The curly tail mouse provides a model for neural tube defects (spina bifida and exencephaly) that are resistant to prevention by folic acid. The major ct gene, responsible for spina bifida, corresponds to a hypomorphic allele of grainyhead-like 3 (Grhl3) but the frequency of NTDs is strongly influenced by modifiers in the genetic background. Moreover, exencephaly in the curly tail strain is not prevented by reinstatement of Grhl3 expression. In the current study we found that expression of Mthfd1L, encoding a key component of mitochondrial folate one-carbon metabolism (FOCM), is significantly reduced in ct/ct embryos compared to a partially congenic wild-type strain. This expression change is not attributable to regulation by Grhl3 or the genetic background at the Mthfd1L locus. Mitochondrial FOCM provides one-carbon units as formate for FOCM reactions in the cytosol. We found that maternal supplementation with formate prevented NTDs in curly tail embryos and also resulted in increased litter size. Analysis of the folate profile of neurulation-stage embryos showed that formate supplementation resulted in an increased proportion of formyl-THF and THF but a reduction in proportion of 5-methyl THF. In contrast, THF decreased and 5-methyl THF was relatively more abundant in the liver of supplemented dams than in controls. In embryos cultured through the period of spinal neurulation, incorporation of labelled thymidine and adenine into genomic DNA was suppressed by supplemental formate, suggesting that de novo folate-dependent biosynthesis of nucleotides (thymidylate and purines) was enhanced. We hypothesise that reduced Mthfd1L expression may contribute to susceptibility to NTDs in the curly tail strain and that formate acts as a one-carbon donor to prevent NTDs.

X-linked cone dysfunction disorders such as Blue Cone Monochromacy and X-linked Cone Dystrophy are characterized by complete loss (of) or reduced L- and M- cone function due to defects in the OPN1LW/OPN1MW gene cluster. Here we investigated 24 affected males from 16 families with either a structurally intact gene cluster or at least one intact single (hybrid) gene but harbouring rare combinations of common SNPs in exon 3 in single or multiple OPN1LW and OPN1MW gene copies. We assessed twelve different OPN1LW/MW exon 3 haplotypes by semi-quantitative minigene splicing assay. Nine haplotypes resulted in aberrant splicing of ≥20% of transcripts including the known pathogenic haplotypes (i.e. 'LIAVA', 'LVAVA') with absent or minute amounts of correctly spliced transcripts, respectively. De novo formation of the 'LIAVA' haplotype derived from an ancestral less deleterious 'LIAVS' haplotype was observed in one family with strikingly different phenotypes among affected family members. We could establish intrachromosomal gene conversion in the male germline as underlying mechanism. Gene conversion in the OPN1LW/OPN1MW genes has been postulated, however, we are first to demonstrate a de novo gene conversion within the lineage of a pedigree.

The clinical utilization of non-invasive prenatal testing (NIPT) for identification of fetal aneuploidies is expanding worldwide. The aim of this study was to gain an increased understanding of pregnant women's awareness, attitudes, preferences for risk information and decision-making concerning prenatal examinations with emphasis on NIPT, before its introduction into Swedish healthcare.

Mitochondria assemble into flexible networks. Here we present a simple method for the simultaneous quantification of mitochondrial membrane potential and network morphology that is based on computational co-localisation analysis of differentially imported fluorescent marker proteins. Established in, but not restricted to, Saccharomyces cerevisiae, MitoLoc reproducibly measures changes in membrane potential induced by the uncoupling agent CCCP, by oxidative stress, in respiratory deficient cells, and in ∆fzo1, ∆ref2, and ∆dnm1 mutants that possess fission and fusion defects. In combination with super-resolution images, MitoLoc uses 3D reconstruction to calculate six geometrical classifiers which differentiate network morphologies in ∆fzo1, ∆ref2, and ∆dnm1 mutants, under oxidative stress and in cells lacking mtDNA, even when the network is fragmented to a similar extent. We find that mitochondrial fission and a decline in membrane potential do regularly, but not necessarily, co-occur. MitoLoc hence simplifies the measurement of mitochondrial membrane potential in parallel to detect morphological changes in mitochondrial networks. Marker plasmid open-source software as well as the mathematical procedures are made openly available.

A challenge in solving the genotype-to-phenotype relationship is to predict a cell's metabolome, believed to correlate poorly with gene expression. Using comparative quantitative proteomics, we found that differential protein expression in 97 Saccharomyces cerevisiae kinase deletion strains is non-redundant and dominated by abundance changes in metabolic enzymes. Associating differential enzyme expression landscapes to corresponding metabolomes using network models provided reasoning for poor proteome-metabolome correlations; differential protein expression redistributes flux control between many enzymes acting in concert, a mechanism not captured by one-to-one correlation statistics. Mapping these regulatory patterns using machine learning enabled the prediction of metabolite concentrations, as well as identification of candidate genes important for the regulation of metabolism. Overall, our study reveals that a large part of metabolism regulation is explained through coordinated enzyme expression changes. Our quantitative data indicate that this mechanism explains more than half of metabolism regulation and underlies the interdependency between enzyme levels and metabolism, which renders the metabolome a predictable phenotype.

Quantitative proteomics is key for basic research, but needs improvements to satisfy an increasing demand for large sample series in diagnostics, academia and industry. A switch from nanoflowrate to microflowrate chromatography can improve throughput and reduce costs. However, concerns about undersampling and coverage have so far hampered its broad application. We used a QTOF mass spectrometer of the penultimate generation (TripleTOF5600), converted a nanoLC system into a microflow platform, and adapted a SWATH regime for large sample series by implementing retention time- and batch correction strategies. From 3 µg to 5 µg of unfractionated tryptic digests that are obtained from proteomics-typical amounts of starting material, microLC-SWATH-MS quantifies up to 4000 human or 1750 yeast proteins in an hour or less. In the acquisition of 750 yeast proteomes, retention times varied between 2% and 5%, and quantified the typical peptide with 5-8% signal variation in replicates, and below 20% in samples acquired over a five-months period. Providing precise quantities without being dependent on the latest hardware, our study demonstrates that the combination of microflow chromatography and data-independent acquisition strategies has the potential to overcome current bottlenecks in academia and industry, enabling the cost-effective generation of precise quantitative proteomes in large scale.

Saccharomyces cerevisiae strain W303 is a widely used model organism. However, little is known about its genetic origins, as it was created in the 1970s from crossing yeast strains of uncertain genealogy. To obtain insights into its ancestry and physiology, we sequenced the genome of its variant W303-K6001, a yeast model of ageing research. The combination of two next-generation sequencing (NGS) technologies (Illumina and Roche/454 sequencing) yielded an 11.8 Mb genome assembly at an N50 contig length of 262 kb. Although sequencing was substantially more precise and sensitive than whole-genome tiling arrays, both NGS platforms produced a number of false positives. At a 378× average coverage, only 74 per cent of called differences to the S288c reference genome were confirmed by both techniques. The consensus W303-K6001 genome differs in 8133 positions from S288c, predicting altered amino acid sequence in 799 proteins, including factors of ageing and stress resistance. The W303-K6001 (85.4%) genome is virtually identical (less than equal to 0.5 variations per kb) to S288c, and thus originates in the same ancestor. Non-S288c regions distribute unequally over the genome, with chromosome XVI the most (99.6%) and chromosome XI the least (54.5%) S288c-like. Several of these clusters are shared with Σ1278B, another widely used S288c-related model, indicating that these strains share a second ancestor. Thus, the W303-K6001 genome pictures details of complex genetic relationships between the model strains that date back to the early days of experimental yeast genetics. Moreover, this study underlines the necessity of combining multiple NGS and genome-assembling techniques for achieving accurate variant calling in genomic studies.

Neural tube defects (NTDs), including spina bifida and anencephaly, are common birth defects of the central nervous system. The complex multigenic causation of human NTDs, together with the large number of possible candidate genes, has hampered efforts to delineate their molecular basis. Function of folate one-carbon metabolism (FOCM) has been implicated as a key determinant of susceptibility to NTDs. The glycine cleavage system (GCS) is a multi-enzyme component of mitochondrial folate metabolism, and GCS-encoding genes therefore represent candidates for involvement in NTDs. To investigate this possibility, we sequenced the coding regions of the GCS genes: AMT, GCSH and GLDC in NTD patients and controls. Two unique non-synonymous changes were identified in the AMT gene that were absent from controls. We also identified a splice acceptor site mutation and five different non-synonymous variants in GLDC, which were found to significantly impair enzymatic activity and represent putative causative mutations. In order to functionally test the requirement for GCS activity in neural tube closure, we generated mice that lack GCS activity, through mutation of AMT. Homozygous Amt(-/-) mice developed NTDs at high frequency. Although these NTDs were not preventable by supplemental folic acid, there was a partial rescue by methionine. Overall, our findings suggest that loss-of-function mutations in GCS genes predispose to NTDs in mice and humans. These data highlight the importance of adequate function of mitochondrial folate metabolism in neural tube closure.

Cranial neural tube defects (NTDs) occur in mice carrying mutant alleles of many different genes, whereas isolated spinal NTDs (spina bifida) occur in fewer models, despite being common human birth defects. Spina bifida occurs at high frequency in the Axial defects (Axd) mouse mutant but the causative gene is not known. In the current study, the Axd mutation was mapped by linkage analysis. Within the critical genomic region, sequencing did not reveal a coding mutation whereas expression analysis demonstrated significant up-regulation of grainyhead-like 2 (Grhl2) in Axd mutant embryos. Expression of other candidate genes did not differ between genotypes. In order to test the hypothesis that over-expression of Grhl2 causes Axd NTDs, we performed a genetic cross to reduce Grhl2 function in Axd heterozygotes. Grhl2 loss of function mutant mice were generated and displayed both cranial and spinal NTDs. Compound heterozygotes carrying both loss (Grhl2 null) and putative gain of function (Axd) alleles exhibited normalization of spinal neural tube closure compared with Axd/+ littermates, which exhibit delayed closure. Grhl2 is expressed in the surface ectoderm and hindgut endoderm in the spinal region, overlapping with grainyhead-like 3 (Grhl3). Axd mutants display delayed eyelid closure, as reported in Grhl3 null embryos. Moreover, Axd mutant embryos exhibited increased ventral curvature of the spinal region and reduced proliferation in the hindgut, reminiscent of curly tail embryos, which carry a hypomorphic allele of Grhl3. Overall, our data suggest that defects in Axd mutant embryos result from over-expression of Grhl2.

Closure of the neural tube during embryogenesis is a crucial step in development of the central nervous system. Failure of this process results in neural tube defects, including spina bifida and anencephaly, which are among the most common birth defects worldwide. Maternal use of folic acid supplements reduces risk of neural tube defects but a proportion of cases are not preventable. Folic acid is thought to act through folate one-carbon metabolism, which transfers one-carbon units for methylation reactions and nucleotide biosynthesis. Hence suboptimal performance of the intervening reactions could limit the efficacy of folic acid. We hypothesized that direct supplementation with nucleotides, downstream of folate metabolism, has the potential to support neural tube closure. Therefore, in a mouse model that exhibits folic acid-resistant neural tube defects, we tested the effect of specific combinations of pyrimidine and purine nucleotide precursors and observed a significant protective effect. Labelling in whole embryo culture showed that nucleotides are taken up by the neurulating embryo and incorporated into genomic DNA. Furthermore, the mitotic index was elevated in neural folds and hindgut of treated embryos, consistent with a proposed mechanism of neural tube defect prevention through stimulation of cellular proliferation. These findings may provide an impetus for future investigations of supplemental nucleotides as a means to prevent a greater proportion of human neural tube defects than can be achieved by folic acid alone.

Yeast has been established as an efficient model system to study biological principles underpinning human health. In this review we focus on yeast models covering two aspects of cancer formation and progression (i) the activity of pyruvate kinase (PK), which recapitulates metabolic features of cancer cells, including the Warburg effect, and (ii) chromosome bridge-induced translocation (BIT) mimiking genome instability in cancer. Saccharomyces cerevisiae is an excellent model to study cancer cell metabolism, as exponentially growing yeast cells exhibit many metabolic similarities with rapidly proliferating cancer cells. The metabolic reconfiguration includes an increase in glucose uptake and fermentation, at the expense of respiration and oxidative phosphorylation (the Warburg effect), and involves a broad reconfiguration of nucleotide and amino acid metabolism. Both in yeast and humans, the regulation of this process seems to have a central player, PK, which is up-regulated in cancer, and to occur mostly on a post-transcriptional and post-translational basis. Furthermore, BIT allows to generate selectable translocation-derived recombinants ("translocants"), between any two desired chromosomal locations, in wild-type yeast strains transformed with a linear DNA cassette carrying a selectable marker flanked by two DNA sequences homologous to different chromosomes. Using the BIT system, targeted non-reciprocal translocations in mitosis are easily inducible. An extensive collection of different yeast translocants exhibiting genome instability and aberrant phenotypes similar to cancer cells has been produced and subjected to analysis. In this review, we hence provide an overview upon two yeast cancer models, and extrapolate general principles for mimicking human disease mechanisms in yeast.

Fluoropyrimidines are the first-line treatment for colorectal cancer, but their efficacy is highly variable between patients. We queried whether gut microbes, a known source of inter-individual variability, impacted drug efficacy. Combining two tractable genetic models, the bacterium E. coli and the nematode C. elegans, we performed three-way high-throughput screens that unraveled the complexity underlying host-microbe-drug interactions. We report that microbes can bolster or suppress the effects of fluoropyrimidines through metabolic drug interconversion involving bacterial vitamin B6, B9, and ribonucleotide metabolism. Also, disturbances in bacterial deoxynucleotide pools amplify 5-FU-induced autophagy and cell death in host cells, an effect regulated by the nucleoside diphosphate kinase ndk-1. Our data suggest a two-way bacterial mediation of fluoropyrimidine effects on host metabolism, which contributes to drug efficacy. These findings highlight the potential therapeutic power of manipulating intestinal microbiota to ensure host metabolic health and treat disease.

Human susceptibility to obesity is mainly genetic, yet the underlying evolutionary drivers causing variation from person to person are not clear. One theory rationalizes that populations that have adapted to warmer climates have reduced their metabolic rates, thereby increasing their propensity to store energy. We uncover here the function of a gene that supports this theory. THADA is one of the genes most strongly selected during evolution as humans settled in different climates. We report here that THADA knockout flies are obese, hyperphagic, have reduced energy production, and are sensitive to the cold. THADA binds the sarco/ER Ca2+ ATPase (SERCA) and acts on it as an uncoupler. Reducing SERCA activity in THADA mutant flies rescues their obesity, pinpointing SERCA as a key effector of THADA function. In sum, this identifies THADA as a regulator of the balance between energy consumption and energy storage, which was selected during human evolution.

Epidiolex™, a form of highly purified cannabidiol (CBD) derived from Cannabis plants, has demonstrated seizure control activity in patients with Dravet syndrome, without a fully elucidated mechanism of action. We have employed an unbiased approach to investigate this mechanism at a cellular level.

Defects in ear canal development can cause severe hearing loss as sound waves fail to reach the middle ear. Here, we reveal new mechanisms that control human canal development and highlight for the first time the complex system of canal closure and reopening. These processes can be perturbed in mutant mice and in explant culture, mimicking the defects associated with canal atresia. The more superficial part of the canal forms from an open primary canal that closes and then reopens. In contrast, the deeper part of the canal forms from an extending solid meatal plate that opens later. Closure and fusion of the primary canal was linked to loss of periderm, with failure in periderm formation in Grhl3 mutant mice associated with premature closure of the canal. Conversely, inhibition of cell death in the periderm resulted in an arrest of closure. Once closed, re-opening of the canal occurred in a wave, triggered by terminal differentiation of the epithelium. Understanding these complex processes involved in canal development sheds light on the underlying causes of canal atresia.

Some species responded successfully to prehistoric changes in climate [1, 2], while others failed to adapt and became extinct [3]. The factors that determine successful climate adaptation remain poorly understood. We constructed a reference genome and studied physiological adaptations in the Alpine marmot (Marmota marmota), a large ground-dwelling squirrel exquisitely adapted to the "ice-age" climate of the Pleistocene steppe [4, 5]. Since the disappearance of this habitat, the rodent persists in large numbers in the high-altitude Alpine meadow [6, 7]. Genome and metabolome showed evidence of adaptation consistent with cold climate, affecting white adipose tissue. Conversely, however, we found that the Alpine marmot has levels of genetic variation that are among the lowest for mammals, such that deleterious mutations are less effectively purged. Our data rule out typical explanations for low diversity, such as high levels of consanguineous mating, or a very recent bottleneck. Instead, ancient demographic reconstruction revealed that genetic diversity was lost during the climate shifts of the Pleistocene and has not recovered, despite the current high population size. We attribute this slow recovery to the marmot's adaptive life history. The case of the Alpine marmot reveals a complicated relationship between climatic changes, genetic diversity, and conservation status. It shows that species of extremely low genetic diversity can be very successful and persist over thousands of years, but also that climate-adapted life history can trap a species in a persistent state of low genetic diversity.

Neural tube defects (NTDs) result from failure of neural tube closure during embryogenesis. These severe birth defects of the central nervous system include anencephaly and spina bifida, and affect 0.5-2 per 1,000 pregnancies worldwide in humans. It has been demonstrated that acetylation plays a pivotal role during neural tube closure, as animal models for defective histone acetyltransferase proteins display NTDs. Acetylation represents an important component of the complex network of posttranslational regulatory interactions, suggesting a possible fundamental role during primary neurulation events. This study aimed to assess protein acetylation contribution to early patterning of the central nervous system both in human and murine specimens.

Eukaryotic genomes express numerous long intergenic non-coding RNAs (lincRNAs) that do not overlap any coding genes. Some lincRNAs function in various aspects of gene regulation, but it is not clear in general to what extent lincRNAs contribute to the information flow from genotype to phenotype. To explore this question, we systematically analysed cellular roles of lincRNAs in Schizosaccharomyces pombe. Using seamless CRISPR/Cas9-based genome editing, we deleted 141 lincRNA genes to broadly phenotype these mutants, together with 238 diverse coding-gene mutants for functional context. We applied high-throughput colony-based assays to determine mutant growth and viability in benign conditions and in response to 145 different nutrient, drug, and stress conditions. These analyses uncovered phenotypes for 47.5% of the lincRNAs and 96% of the protein-coding genes. For 110 lincRNA mutants, we also performed high-throughput microscopy and flow cytometry assays, linking 37% of these lincRNAs with cell-size and/or cell-cycle control. With all assays combined, we detected phenotypes for 84 (59.6%) of all lincRNA deletion mutants tested. For complementary functional inference, we analysed colony growth of strains ectopically overexpressing 113 lincRNA genes under 47 different conditions. Of these overexpression strains, 102 (90.3%) showed altered growth under certain conditions. Clustering analyses provided further functional clues and relationships for some of the lincRNAs. These rich phenomics datasets associate lincRNA mutants with hundreds of phenotypes, indicating that most of the lincRNAs analysed exert cellular functions in specific environmental or physiological contexts. This study provides groundwork to further dissect the roles of these lincRNAs in the relevant conditions.

Severe COVID-19 is linked to both dysfunctional immune response and unrestrained immunopathology, and it remains unclear whether T cells contribute to disease pathology. Here, we combined single-cell transcriptomics and single-cell proteomics with mechanistic studies to assess pathogenic T cell functions and inducing signals. We identified highly activated CD16+ T cells with increased cytotoxic functions in severe COVID-19. CD16 expression enabled immune-complex-mediated, T cell receptor-independent degranulation and cytotoxicity not found in other diseases. CD16+ T cells from COVID-19 patients promoted microvascular endothelial cell injury and release of neutrophil and monocyte chemoattractants. CD16+ T cell clones persisted beyond acute disease maintaining their cytotoxic phenotype. Increased generation of C3a in severe COVID-19 induced activated CD16+ cytotoxic T cells. Proportions of activated CD16+ T cells and plasma levels of complement proteins upstream of C3a were associated with fatal outcome of COVID-19, supporting a pathological role of exacerbated cytotoxicity and complement activation in COVID-19.

To discover genes implicated in human congenital disorders, we performed ENU mutagenesis in the mouse and screened for mutations affecting embryonic development. In this work, we report defects of heart development in mice homozygous for a mutation of coactivator-associated arginine methyltransferase 1 (Carm1). While Carm1 has been extensively studied, it has never been previously associated with a role in heart development. Phenotype analysis combining histology and microcomputed tomography imaging shows a range of cardiac defects. Most notably, many affected midgestation embryos appear to have cardiac rupture and hemorrhaging in the thorax. Mice that survive to late gestation show a variety of cardiac defects, including ventricular septal defects, double outlet right ventricle, and persistent truncus arteriosus. Transcriptome analyses of the mutant embryos by mRNA-seq reveal the perturbation of several genes involved in cardiac morphogenesis and muscle development and function. In addition, we observe the mislocalization of cardiac neural crest cells at E12.5 in the outflow tract. The cardiac phenotype of Carm1 mutant embryos is similar to that of Pax3 null mutants, and PAX3 is a putative target of CARM1. However, our analysis does not support the hypothesis that developmental defects in Carm1 mutant embryos are primarily due to a functional defect of PAX3.