Unravelling the Genetic Landscape of Hemiplegic Migraine: Exploring Innovative Strategies and Emerging Approaches.

Migraine is a severe, debilitating neurovascular disorder. Hemiplegic migraine (HM) is a rare and debilitating neurological condition with a strong genetic basis. Sequencing technologies have improved the diagnosis and our understanding of the molecular pathophysiology of HM. Linkage analysis and sequencing studies in HM families have identified pathogenic variants in ion channels and related genes, including CACNA1A, ATP1A2, and SCN1A, that cause HM. However, approximately 75% of HM patients are negative for these mutations, indicating there are other genes involved in disease causation. In this review, we explored our current understanding of the genetics of HM. The evidence presented herein summarises the current knowledge of the genetics of HM, which can be expanded further to explain the remaining heritability of this debilitating condition. Innovative bioinformatics and computational strategies to cover the entire genetic spectrum of HM are also discussed in this review.

Multitrait engineering of Hassawi red rice for sustainable cultivation.

Sustainable agriculture requires locally adapted varieties that produce nutritious food with limited agricultural inputs. Genome engineering represents a viable approach to develop cultivars that fulfill these criteria. For example, the red Hassawi rice, a native landrace of Saudi Arabia, tolerates local drought and high-salinity conditions and produces grain with diverse health-promoting phytochemicals. However, Hassawi has a long growth cycle, high cultivation costs, low productivity, and susceptibility to lodging. Here, to improve these undesirable traits via genome editing, we established efficient regeneration and Agrobacterium-mediated transformation protocols for Hassawi. In addition, we generated the first high-quality reference genome and targeted the key flowering repressor gene, Hd4, thus shortening the plant's lifecycle and height. Using CRISPR/Cas9 multiplexing, we simultaneously disrupted negative regulators of flowering time (Hd2, Hd4, and Hd5), grain size (GS3), grain number (GN1a), and plant height (Sd1). The resulting homozygous mutant lines flowered extremely early (∼56 days) and had shorter stems (approximately 107 cm), longer grains (by 5.1%), and more grains per plant (by 50.2%), thereby enhancing overall productivity. Furthermore, the awns of grains were 86.4% shorter compared to unedited plants. Moreover, the modified rice grain displayed improved nutritional attributes. As a result, the modified Hassawi rice combines several desirable traits that can incentivize large-scale cultivation and reduce malnutrition.

A high-performance computational workflow to accelerate GATK SNP detection across a 25-genome dataset.

BACKGROUND: Single-nucleotide polymorphisms (SNPs) are the most widely used form of molecular genetic variation studies. As reference genomes and resequencing data sets expand exponentially, tools must be in place to call SNPs at a similar pace. The genome analysis toolkit (GATK) is one of the most widely used SNP calling software tools publicly available, but unfortunately, high-performance computing versions of this tool have yet to become widely available and affordable. RESULTS: Here we report an open-source high-performance computing genome variant calling workflow (HPC-GVCW) for GATK that can run on multiple computing platforms from supercomputers to desktop machines. We benchmarked HPC-GVCW on multiple crop species for performance and accuracy with comparable results with previously published reports (using GATK alone). Finally, we used HPC-GVCW in production mode to call SNPs on a "subpopulation aware" 16-genome rice reference panel with ~ 3000 resequenced rice accessions. The entire process took ~ 16 weeks and resulted in the identification of an average of 27.3 M SNPs/genome and the discovery of ~ 2.3 million novel SNPs that were not present in the flagship reference genome for rice (i.e., IRGSP RefSeq). CONCLUSIONS: This study developed an open-source pipeline (HPC-GVCW) to run GATK on HPC platforms, which significantly improved the speed at which SNPs can be called. The workflow is widely applicable as demonstrated successfully for four major crop species with genomes ranging in size from 400 Mb to 2.4 Gb. Using HPC-GVCW in production mode to call SNPs on a 25 multi-crop-reference genome data set produced over 1.1 billion SNPs that were publicly released for functional and breeding studies. For rice, many novel SNPs were identified and were found to reside within genes and open chromatin regions that are predicted to have functional consequences. Combined, our results demonstrate the usefulness of combining a high-performance SNP calling architecture solution with a subpopulation-aware reference genome panel for rapid SNP discovery and public deployment.

High-Quality Genome Assemblies Reveal Evolutionary Dynamics of Repetitive DNA and Structural Rearrangements in the Drosophila virilis Subgroup.

High-quality genome assemblies across a range of nontraditional model organisms can accelerate the discovery of novel aspects of genome evolution. The Drosophila virilis group has several attributes that distinguish it from more highly studied species in the Drosophila genus, such as an unusual abundance of repetitive elements and extensive karyotype evolution, in addition to being an attractive model for speciation genetics. Here, we used long-read sequencing to assemble five genomes of three virilis group species and characterized sequence and structural divergence and repetitive DNA evolution. We find that our contiguous genome assemblies allow characterization of chromosomal arrangements with ease and can facilitate analysis of inversion breakpoints. We also leverage a small panel of resequenced strains to explore the genomic pattern of divergence and polymorphism in this species and show that known demographic histories largely predicts the extent of genome-wide segregating polymorphism. We further find that a neo-X chromosome in Drosophila americana displays X-like levels of nucleotide diversity. We also found that unusual repetitive elements were responsible for much of the divergence in genome composition among species. Helitron-derived tandem repeats tripled in abundance on the Y chromosome in D. americana compared to Drosophila novamexicana, accounting for most of the difference in repeat content between these sister species. Repeats with characteristics of both transposable elements and satellite DNAs expanded by 3-fold, mostly in euchromatin, in both D. americana and D. novamexicana compared to D. virilis. Our results represent a major advance in our understanding of genome biology in this emerging model clade.

Oryza glumaepatula: A wild relative to improve drought tolerance in cultivated rice.

Developing drought-resistant rice (Oryza sativa, L.) is essential for improving field productivity, especially in rain-fed areas affected by climate change. Wild relatives of rice are potential sources for drought-resistant traits. Therefore, we compared root growth and drought response among 22 wild Oryza species, from which Oryza glumaepatula was selected as a promising source for further exploration. A geographically diverse panel of 69 O. glumaepatula accessions was then screened for drought stress-related traits, and 6 of these accessions showed lower shoot dry weight (SDW) reduction, greater percentage of deep roots, and lower stomatal density (STO) under drought than the drought tolerant O. sativa variety, Sahbhagi dhan. Based on whole-genome resequencing of all 69 O. glumaepatula accessions and variant calling to a high-quality O. glumaepatula reference genome, we detected multiple genomic loci colocating for SDW, root dry weight at 30 to 45 cm depth, and STO in consecutive drought trials. Geo-referencing indicated that the potential drought donors originated in flood-prone locations, corroborating previous hypotheses about the coexistence of flood and drought tolerance within individual Oryza genomes. These findings present potential donor accessions, traits, and genomic loci from an AA genome wild relative of rice that, together with the recently developed reference genome, may be useful for further introgression of drought tolerance into the O. sativa backgrounds.

Development and implementation of the lung volume reduction pulmonary rehabilitation tool to identify eligibility for lung volume reduction in people with chronic obstructive pulmonary disease during pulmonary rehabilitation.

BACKGROUND: Completion of pulmonary rehabilitation is recognised in chronic obstructive pulmonary disease (COPD) guidelines as a key opportunity to consider systematically whether a respiratory review to assess potential suitability for a lung volume reduction (LVR) procedure might be appropriate. We describe the development of a simple decision-support tool (the LVR-PR tool) to aid clinicians working in pulmonary rehabilitation, to operationalise this process. METHODS: We took an iterative mixed methods approach, which was partnership-based and involved an initial consensus survey, focus groups and an observational study cohort at multiple pulmonary rehabilitation centres. RESULTS: Diagnosis (97%), exercise capacity (84%), breathlessness (78%) and co-morbidities (76%) were acknowledged to be essential items for assessing basic LVR eligibility. Collating prior investigations and assessing patient understanding were considered useful but not essential. Clinician concerns included; streamlining the tool; access to clinical information and investigations; and care needed around introducing LVR therapies to patients in a PR setting. Access to clearer information about LVR procedures, the clinician's role in considering eligibility and how educational resources should be delivered were identified as important themes from patient group discussions. The LVR-PR tool was considered to be feasible and valid for implementation in a variety of PR services across the UK subject to the provision of appropriate health professional training. Clinicians working in specialist LVR centres across the UK who were not otherwise involved in the development process confirmed the tool's validity using the content validity index (CVI). INTERPRETATION: The LVR-PR tool appears to be an acceptable tool that can be feasibly implemented in PR services subject to good quality educational resources for both patients and healthcare professionals.

Genome-wide association analysis identifies natural allelic variants associated with panicle architecture variation in African rice, Oryza glaberrima Steud.

African rice (Oryza glaberrima Steud), a short-day cereal crop closely related to Asian rice (Oryza sativa L.), has been cultivated in Sub-Saharan Africa for ∼ 3,000 years. Although less cultivated globally, it is a valuable genetic resource in creating high-yielding cultivars that are better adapted to diverse biotic and abiotic stresses. While inflorescence architecture, a key trait for rice grain yield improvement, has been extensively studied in Asian rice, the morphological and genetic determinants of this complex trait are less understood in African rice. In this study, using a previously developed association panel of 162 O. glaberrima accessions and new SNP variants characterized through mapping to a new version of the O. glaberrima reference genome, we conducted a genome-wide association study of four major morphological panicle traits. We have found a total of 41 stable genomic regions that are significantly associated with these traits, of which 13 co-localized with previously identified QTLs in O. sativa populations and 28 were unique for this association panel. Additionally, we found a genomic region of interest on chromosome 3 that was associated with the number of spikelets and primary and secondary branches. Within this region was localized the O. sativa ortholog of the PHYTOCHROME B gene (Oglab_006903/OgPHYB). Haplotype analysis revealed the occurrence of natural sequence variants at the OgPHYB locus associated with panicle architecture variation through modulation of the flowering time phenotype, whereas no equivalent alleles were found in O. sativa. The identification in this study of genomic regions specific to O. glaberrima indicates panicle-related intra-specific genetic variation in this species, increasing our understanding of the underlying molecular processes governing panicle architecture. Identified candidate genes and major haplotypes may facilitate the breeding of new African rice cultivars with preferred panicle traits.

High quality genome assemblies reveal evolutionary dynamics of repetitive DNA and structural rearrangements in the Drosophila virilis sub-group.

High-quality genome assemblies across a range of non-traditional model organisms can accelerate the discovery of novel aspects of genome evolution. The Drosophila virilis group has several attributes that distinguish it from more highly studied species in the Drosophila genus, such as an unusual abundance of repetitive elements and extensive karyotype evolution, in addition to being an attractive model for speciation genetics. Here we used long-read sequencing to assemble five genomes of three virilis group species and characterized sequence and structural divergence and repetitive DNA evolution. We find that our contiguous genome assemblies allow characterization of chromosomal arrangements with ease and can facilitate analysis of inversion breakpoints. We also leverage a small panel of resequenced strains to explore the genomic pattern of divergence and polymorphism in this species and show that known demographic histories largely predicts the extent of genome-wide segregating polymorphism. We further find that a neo-X chromosome in D. americana displays X-like levels of nucleotide diversity. We also found that unusual repetitive elements were responsible for much of the divergence in genome composition among species. Helitron-derived tandem repeats tripled in abundance on the Y chromosome in D. americana compared to D. novamexicana, accounting for most of the difference in repeat content between these sister species. Repeats with characteristics of both transposable elements and satellite DNAs expanded by three-fold, mostly in euchromatin, in both D. americana and D. novamexicana compared to D. virilis. Our results represent a major advance in our understanding of genome biology in this emerging model clade.

Protein familiarity is a fundamental but rarely operationalized concept in the safety assessment of genetically modified crops: example of phosphomannose isomerase (PMI).

Fundamental to the safety assessment of genetically modified (GM) crops is the concept of negligible risk for newly expressed proteins for which there is a history of safe use. Although this simple concept has been stated in international and regional guidance for assessing the risk of newly expressed proteins in GM crops, its full implementation by regulatory authorities has been lacking. As a result, safety studies are often repeated at a significant expenditure of resources by developers, study results are repeatedly reviewed by regulators, and animals are sacrificed needlessly to complete redundant animal toxicity studies. This situation is illustrated using the example of the selectable marker phosphomannose isomerase (PMI) for which familiarity has been established. Reviewed is the history of safe use for PMI and predictable results of newly conducted safety studies including bioinformatic comparisons, resistance to digestion, and acute toxicity that were repeated to gain regulatory reapproval of PMI expressed from constructs in recently developed GM maize. As expected, the results of these newly repeated hazard-identification and characterization studies for PMI indicate negligible risk. PMI expressed in recently developed GM crops provides an opportunity to use the concept of familiarity by regulatory authorities to reduce risk-disproportionate regulation of these new events and lessen the resulting waste of both developer and regulator resources, as well as eliminate unnecessary animal testing. This would also correctly imply that familiar proteins like PMI have negligible risk. Together, such modernization of regulations would benefit society through enabling broader and faster access to needed technologies.

Lung injury induces a polarized immune response by self-antigen-specific CD4+ Foxp3+ regulatory T cells.

Self-antigen-specific T cells are prevalent in the mature adaptive immune system but are regulated through multiple mechanisms of tolerance. However, inflammatory conditions such as tissue injury may allow these T cells to break tolerance and trigger autoimmunity. To understand how the T cell repertoire responds to the presentation of self-antigen under highly stimulatory conditions, we use peptide:major histocompatibility complex (MHC) class II tetramers to track the behavior of endogenous CD4+ T cells with specificity to a lung-expressed self-antigen in mouse models of immune-mediated lung injury. Acute injury results in the exclusive expansion of CD4+ regulatory T cells (Tregs) that is dependent on self-antigen recognition and interleukin-2 (IL-2). Conversely, conventional CD4+ T cells of the same self-antigen specificity remain unresponsive even following Treg ablation. Thus, the self-antigen-specific CD4+ T cell repertoire is poised to serve a regulatory function during acute tissue damage to limit further damage and the possibility of autoimmunity.

Non-seed plants are emerging gene sources for agriculture and insect control proteins.

The non-seed plants (e.g., charophyte algae, bryophytes, and ferns) have multiple human uses, but their contributions to agriculture and research have lagged behind seed plants. While sharing broadly conserved biology with seed plants and the major crops, non-seed plants sometimes possess alternative molecular and physiological adaptations. These adaptations may guide crop improvements. One such area is the presence of multiple classes of insecticidal proteins found in non-seed plant genomes which are either absent or widely diverged in seed plants. There are documented uses of non-seed plants, and ferns for example have been used in human diets. Among the occasional identifiable toxins or antinutritive components present in non-seed plants, none include these insecticidal proteins. Apart from these discrete risk factors which can be addressed in the safety assessment, there should be no general safety concern about sourcing genes from non-seed plant species.

Using a Combination of Indirect Calorimetry, Infrared Thermography, and Blood Glucose Levels to Measure Brown Adipose Tissue Thermogenesis in Humans.

In mammals, brown adipose tissue (BAT) is activated rapidly in response to cold in order to maintain body temperature. Although BAT has been studied greatly in small animals, it is difficult to measure the activity of BAT in humans. Therefore, little is known about the heat-generating capacity and physiological significance of BAT in humans, including the degree to which components of the diet can activate BAT. This is due to the limitations in the currently most used method to assess the activation of BAT-radiolabeled glucose (fluorodeoxyglucose or 18FDG) measured by positron emission tomography-computerized tomography (PET-CT). This method is usually performed in fasted subjects, as feeding induces glucose uptake by the muscles, which can mask the glucose uptake into the BAT. This paper describes a detailed protocol for quantifying total-body human energy expenditure and substrate utilization from BAT thermogenesis by combining indirect calorimetry, infrared thermography, and blood glucose monitoring in carbohydrate-loaded adult males. To characterize the physiological significance of BAT, measures of the impact of BAT activity on human health are critical. We demonstrate a protocol to achieve this by combining carbohydrate loading and indirect calorimetry with measurements of supraclavicular changes in temperature. This novel approach will help to understand the physiology and pharmacology of BAT thermogenesis in humans.

A human model of asthma exacerbation reveals transcriptional programs and cell circuits specific to allergic asthma.

Asthma is a chronic disease most commonly associated with allergy and type 2 inflammation. However, the mechanisms that link airway inflammation to the structural changes that define asthma are incompletely understood. Using a human model of allergen-induced asthma exacerbation, we compared the lower airway mucosa in allergic asthmatics and allergic non-asthmatic controls using single-cell RNA sequencing. In response to allergen, the asthmatic airway epithelium was highly dynamic and up-regulated genes involved in matrix degradation, mucus metaplasia, and glycolysis while failing to induce injury-repair and antioxidant pathways observed in controls. IL9-expressing pathogenic TH2 cells were specific to asthmatic airways and were only observed after allergen challenge. Additionally, conventional type 2 dendritic cells (DC2 that express CD1C) and CCR2-expressing monocyte-derived cells (MCs) were uniquely enriched in asthmatics after allergen, with up-regulation of genes that sustain type 2 inflammation and promote pathologic airway remodeling. In contrast, allergic controls were enriched for macrophage-like MCs that up-regulated tissue repair programs after allergen challenge, suggesting that these populations may protect against asthmatic airway remodeling. Cellular interaction analyses revealed a TH2-mononuclear phagocyte-basal cell interactome unique to asthmatics. These pathogenic cellular circuits were characterized by type 2 programming of immune and structural cells and additional pathways that may sustain and amplify type 2 signals, including TNF family signaling, altered cellular metabolism, failure to engage antioxidant responses, and loss of growth factor signaling. Our findings therefore suggest that pathogenic effector circuits and the absence of proresolution programs drive structural airway disease in response to type 2 inflammation.

Multi-omics resources for targeted agronomic improvement of pigmented rice.

Pigmented rice (Oryza sativa L.) is a rich source of nutrients, but pigmented lines typically have long life cycles and limited productivity. Here we generated genome assemblies of 5 pigmented rice varieties and evaluated the genetic variation among 51 pigmented rice varieties by resequencing an additional 46 varieties. Phylogenetic analyses divided the pigmented varieties into four varietal groups: Geng-japonica, Xian-indica, circum-Aus and circum-Basmati. Metabolomics and ionomics profiling revealed that black rice varieties are rich in aromatic secondary metabolites. We established a regeneration and transformation system and used CRISPR-Cas9 to knock out three flowering time repressors (Hd2, Hd4 and Hd5) in the black Indonesian rice Cempo Ireng, resulting in an early maturing variety with shorter stature. Our study thus provides a multi-omics resource for understanding and improving Asian pigmented rice.

Pan-genome inversion index reveals evolutionary insights into the subpopulation structure of Asian rice.

Understanding and exploiting genetic diversity is a key factor for the productive and stable production of rice. Here, we utilize 73 high-quality genomes that encompass the subpopulation structure of Asian rice (Oryza sativa), plus the genomes of two wild relatives (O. rufipogon and O. punctata), to build a pan-genome inversion index of 1769 non-redundant inversions that span an average of ~29% of the O. sativa cv. Nipponbare reference genome sequence. Using this index, we estimate an inversion rate of ~700 inversions per million years in Asian rice, which is 16 to 50 times higher than previously estimated for plants. Detailed analyses of these inversions show evidence of their effects on gene expression, recombination rate, and linkage disequilibrium. Our study uncovers the prevalence and scale of large inversions (≥100 bp) across the pan-genome of Asian rice and hints at their largely unexplored role in functional biology and crop performance.

Lung injury induces a polarized immune response by self antigen-specific Foxp3 + regulatory T cells.

Self antigen-specific T cells are prevalent in the mature adaptive immune system, but are regulated through multiple mechanisms of tolerance. However, inflammatory conditions such as tissue injury may provide these T cells with an opportunity to break tolerance and trigger autoimmunity. To understand how the T cell repertoire responds to the presentation of self antigen under highly stimulatory conditions, we used peptide:MHCII tetramers to track the behavior of endogenous CD4 + T cells with specificity to a lung-expressed self antigen in mouse models of immune-mediated lung injury. Acute injury resulted in the exclusive expansion of regulatory T cells (Tregs) that was dependent on self antigen recognition and IL-2. Conversely, conventional T cells of the same self antigen specificity remained unresponsive, even following Treg ablation. Thus, the self antigen-specific T cell repertoire is poised to serve a regulatory function during acute tissue damage to limit further damage and the possibility of autoimmunity.

Prediction of crossover recombination using parental genomes.

Meiotic recombination is a crucial cellular process, being one of the major drivers of evolution and adaptation of species. In plant breeding, crossing is used to introduce genetic variation among individuals and populations. While different approaches to predict recombination rates for different species have been developed, they fail to estimate the outcome of crossings between two specific accessions. This paper builds on the hypothesis that chromosomal recombination correlates positively to a measure of sequence identity. It presents a model that uses sequence identity, combined with other features derived from a genome alignment (including the number of variants, inversions, absent bases, and CentO sequences) to predict local chromosomal recombination in rice. Model performance is validated in an inter-subspecific indica x japonica cross, using 212 recombinant inbred lines. Across chromosomes, an average correlation of about 0.8 between experimental and prediction rates is achieved. The proposed model, a characterization of the variation of the recombination rates along the chromosomes, can enable breeding programs to increase the chances of creating novel allele combinations and, more generally, to introduce new varieties with a collection of desirable traits. It can be part of a modern panel of tools that breeders can use to reduce costs and execution times of crossing experiments.

FrangiPANe, a tool for creating a panreference using left behind reads.

We present here FrangiPANe, a pipeline developed to build panreference using short reads through a map-then-assemble strategy. Applying it to 248 African rice genomes using an improved CG14 reference genome, we identified an average of 8 Mb of new sequences and 5290 new contigs per individual. In total, 1.4 G of new sequences, consisting of 1 306 676 contigs, were assembled. We validated 97.7% of the contigs of the TOG5681 cultivar individual assembly from short reads on a newly long reads genome assembly of the same TOG5681 cultivar. FrangiPANe also allowed the anchoring of 31.5% of the new contigs within the CG14 reference genome, with a 92.5% accuracy at 2 kb span. We annotated in addition 3252 new genes absent from the reference. FrangiPANe was developed as a modular and interactive application to simplify the construction of a panreference using the map-then-assemble approach. It is available as a Docker image containing (i) a Jupyter notebook centralizing codes, documentation and interactive visualization of results, (ii) python scripts and (iii) all the software and libraries requested for each step of the analysis. We foreseen our approach will help leverage large-scale illumina dataset for pangenome studies in GWAS or detection of selection.

The gyrfalcon (Falco rusticolus) genome.

High-quality genome assemblies are characterized by high-sequence contiguity, completeness, and a low error rate, thus providing the basis for a wide array of studies focusing on natural species ecology, conservation, evolution, and population genomics. To provide this valuable resource for conservation projects and comparative genomics studies on gyrfalcon (Falco rusticolus), we sequenced and assembled the genome of this species using third-generation sequencing strategies and optical maps. Here, we describe a highly contiguous and complete genome assembly comprising 20 scaffolds and 13 contigs with a total size of 1.193 Gbp, including 8,064 complete Benchmarking Universal Single-Copy Orthologs (BUSCOs) of the total 8,338 BUSCO groups present in the library aves_odb10. Of these BUSCO genes, 96.7% were complete, 96.1% were present as a single copy, and 0.6% were duplicated. Furthermore, 0.8% of BUSCO genes were fragmented and 2.5% (210) were missing. A de novo search for transposable elements (TEs) identified 5,716 TEs that masked 7.61% of the F. rusticolus genome assembly when combined with publicly available TE collections. Long interspersed nuclear elements, in particular, the element Chicken-repeat 1 (CR1), were the most abundant TEs in the F. rusticolus genome. A de novo first-pass gene annotation was performed using 293,349 PacBio Iso-Seq transcripts and 496,195 transcripts derived from the assembly of 42,429,525 Illumina PE RNA-seq reads. In all, 19,602 putative genes, of which 59.31% were functionally characterized and associated with Gene Ontology terms, were annotated. A comparison of the gyrfalcon genome assembly with the publicly available assemblies of the domestic chicken (Gallus gallus), zebra finch (Taeniopygia guttata), and hummingbird (Calypte anna) revealed several genome rearrangements. In particular, nine putative chromosome fusions were identified in the gyrfalcon genome assembly compared with those in the G. gallus genome assembly. This genome assembly, its annotation for TEs and genes, and the comparative analyses presented, complement and strength the base of high-quality genome assemblies and associated resources available for comparative studies focusing on the evolution, ecology, and conservation of Aves.