Discovery of recessive effect of human polymerase δ proofreading deficiency through mutational analysis of POLD1-mutated normal and cancer cells.

Constitutional heterozygous pathogenic variants in the exonuclease domain of POLE and POLD1, which affect the proofreading activity of the corresponding polymerases, cause a cancer predisposition syndrome characterized by increased risk of gastrointestinal polyposis, colorectal cancer, endometrial cancer and other tumor types. The generally accepted explanation for the connection between the disruption of the proofreading activity of polymerases epsilon and delta and cancer development is through an increase in the somatic mutation rate. Here we studied an extended family with multiple members heterozygous for the pathogenic POLD1 variant c.1421T>C p.(Leu474Pro), which segregates with the polyposis and cancer phenotypes. Through the analysis of mutational patterns of patient-derived fibroblasts colonies and de novo mutations obtained by parent-offspring comparisons, we concluded that heterozygous POLD1 L474P just subtly increases the somatic and germline mutation burden. In contrast, tumors developed in individuals with a heterozygous mutation in the exonuclease domain of POLD1, including L474P, have an extremely high mutation rate (>100 mut/Mb) associated with signature SBS10d. We solved this contradiction through the observation that tumorigenesis involves somatic inactivation of the wildtype POLD1 allele. These results imply that exonuclease deficiency of polymerase delta has a recessive effect on mutation rate.

A hybrid pathway for self-sustained luminescence.

The fungal bioluminescence pathway can be reconstituted in other organisms allowing luminescence imaging without exogenously supplied substrate. The pathway starts from hispidin biosynthesis-a step catalyzed by a large fungal polyketide synthase that requires a posttranslational modification for activity. Here, we report identification of alternative compact hispidin synthases encoded by a phylogenetically diverse group of plants. A hybrid bioluminescence pathway that combines plant and fungal genes is more compact, not dependent on availability of machinery for posttranslational modifications, and confers autonomous bioluminescence in yeast, mammalian, and plant hosts. The compact size of plant hispidin synthases enables additional modes of delivery of autoluminescence, such as delivery with viral vectors.

Speciation and repeated origins of hypertrophied lips in parallel adaptive radiations of cyprinid fish from East Africa.

The evolution of convergent phenotypes is one of the most interesting phenomena of repeated adaptive radiations. Here, we examined the repeated patterns of thick-lipped or "rubberlip" phenotype of cyprinid fish of the genus Labeobarbus discovered in riverine environments of the Ethiopian Highlands, East Africa. To test the adaptive value of thickened lips, identify the ecological niche of the thick-lipped ecomorphs, and test whether these ecomorphs are the products of adaptive divergence, we studied six sympatric pairs of ecomorphs with hypertrophied lips and the normal lip structure from different riverine basins. Trophic morphology, diet, stable isotope (δ15N and δ13C) signatures, as well as mtDNA markers and genome-wide SNP variation, were analyzed. Our results show that thick-lipped ecomorphs partition trophic resources with generalized ecomorphs in only one-half of the examined sympatric pairs despite the pronounced divergence in lip structure. In these thick-lipped ecomorphs that were trophically diverged, the data on their diet along with the elevated 15N values suggest an invertivorous specialization different from the basal omnivorous-detritivouros feeding mode of the generalized ecomorphs. Genetic data confirmed an independent and parallel origin of all six lipped ecomorphs. Yet, only one of those six thick-lipped ecomorphs had a notable genetic divergence with sympatric non-lipped ecomorphs based on nuclear SNPs data (F ST = 0.21). Sympatric pairs can be sorted by combinations of phenotypic, ecological, and genetic divergence from an ecologically non-functional mouth polymorphism via ecologically functional polymorphism to a matured speciation stage via divergent evolution.

Divergent molecular signatures in fish Bouncer proteins define cross-fertilization boundaries.

Molecular compatibility between gametes is a prerequisite for successful fertilization. As long as a sperm and egg can recognize and bind each other via their surface proteins, gamete fusion may occur even between members of separate species, resulting in hybrids that can impact speciation. The egg membrane protein Bouncer confers species specificity to gamete interactions between medaka and zebrafish, preventing their cross-fertilization. Here, we leverage this specificity to uncover distinct amino acid residues and N-glycosylation patterns that differentially influence the function of medaka and zebrafish Bouncer and contribute to cross-species incompatibility. Curiously, in contrast to the specificity observed for medaka and zebrafish Bouncer, seahorse and fugu Bouncer are compatible with both zebrafish and medaka sperm, in line with the pervasive purifying selection that dominates Bouncer's evolution. The Bouncer-sperm interaction is therefore the product of seemingly opposing evolutionary forces that, for some species, restrict fertilization to closely related fish, and for others, allow broad gamete compatibility that enables hybridization.

Using AlphaFold to predict the impact of single mutations on protein stability and function.

AlphaFold changed the field of structural biology by achieving three-dimensional (3D) structure prediction from protein sequence at experimental quality. The astounding success even led to claims that the protein folding problem is "solved". However, protein folding problem is more than just structure prediction from sequence. Presently, it is unknown if the AlphaFold-triggered revolution could help to solve other problems related to protein folding. Here we assay the ability of AlphaFold to predict the impact of single mutations on protein stability (ΔΔG) and function. To study the question we extracted the pLDDT and metrics from AlphaFold predictions before and after single mutation in a protein and correlated the predicted change with the experimentally known ΔΔG values. Additionally, we correlated the same AlphaFold pLDDT metrics with the impact of a single mutation on structure using a large scale dataset of single mutations in GFP with the experimentally assayed levels of fluorescence. We found a very weak or no correlation between AlphaFold output metrics and change of protein stability or fluorescence. Our results imply that AlphaFold may not be immediately applied to other problems or applications in protein folding.

Relation Between the Number of Peaks and the Number of Reciprocal Sign Epistatic Interactions.

Empirical essays of fitness landscapes suggest that they may be rugged, that is having multiple fitness peaks. Such fitness landscapes, those that have multiple peaks, necessarily have special local structures, called reciprocal sign epistasis (Poelwijk et al. in J Theor Biol 272:141-144, 2011). Here, we investigate the quantitative relationship between the number of fitness peaks and the number of reciprocal sign epistatic interactions. Previously, it has been shown (Poelwijk et al. in J Theor Biol 272:141-144, 2011) that pairwise reciprocal sign epistasis is a necessary but not sufficient condition for the existence of multiple peaks. Applying discrete Morse theory, which to our knowledge has never been used in this context, we extend this result by giving the minimal number of reciprocal sign epistatic interactions required to create a given number of peaks.

Heterogeneity of the GFP fitness landscape and data-driven protein design.

Studies of protein fitness landscapes reveal biophysical constraints guiding protein evolution and empower prediction of functional proteins. However, generalisation of these findings is limited due to scarceness of systematic data on fitness landscapes of proteins with a defined evolutionary relationship. We characterized the fitness peaks of four orthologous fluorescent proteins with a broad range of sequence divergence. While two of the four studied fitness peaks were sharp, the other two were considerably flatter, being almost entirely free of epistatic interactions. Mutationally robust proteins, characterized by a flat fitness peak, were not optimal templates for machine-learning-driven protein design - instead, predictions were more accurate for fragile proteins with epistatic landscapes. Our work paves insights for practical application of fitness landscape heterogeneity in protein engineering.

Chromosome-encoded IpaH ubiquitin ligases indicate non-human enteroinvasive Escherichia.

Until recently, Shigella and enteroinvasive Escherichia coli were thought to be primate-restricted pathogens. The base of their pathogenicity is the type 3 secretion system (T3SS) encoded by the pINV virulence plasmid, which facilitates host cell invasion and subsequent proliferation. A large family of T3SS effectors, E3 ubiquitin-ligases encoded by the ipaH genes, have a key role in the Shigella pathogenicity through the modulation of cellular ubiquitination that degrades host proteins. However, recent genomic studies identified ipaH genes in the genomes of Escherichia marmotae, a potential marmot pathogen, and an E. coli extracted from fecal samples of bovine calves, suggesting that non-human hosts may also be infected by these strains, potentially pathogenic to humans. We performed a comparative genomic study of the functional repertoires in the ipaH gene family in Shigella and enteroinvasive Escherichia from human and predicted non-human hosts. We found that fewer than half of Shigella genomes had a complete set of ipaH genes, with frequent gene losses and duplications that were not consistent with the species tree and nomenclature. Non-human host IpaH proteins had a diverse set of substrate-binding domains and, in contrast to the Shigella proteins, two variants of the NEL C-terminal domain. Inconsistencies between strains phylogeny and composition of effectors indicate horizontal gene transfer between E. coli adapted to different hosts. These results provide a framework for understanding of ipaH-mediated host-pathogens interactions and suggest a need for a genomic study of fecal samples from diseased animals.

Scientists without borders: lessons from Ukraine.

Conflicts and natural disasters affect entire populations of the countries involved and, in addition to the thousands of lives destroyed, have a substantial negative impact on the scientific advances these countries provide. The unprovoked invasion of Ukraine by Russia, the devastating earthquake in Turkey and Syria, and the ongoing conflicts in the Middle East are just a few examples. Millions of people have been killed or displaced, their futures uncertain. These events have resulted in extensive infrastructure collapse, with loss of electricity, transportation, and access to services. Schools, universities, and research centers have been destroyed along with decades' worth of data, samples, and findings. Scholars in disaster areas face short- and long-term problems in terms of what they can accomplish now for obtaining grants and for employment in the long run. In our interconnected world, conflicts and disasters are no longer a local problem but have wide-ranging impacts on the entire world, both now and in the future. Here, we focus on the current and ongoing impact of war on the scientific community within Ukraine and from this draw lessons that can be applied to all affected countries where scientists at risk are facing hardship. We present and classify examples of effective and feasible mechanisms used to support researchers in countries facing hardship and discuss how these can be implemented with help from the international scientific community and what more is desperately needed. Reaching out, providing accessible training opportunities, and developing collaborations should increase inclusion and connectivity, support scientific advancements within affected communities, and expedite postwar and disaster recovery.

Rates of SARS-CoV-2 transmission and vaccination impact the fate of vaccine-resistant strains.

Vaccines are thought to be the best available solution for controlling the ongoing SARS-CoV-2 pandemic. However, the emergence of vaccine-resistant strains may come too rapidly for current vaccine developments to alleviate the health, economic and social consequences of the pandemic. To quantify and characterize the risk of such a scenario, we created a SIR-derived model with initial stochastic dynamics of the vaccine-resistant strain to study the probability of its emergence and establishment. Using parameters realistically resembling SARS-CoV-2 transmission, we model a wave-like pattern of the pandemic and consider the impact of the rate of vaccination and the strength of non-pharmaceutical intervention measures on the probability of emergence of a resistant strain. As expected, we found that a fast rate of vaccination decreases the probability of emergence of a resistant strain. Counterintuitively, when a relaxation of non-pharmaceutical interventions happened at a time when most individuals of the population have already been vaccinated the probability of emergence of a resistant strain was greatly increased. Consequently, we show that a period of transmission reduction close to the end of the vaccination campaign can substantially reduce the probability of resistant strain establishment. Our results suggest that policymakers and individuals should consider maintaining non-pharmaceutical interventions and transmission-reducing behaviours throughout the entire vaccination period.

The limits of normal approximation for adult height.

Adult height inspired the first biometrical and quantitative genetic studies and is a test-case trait for understanding heritability. The studies of height led to formulation of the classical polygenic model, that has a profound influence on the way we view and analyse complex traits. An essential part of the classical model is an assumption of additivity of effects and normality of the distribution of the residuals. However, it may be expected that the normal approximation will become insufficient in bigger studies. Here, we demonstrate that when the height of hundreds of thousands of individuals is analysed, the model complexity needs to be increased to include non-additive interactions between sex, environment and genes. Alternatively, the use of log-normal approximation allowed us to still use the additive effects model. These findings are important for future genetic and methodologic studies that make use of adult height as an exemplar trait.

Author Correction: Plants with genetically encoded autoluminescence.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A Method for Identification of the Methylation Level of CpG Islands From NGS Data.

In the course of sample preparation for Next Generation Sequencing (NGS), DNA is fragmented by various methods. Fragmentation shows a persistent bias with regard to the cleavage rates of various dinucleotides. With the exception of CpG dinucleotides the previously described biases were consistent with results of the DNA cleavage in solution. Here we computed cleavage rates of all dinucleotides including the methylated CpG and unmethylated CpG dinucleotides using data of the Whole Genome Sequencing datasets of the 1000 Genomes project. We found that the cleavage rate of CpG is significantly higher for the methylated CpG dinucleotides. Using this information, we developed a classifier for distinguishing cancer and healthy tissues based on their CpG islands statuses of the fragmentation. A simple Support Vector Machine classifier based on this algorithm shows an accuracy of 84%. The proposed method allows the detection of epigenetic markers purely based on mechanochemical DNA fragmentation, which can be detected by a simple analysis of the NGS sequencing data.

Plants with genetically encoded autoluminescence.

Autoluminescent plants engineered to express a bacterial bioluminescence gene cluster in plastids have not been widely adopted because of low light output. We engineered tobacco plants with a fungal bioluminescence system that converts caffeic acid (present in all plants) into luciferin and report self-sustained luminescence that is visible to the naked eye. Our findings could underpin development of a suite of imaging tools for plants.

Large multiple sequence alignments with a root-to-leaf regressive method.

Multiple sequence alignments (MSAs) are used for structural1,2 and evolutionary predictions1,2, but the complexity of aligning large datasets requires the use of approximate solutions3, including the progressive algorithm4. Progressive MSA methods start by aligning the most similar sequences and subsequently incorporate the remaining sequences, from leaf to root, based on a guide tree. Their accuracy declines substantially as the number of sequences is scaled up5. We introduce a regressive algorithm that enables MSA of up to 1.4 million sequences on a standard workstation and substantially improves accuracy on datasets larger than 10,000 sequences. Our regressive algorithm works the other way around from the progressive algorithm and begins by aligning the most dissimilar sequences. It uses an efficient divide-and-conquer strategy to run third-party alignment methods in linear time, regardless of their original complexity. Our approach will enable analyses of extremely large genomic datasets such as the recently announced Earth BioGenome Project, which comprises 1.5 million eukaryotic genomes6.

HypercubeME: two hundred million combinatorially complete datasets from a single experiment.

MOTIVATION: Epistasis, the context-dependence of the contribution of an amino acid substitution to fitness, is common in evolution. To detect epistasis, fitness must be measured for at least four genotypes: the reference genotype, two different single mutants and a double mutant with both of the single mutations. For higher-order epistasis of the order n, fitness has to be measured for all 2n genotypes of an n-dimensional hypercube in genotype space forming a "combinatorially complete dataset". So far, only a handful of such datasets have been produced by manual curation. Concurrently, random mutagenesis experiments have produced measurements of fitness and other phenotypes in a high-throughput manner, potentially containing a number of combinatorially complete datasets. RESULTS: We present an effective recursive algorithm for finding all hypercube structures in random mutagenesis experimental data. To test the algorithm, we applied it to the data from a recent HIS3 protein dataset and found all 199,847,053 unique combinatorially complete genotype combinations of dimensionality ranging from two to twelve. The algorithm may be useful for researchers looking for higher-order epistasis in their high-throughput experimental data. AVAILABILITY: https://github.com/ivankovlab/HypercubeME.git. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

An experimental assay of the interactions of amino acids from orthologous sequences shaping a complex fitness landscape.

Characterizing the fitness landscape, a representation of fitness for a large set of genotypes, is key to understanding how genetic information is interpreted to create functional organisms. Here we determined the evolutionarily-relevant segment of the fitness landscape of His3, a gene coding for an enzyme in the histidine synthesis pathway, focusing on combinations of amino acid states found at orthologous sites of extant species. Just 15% of amino acids found in yeast His3 orthologues were always neutral while the impact on fitness of the remaining 85% depended on the genetic background. Furthermore, at 67% of sites, amino acid replacements were under sign epistasis, having both strongly positive and negative effect in different genetic backgrounds. 46% of sites were under reciprocal sign epistasis. The fitness impact of amino acid replacements was influenced by only a few genetic backgrounds but involved interaction of multiple sites, shaping a rugged fitness landscape in which many of the shortest paths between highly fit genotypes are inaccessible.