Development of a Comprehensive Genotype-to-Fitness Map of Adaptation-Driving Mutations in Yeast.

Adaptive evolution plays a large role in generating the phenotypic diversity observed in nature, yet current methods are impractical for characterizing the molecular basis and fitness effects of large numbers of individual adaptive mutations. Here, we used a DNA barcoding approach to generate the genotype-to-fitness map for adaptation-driving mutations from a Saccharomyces cerevisiae population experimentally evolved by serial transfer under limiting glucose. We isolated and measured the fitness of thousands of independent adaptive clones and sequenced the genomes of hundreds of clones. We found only two major classes of adaptive mutations: self-diploidization and mutations in the nutrient-responsive Ras/PKA and TOR/Sch9 pathways. Our large sample size and precision of measurement allowed us to determine that there are significant differences in fitness between mutations in different genes, between different paralogs, and even between different classes of mutations within the same gene.

A microbiome-targeting fibre-enriched nutritional formula is well tolerated and improves quality of life and haemoglobin A1c in type 2 diabetes: A double-blind, randomized, placebo-controlled trial.

AIMS: To investigate a prebiotic fibre-enriched nutritional formula on health-related quality of life and metabolic control in type 2 diabetes. MATERIALS AND METHODS: This was a 12-week, double-blind, placebo-controlled study with an unblinded dietary advice only comparator arm. Participants were randomized 2:1:1 to a prebiotic fibre-enriched nutritional formula (Active), a placebo fibre-absent nutritional formula (Placebo), or non-blinded dietary advice alone (Diet). Primary endpoint was change in core Type 2 Diabetes Distress Assessment System (cT2-DDAS) at week 12. Glycated haemoglobin (HbA1c) change was a key secondary endpoint. RESULTS: In total, 192 participants were randomized. Mean age was 54.3 years, HbA1c 7.8%, and body mass index 35.9 kg/m2 . At week 12, cT2-DDAS reduced significantly in Active versus Placebo (-0.4, p = .03), and HbA1c was reduced significantly in Active vs Placebo (-0.64%, p = .01). Gut microbiome sequencing revealed that the relative abundance of two species of butyrate-producing bacteria (Roseburia faecis and Anaerostipes hadrus) increased significantly in Active vs. Placebo. CONCLUSIONS: A microbiome-targeting nutritional formula significantly improved cT2-DDAS and HbA1c, suggesting the potential for prebiotic fibre as a complement to lifestyle and/or pharmaceutical interventions for managing type 2 diabetes.

A common polymorphism in the mechanosensitive ion channel PIEZO1 is associated with protection from severe malaria in humans.

Malaria caused by the apicomplexan parasite Plasmodium falciparum has served as a strong evolutionary force throughout human history, selecting for red blood cell polymorphisms that confer innate protection against severe disease. Recently, gain-of-function mutations in the mechanosensitive ion channel PIEZO1 were shown to ameliorate Plasmodium parasite growth, blood-brain barrier dysfunction, and mortality in a mouse model of malaria. In humans, the gain-of-function allele PIEZO1 E756del is highly prevalent and enriched in Africans, raising the possibility that it is under positive selection due to malaria. Here we used a case-control study design to test for an association between PIEZO1 E756del and malaria severity among children in Gabon. We found that the E756del variant is strongly associated with protection against severe malaria in heterozygotes. In subjects with sickle cell trait, heterozygosity for PIEZO1 E756del did not confer additive protection and homozygosity was associated with an elevated risk of severe disease, suggesting an epistatic relationship between hemoglobin S and PIEZO1 E756del. Using donor blood samples, we show that red cells heterozygous for PIEZO1 E756del are not dehydrated and can support the intracellular growth of P. falciparum similar to wild-type cells. However, surface expression of the P. falciparum virulence protein PfEMP-1 was significantly reduced in infected cells heterozygous for PIEZO1 756del, a phenomenon that has been observed with other protective polymorphisms, such as hemoglobin C. Our findings demonstrate that PIEZO1 is an important innate determinant of malaria susceptibility in humans and suggest that the mechanism of protection may be related to impaired export of P. falciparum virulence proteins.

Historical trends and new surveillance of Plasmodium falciparum drug resistance markers in Angola.

BACKGROUND: Plasmodium falciparum resistance to chloroquine (CQ) and sulfadoxine-pyrimethamine (SP) has historically posed a major threat to malaria control throughout the world. The country of Angola officially replaced CQ with artemisinin-based combination therapy (ACT) as a first-line treatment in 2006, but malaria cases and deaths have recently been rising. Many classic resistance mutations are relevant for the efficacy of currently available drugs, making it important to continue monitoring their frequency in Angola. METHODS: Plasmodium falciparum DNA was sampled from the blood of 50 hospital patients in Cabinda, Angola from October-December of 2018. Each infection was genotyped for 13 alleles in the genes crt, mdr1, dhps, dhfr, and kelch13, which are collectively involved in resistance to six common anti-malarials. To compare frequency patterns over time, P. falciparum genotype data were also collated from studies published from across Angola in the last two decades. RESULTS: The two most important alleles for CQ resistance, crt 76T and mdr1 86Y, were found at respective frequencies of 71.4% and 6.5%. Historical data suggest that mdr1 N86 has been steadily replacing 86Y throughout Angola in the last decade, while the frequency of crt 76T has been more variable across studies. Over a third of new samples from Cabinda were 'quintuple mutants' for SP resistance in dhfr/dhps, with a sixth mutation at dhps A581G present at 9.6% frequency. The markers dhfr 51I, dhfr 108N, and dhps 437G have been nearly fixed in Angola since the early 2000s, whereas dhfr 59R may have risen to high frequency more recently. Finally, no non-synonymous polymorphisms were detected in kelch13, which is involved in artemisinin resistance in Southeast Asia. CONCLUSIONS: Genetic markers of P. falciparum resistance to CQ are likely declining in frequency in Angola, consistent with the official discontinuation of CQ in 2006. The high frequency of multiple genetic markers of SP resistance is consistent with the continued public and private use of SP. In the future, more complete haplotype data from mdr1, dhfr, and dhps will be critical for understanding the changing efficacy of multiple anti-malarial drugs. These data can be used to support effective drug policy decisions in Angola.

High rate of adaptation of mammalian proteins that interact with Plasmodium and related parasites.

Plasmodium parasites, along with their Piroplasm relatives, have caused malaria-like illnesses in terrestrial mammals for millions of years. Several Plasmodium-protective alleles have recently evolved in human populations, but little is known about host adaptation to blood parasites over deeper evolutionary timescales. In this work, we analyze mammalian adaptation in ~500 Plasmodium- or Piroplasm- interacting proteins (PPIPs) manually curated from the scientific literature. We show that (i) PPIPs are enriched for both immune functions and pleiotropy with other pathogens, and (ii) the rate of adaptation across mammals is significantly elevated in PPIPs, compared to carefully matched control proteins. PPIPs with high pathogen pleiotropy show the strongest signatures of adaptation, but this pattern is fully explained by their immune enrichment. Several pieces of evidence suggest that blood parasites specifically have imposed selection on PPIPs. First, even non-immune PPIPs that lack interactions with other pathogens have adapted at twice the rate of matched controls. Second, PPIP adaptation is linked to high expression in the liver, a critical organ in the parasite life cycle. Finally, our detailed investigation of alpha-spectrin, a major red blood cell membrane protein, shows that domains with particularly high rates of adaptation are those known to interact specifically with P. falciparum. Overall, we show that host proteins that interact with Plasmodium and Piroplasm parasites have experienced elevated rates of adaptation across mammals, and provide evidence that some of this adaptation has likely been driven by blood parasites.

Intrinsic differences between males and females determine sex-specific consequences of inbreeding.

BACKGROUND: Inbreeding increases homozygosity and exposes deleterious recessive alleles, generally decreasing the fitness of inbred individuals. Interestingly, males and females are usually affected differently by inbreeding, though the more vulnerable sex depends on the species and trait measured. RESULTS: We used the soil-dwelling nematode Caenorhabditis remanei to examine sex-specific inbreeding depression across nine lineages, five levels of inbreeding, and hundreds of thousands of progeny. Female nematodes consistently suffered greater fitness losses than their male counterparts, especially at high levels of inbreeding. CONCLUSIONS: These results suggest that females experience stronger selection on genes contributing to reproductive traits. Inbreeding depression in males may be further reduced by sex chromosome hemizygosity, which affects the dominance of some mutations, as well as by the absence of sexual selection. Determining the relative contributions of sex-specific expression, genes on the sex chromosomes, and the environment they are filtered through-including opportunities for sexual selection-may explain the frequent though inconsistent records of sex differences in inbreeding depression, along with their implications for conservation and the evolution of mating systems.

Rapid diversification associated with ecological specialization in Neotropical Adelpha butterflies.

Rapid diversification is often associated with morphological or ecological adaptations that allow organisms to radiate into novel niches. Neotropical Adelpha butterflies, which comprise over 200 species and subspecies, are characterized by extraordinary breadth in host plant use and wing colour patterns compared to their closest relatives. To examine the relationship between phenotypic and species diversification, we reconstructed the phylogenetic history of Adelpha and its temperate sister genus Limenitis using genomewide restriction-site-associated DNA (RAD) sequencing. Despite a declining fraction of shared markers with increasing evolutionary distance, the RAD-Seq data consistently generated well-supported trees using a variety of phylogenetic methods. These well-resolved phylogenies allow the identification of an ecologically important relationship with a toxic host plant family, as well as the confirmation of widespread, convergent wing pattern mimicry throughout the genus. Taken together, our results support the hypothesis that evolutionary innovations in both larvae and adults have permitted the colonization of novel host plants and fuelled adaptive diversification within this large butterfly radiation.

Antigenic diversity in malaria parasites is maintained on extrachromosomal DNA.

Sequence variation among antigenic var genes enables Plasmodium falciparum malaria parasites to evade host immunity. Using long sequence reads from haploid clones from a mutation accumulation experiment, we detect var diversity inconsistent with simple chromosomal inheritance. We discover putatively circular DNA that is strongly enriched for var genes, which exist in multiple alleles per locus separated by recombination and indel events. Extrachromosomal DNA likely contributes to rapid antigenic diversification in P. falciparum.

Direct long read visualization reveals metabolic interplay between two antimalarial drug targets.

Increases in the copy number of large genomic regions, termed genome amplification, are an important adaptive strategy for malaria parasites. Numerous amplifications across the Plasmodium falciparum genome contribute directly to drug resistance or impact the fitness of this protozoan parasite. During the characterization of parasite lines with amplifications of the dihydroorotate dehydrogenase (DHODH) gene, we detected increased copies of an additional genomic region that encompassed 3 genes (~5 kb) including GTP cyclohydrolase I (GCH1 amplicon). While this gene is reported to increase the fitness of antifolate resistant parasites, GCH1 amplicons had not previously been implicated in any other antimalarial resistance context. Here, we further explored the association between GCH1 and DHODH copy number. Using long read sequencing and single read visualization, we directly observed a higher number of tandem GCH1 amplicons in parasites with increased DHODH copies (up to 9 amplicons) compared to parental parasites (3 amplicons). While all GCH1 amplicons shared a consistent structure, expansions arose in 2-unit steps (from 3 to 5 to 7, etc copies). Adaptive evolution of DHODH and GCH1 loci was further bolstered when we evaluated prior selection experiments; DHODH amplification was only successful in parasite lines with pre-existing GCH1 amplicons. These observations, combined with the direct connection between metabolic pathways that contain these enzymes, lead us to propose that the GCH1 locus is beneficial for the fitness of parasites exposed to DHODH inhibitors. This finding highlights the importance of studying variation within individual parasite genomes as well as biochemical connections of drug targets as novel antimalarials move towards clinical approval.

Cardiometabolic Effects of Omnivorous vs Vegan Diets in Identical Twins: A Randomized Clinical Trial.

Importance: Increasing evidence suggests that, compared with an omnivorous diet, a vegan diet confers potential cardiovascular benefits from improved diet quality (ie, higher consumption of vegetables, legumes, fruits, whole grains, nuts, and seeds). Objective: To compare the effects of a healthy vegan vs healthy omnivorous diet on cardiometabolic measures during an 8-week intervention. Design, Setting, and Participants: This single-center, population-based randomized clinical trial of 22 pairs of twins (N = 44) randomized participants to a vegan or omnivorous diet (1 twin per diet). Participant enrollment began March 28, 2022, and continued through May 5, 2022. The date of final follow-up data collection was July 20, 2022. This 8-week, open-label, parallel, dietary randomized clinical trial compared the health impact of a vegan diet vs an omnivorous diet in identical twins. Primary analysis included all available data. Intervention: Twin pairs were randomized to follow a healthy vegan diet or a healthy omnivorous diet for 8 weeks. Diet-specific meals were provided via a meal delivery service from baseline through week 4, and from weeks 5 to 8 participants prepared their own diet-appropriate meals and snacks. Main Outcomes and Measures: The primary outcome was difference in low-density lipoprotein cholesterol concentration from baseline to end point (week 8). Secondary outcome measures were changes in cardiometabolic factors (plasma lipids, glucose, and insulin levels and serum trimethylamine N-oxide level), plasma vitamin B12 level, and body weight. Exploratory measures were adherence to study diets, ease or difficulty in following the diets, participant energy levels, and sense of well-being. Results: A total of 22 pairs (N = 44) of twins (34 [77.3%] female; mean [SD] age, 39.6 [12.7] years; mean [SD] body mass index, 25.9 [4.7]) were enrolled in the study. After 8 weeks, compared with twins randomized to an omnivorous diet, the twins randomized to the vegan diet experienced significant mean (SD) decreases in low-density lipoprotein cholesterol concentration (-13.9 [5.8] mg/dL; 95% CI, -25.3 to -2.4 mg/dL), fasting insulin level (-2.9 [1.3] µIU/mL; 95% CI, -5.3 to -0.4 µIU/mL), and body weight (-1.9 [0.7] kg; 95% CI, -3.3 to -0.6 kg). Conclusions and Relevance: In this randomized clinical trial of the cardiometabolic effects of omnivorous vs vegan diets in identical twins, the healthy vegan diet led to improved cardiometabolic outcomes compared with a healthy omnivorous diet. Clinicians can consider this dietary approach as a healthy alternative for their patients. Trial Registration: ClinicalTrials.gov Identifier: NCT05297825.

Revisiting the malaria hypothesis: accounting for polygenicity and pleiotropy.

The malaria hypothesis predicts local, balancing selection of deleterious alleles that confer strong protection from malaria. Three protective variants, recently discovered in red cell genes, are indeed more common in African than European populations. Still, up to 89% of the heritability of severe malaria is attributed to many genome-wide loci with individually small effects. Recent analyses of hundreds of genome-wide association studies (GWAS) in humans suggest that most functional, polygenic variation is pleiotropic for multiple traits. Interestingly, GWAS alleles and red cell traits associated with small reductions in malaria risk are not enriched in African populations. We propose that other selective and neutral forces, in addition to malaria prevalence, explain the global distribution of most genetic variation impacting malaria risk.

Common host variation drives malaria parasite fitness in healthy human red cells.

The replication of Plasmodium falciparum parasites within red blood cells (RBCs) causes severe disease in humans, especially in Africa. Deleterious alleles like hemoglobin S are well-known to confer strong resistance to malaria, but the effects of common RBC variation are largely undetermined. Here, we collected fresh blood samples from 121 healthy donors, most with African ancestry, and performed exome sequencing, detailed RBC phenotyping, and parasite fitness assays. Over one-third of healthy donors unknowingly carried alleles for G6PD deficiency or hemoglobinopathies, which were associated with characteristic RBC phenotypes. Among non-carriers alone, variation in RBC hydration, membrane deformability, and volume was strongly associated with P. falciparum growth rate. Common genetic variants in PIEZO1, SPTA1/SPTB, and several P. falciparum invasion receptors were also associated with parasite growth rate. Interestingly, we observed little or negative evidence for divergent selection on non-pathogenic RBC variation between Africans and Europeans. These findings suggest a model in which globally widespread variation in a moderate number of genes and phenotypes modulates P. falciparum fitness in RBCs.