, as well as cholesterol), protein, and protein subtype (animal or vegetable based). RESULTS: The cumulative incidence of new biliary sludge/stones or progression of baseline sludge to stones was 10.2% by 4-6 weeks postpartum. There was no association between total dietary fat (odds ratio [OR], 1.18; 95% confidence interval [CI], 0.64-2.18 comparing lowest and highest quartiles) or protein intake (OR, 0.83; 95% CI, 0.44-1.22 comparing lowest and highest quartiles) and incident gallbladder disease. There was also no association between individual fatty acids or protein subtype and gallbladder disease. CONCLUSIONS: Neither total nor subtype of dietary fat or protein was associated with incident biliary stone or sludge formation in this cohort of pregnant women.