Synonymous mutations in representative yeast genes are mostly strongly non-neutral.

Synonymous mutations in protein-coding genes do not alter protein sequences and are thus generally presumed to be neutral or nearly neutral1-5. Here, to experimentally verify this presumption, we constructed 8,341 yeast mutants each carrying a synonymous, nonsynonymous or nonsense mutation in one of 21 endogenous genes with diverse functions and expression levels and measured their fitness relative to the wild type in a rich medium. Three-quarters of synonymous mutations resulted in a significant reduction in fitness, and the distribution of fitness effects was overall similar-albeit nonidentical-between synonymous and nonsynonymous mutations. Both synonymous and nonsynonymous mutations frequently disturbed the level of mRNA expression of the mutated gene, and the extent of the disturbance partially predicted the fitness effect. Investigations in additional environments revealed greater across-environment fitness variations for nonsynonymous mutants than for synonymous mutants despite their similar fitness distributions in each environment, suggesting that a smaller proportion of nonsynonymous mutants than synonymous mutants are always non-deleterious in a changing environment to permit fixation, potentially explaining the common observation of substantially lower nonsynonymous than synonymous substitution rates. The strong non-neutrality of most synonymous mutations, if it holds true for other genes and in other organisms, would require re-examination of numerous biological conclusions about mutation, selection, effective population size, divergence time and disease mechanisms that rely on the assumption that synoymous mutations are neutral.

Contraception ends the genetic maintenance of human same-sex sexual behavior.

Because human same-sex sexual behavior (SSB) is heritable and leads to fewer offspring, it is puzzling why SSB-associated alleles have not been selectively purged. Current evidence supports the antagonistic pleiotropy hypothesis that SSB-associated alleles benefit individuals exclusively performing opposite-sex sexual behavior by increasing their number of sexual partners and consequently their number of offspring. However, by analyzing the UK Biobank, here, we show that having more sexual partners no longer predicts more offspring since the availability of oral contraceptives in the 1960s and that SSB is now genetically negatively correlated with the number of offspring, suggesting a loss of SSB's genetic maintenance in modern societies.

Ascertainment Bias in the Genomic Test of Positive Selection on Regulatory Sequences.

Evolution of gene expression mediated by cis-regulatory changes is thought to be an important contributor to organismal adaptation, but identifying adaptive cis-regulatory changes is challenging due to the difficulty in knowing the expectation under no positive selection. A new approach for detecting positive selection on transcription factor binding sites (TFBSs) was recently developed, thanks to the application of machine learning in predicting transcription factor (TF) binding affinities of DNA sequences. Given a TFBS sequence from a focal species and the corresponding inferred ancestral sequence that differs from the former at n sites, one can predict the TF-binding affinities of many n-step mutational neighbors of the ancestral sequence and obtain a null distribution of the derived binding affinity, which allows testing whether the binding affinity of the real derived sequence deviates significantly from the null distribution. Applying this test genomically to all experimentally identified binding sites of 3 TFs in humans, a recent study reported positive selection for elevated binding affinities of TFBSs. Here, we show that this genomic test suffers from an ascertainment bias because, even in the absence of positive selection for strengthened binding, the binding affinities of known human TFBSs are more likely to have increased than decreased in evolution. We demonstrate by computer simulation that this bias inflates the false positive rate of the selection test. We propose several methods to mitigate the ascertainment bias and show that almost all previously reported positive selection signals disappear when these methods are applied.

Gene product diversity: adaptive or not?

One gene does not equal one RNA or protein. The genomic revolution has revealed numerous different RNA and protein molecules that can be produced from one gene, such as circular RNAs generated by back-splicing, proteins with residues mismatching the genomic encoding because of RNA editing, and proteins extended in the C terminus via stop codon readthrough in translation. Are these diverse products results of exquisite gene regulations or imprecise biological processes? While there are cases where the gene product diversity appears beneficial, genome-scale patterns suggest that much of this diversity arises from nonadaptive, molecular errors. This finding has important implications for studying the functions of diverse gene products and for understanding the fundamental properties and evolution of cellular life.

On the Decoupling of Evolutionary Changes in mRNA and Protein Levels.

Variation in gene expression across lineages is thought to explain much of the observed phenotypic variation and adaptation. The protein is closer to the target of natural selection but gene expression is typically measured as the amount of mRNA. The broad assumption that mRNA levels are good proxies for protein levels has been undermined by a number of studies reporting moderate or weak correlations between the two measures across species. One biological explanation for this discrepancy is that there has been compensatory evolution between the mRNA level and regulation of translation. However, we do not understand the evolutionary conditions necessary for this to occur nor the expected strength of the correlation between mRNA and protein levels. Here, we develop a theoretical model for the coevolution of mRNA and protein levels and investigate the dynamics of the model over time. We find that compensatory evolution is widespread when there is stabilizing selection on the protein level; this observation held true across a variety of regulatory pathways. When the protein level is under directional selection, the mRNA level of a gene and the translation rate of the same gene were negatively correlated across lineages but positively correlated across genes. These findings help explain results from comparative studies of gene expression and potentially enable researchers to disentangle biological and statistical hypotheses for the mismatch between transcriptomic and proteomic data.

Short-term and Long-term Efficacy Evaluation of Drug-Loaded Osteoset Artificial Bone Grafting Fusion in the Treatment of Sacroiliac Joint Tuberculosis.

Objective: To analyze the short-term and long-term efficacy of Osteoset artificial bone graft fusion mixed with rifampicin for injection in the treatment of sacroiliac joint tuberculosis. Methods: A retrospective analysis was carried out on 70 patients diagnosed with sacroiliac joint tuberculosis who were admitted and underwent surgical treatment in our orthopedics department between April 2014 and May 2020. The patients were divided into three groups based on the different bone graft materials used: autogenous bone graft group (25 cases), simple lesion removal group (18 cases), and drug-loaded calcium sulfate bone graft group (27 cases). General information and surgical details of the three groups were compared. Sacroiliac X-ray and CT scans were performed at regular intervals to record pre- and post-treatment erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) levels, bone graft fusion rates at 6, 12, and 18 months post-surgery, Majeed score for functional evaluation, and postoperative complications. Results: There was no statistically significant difference in operation time, intraoperative bleeding, and intraoperative pus removal volume among the three groups of patients (P > .05). Postoperatively, 70 patients were followed up, and the serum levels of ESR and CRP in all three groups of patients were significantly reduced at 3 months after surgery (P < .05). In the autogenous bone graft group, the bone graft fusion rates were 24.00% (6/25) at 6 months postoperatively, 76.00% (18/25) at 12 months, and 96.00% (24/25) at 18 months. In the simple lesion removal group, the bone graft fusion rates were 16.67% (3/18) at 6 months postoperatively, 27.78% (5/18) at 12 months, and 55.56% (10/18) at 18 months. In the drug-loaded calcium sulfate bone graft group, the bone graft fusion rates were 18.52% (5/27) at 6 months postoperatively, 55.56% (15/27) at 12 months, and 81.48% (22/27) at 18 months. In the autogenous bone graft group, the postoperative Majeed score averaged (91.47±4.13) points, with 13 cases rated as excellent and 10 cases rated as good, resulting in an excellent and good rate of 92.00% (23/25). The Majeed scores at 6, 12, and 18 months postoperatively were (67.19±4.22) points, (80.28±5.83) points, and (91.47±4.13) points, respectively. Among them, there were 4 excellent and 3 good cases at 6 months postoperatively, with an excellent and good rate of 28.00% (7/25). At 12 months postoperatively, there were 8 excellent and 10 good cases, with an excellent and good rate of 72.00% (18/25). At 18 months postoperatively, there were 13 excellent and 10 good cases, with an excellent and good rate of 92.00% (23/25). In the simple lesion removal group, the Majeed scores at 6, 12, and 18 months postoperatively were (59.17±3.95) points, (69.84±5.16) points, and (76.22±8.76) points, respectively. There were 2 excellent and 2 good cases at 6 months postoperatively, with an excellent and good rate of 22.22% (4/18). At 12 months postoperatively, there were 4 excellent and 3 good cases, with an excellent and good rate of 38.89% (7/18). At 18 months postoperatively, there were 5 excellent and 5 good cases, with an excellent and good rate of 55.56% (10/18). In the drug-loaded calcium sulfate bone graft group, the Majeed scores at 6, 12, and 18 months postoperatively were (63.24±4.17) points, (77.39±5.50) points, and (86.64±7.03) points, respectively. There were 3 excellent and 3 good cases at 6 months postoperatively, with an excellent and good rate of 22.22% (6/27). At 12 months postoperatively, there were 9 excellent and 7 good cases, with an excellent and good rate of 59.26% (16/27). At 18 months postoperatively, there were 10 excellent and 12 good cases, with an excellent and good rate of 81.48% (22/27). The Majeed scores for all three groups of patients showed a significant increase in the three follow-up evaluations compared to pre-treatment (P < .05). Conclusion: Drug-loaded Osteoset artificial bone graft fusion is a safe and effective method for treating bone defects after the debridement of sacroiliac joint tuberculosis lesions. It has fewer postoperative complications and achieves bone graft fusion in a shorter time compared to simple lesion removal methods.

Selective recycling of lithium from spent LiNixCoyMn1-x-yO2 cathode via constructing a synergistic leaching environment.

The global response to lithium scarcity is overstretched, and it is imperative to explore a green process to sustainably and selectively recover lithium from spent lithium-ion battery (LIB) cathodes. This work investigates the distinct leaching behaviors between lithium and transition metals in pure formic acid and the auxiliary effect of acetic acid as a solvent in the leaching reaction. A formic acid-acetic acid (FA-AA) synergistic system was constructed to selectively recycle 96.81% of lithium from spent LIB cathodes by regulating the conditions of the reaction environment to inhibit the leaching of non-target metals. Meanwhile, the transition metals generate carboxylate precipitates enriched in the leaching residue. The inhibition mechanism of manganese leaching by acetic acid and the leaching behavior of nickel or cobalt being precipitated after release was revealed by characterizations such as XPS, SEM, and FTIR. After the reaction, 90.50% of the acid can be recycled by distillation, and small amounts of the residual Li-containing concentrated solution are converted to battery-grade lithium carbonate by roasting and washing (91.62% recovery rate). This recycling process possesses four significant advantages: i) no additional chemicals are required, ii) the lithium sinking step is eliminated, iii) no waste liquid is discharged, and iv) there is the potential for profitability. Overall, this study provides a novel approach to the waste management technology of lithium batteries and sustainable recycling of lithium resources.

Is the Mutation Rate Lower in Genomic Regions of Stronger Selective Constraints?

A study of the plant Arabidopsis thaliana detected lower mutation rates in genomic regions where mutations are more likely to be deleterious, challenging the principle that mutagenesis is blind to its consequence. To examine the generality of this finding, we analyze large mutational data from baker's yeast and humans. The yeast data do not exhibit this trend, whereas the human data show an opposite trend that disappears upon the control of potential confounders. We find that the Arabidopsis study identified substantially more mutations than reported in the original data-generating studies and expected from Arabidopsis' mutation rate. These extra mutations are enriched in polynucleotide tracts and have relatively low sequencing qualities so are likely sequencing errors. Furthermore, the polynucleotide "mutations" can produce the purported mutational trend in Arabidopsis. Together, our results do not support lower mutagenesis of genomic regions of stronger selective constraints in the plant, fungal, and animal models examined.

Genomic estimates of mutation and substitution rates contradict the evolutionary speed hypothesis of the latitudinal diversity gradient.

The latitudinal diversity gradient (LDG) refers to a decrease in biodiversity from the equator to the poles. The evolutionary speed hypothesis, backed by the metabolic theory of ecology, asserts that nucleotide mutation and substitution rates per site per year are higher and thereby speciation rates are higher at higher temperatures, generating the LDG. However, prior empirical investigations of the relationship between the temperature and mutation or substitution rate were based on a few genes and the results were mixed. We here revisit this relationship using genomic data. No significant correlation between the temperature and mutation rate is found in 13 prokaryotes or in 107 eukaryotes. An analysis of 234 diverse trios of bacterial taxa indicates that the synonymous substitution rate is not significantly associated with the growth temperature. The same data, however, reveal a significant negative association between the nonsynonymous substitution rate and temperature, which is explainable by a larger fraction of detrimental nonsynonymous mutations at higher temperatures due to a stronger demand for protein stability. We conclude that the evolutionary speed hypothesis of the LDG is unsupported by genomic data and advise that future mechanistic studies of the LDG should focus on other hypotheses.

Systematic genetic modifications of cell wall biosynthesis enhanced the secretion and surface-display of polysaccharide degrading enzymes in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is a robust cell factory to secrete or surface-display cellulase and amylase for the conversion of agricultural residues into valuable chemicals. Engineering the secretory pathway is a well-known strategy for overproducing these enzymes. Although cell wall biosynthesis can be tightly linked to the secretory pathway by regulation of all involved processes, the effect of its modifications on protein production has not been extensively studied. In this study, we systematically studied the effect of engineering cell wall biosynthesis on the activity of cellulolytic enzyme ß-glucosidase (BGL1) by comparing seventy-nine gene knockout S. cerevisiae strains and newly identified that inactivation of DFG5, YPK1, FYV5, CCW12 and KRE1 obviously improved BGL1 secretion and surface-display. Combinatorial modifications of these genes, particularly double deletion of FVY5 and CCW12, along with the use of rich medium, increased the activity of secreted and surface-displayed BGL1 by 6.13-fold and 7.99-fold, respectively. Additionally, we applied this strategy to improve the activity of the cellulolytic cellobiohydrolase and amylolytic α-amylase. Through proteomic analysis coupled with reverse engineering, we found that in addition to the secretory pathway, regulation of translation processes may also involve in improving enzyme activity by engineering cell wall biosynthesis. Our work provides new insight into the construction of a yeast cell factory for efficient production of polysaccharide degrading enzymes.

Long noncoding RNA LINC01088 inhibits esophageal squamous cell carcinoma progression by targeting the NPM1-HDM2-p53 axis.

Esophageal squamous cell carcinoma (ESCC) is characterized by extensive metastasis and poor prognosis. Long noncoding RNAs (lncRNAs) have been shown to play important roles in ESCC. However, the specific roles of lncRNAs in ESCC tumorigenesis and metastasis remain largely unknown. Here, we investigate LINC01088 in ESCC. Differentially expressed LINC01088 levels are screened from the GEO database. We find that LINC01088 is expressed at low level in collected clinical samples and is correlated with vascular tumor emboli and poor overall survival time of patients after surgery. LINC01088 inhibits not only ESCC cell migration and invasion in vitro, but also tumorigenesis and metastasis in vivo. Mechanistically, LINC01088 directly interacts with nucleophosmin (NPM1) and increases the expression of NPM1 in the nucleoplasm compared to that in the nucleolar region. LINC01088 decreases mutant p53 (mut-p53) expression and rescues the transcriptional activity of p53 by targeting the NPM1-HDM2-p53 axis. LINC01088 may also interfere with the DNA repair function of NPM1 by affecting its translocation. Our results highlight the potential of LINC01088 as a prognostic biomarker and therapeutic target of ESCC.

Biosynthesis of Hybrid Neutral Lipids with Archaeal and Eukaryotic Characteristics in Engineered Saccharomyces cerevisiae.

Discovery of the Asgard superphylum of archaea provides new evidence supporting the two-domain model of life: eukaryotes originated from an Asgard-related archaeon that engulfed a bacterial endosymbiont. However, how eukaryotes acquired bacterial-like membrane lipids with a sn-glycerol-3-phosphate (G3P) backbone instead of the archaeal-like sn-glycerol-1-phosphate (G1P) backbone remains unknown. In this study, we reconstituted archaeal lipid production in Saccharomyces cerevisiae by expressing unsaturated archaeol-synthesizing enzymes. Using Golden Gate cloning for pathway assembly, modular gene replacement was performed, revealing the potential biosynthesis of both G1P- and G3P-based unsaturated archaeol by uncultured Asgard archaea. Unexpectedly, hybrid neutral lipids containing both archaeal isoprenoids and eukaryotic fatty acids were observed in recombinant S. cerevisiae. The ability of yeast and archaeal diacylglycerol acyltransferases to synthesize such hybrid lipids was demonstrated.

Rampant False Detection of Adaptive Phenotypic Optimization by ParTI-Based Pareto Front Inference.

Organisms face tradeoffs in performing multiple tasks. Identifying the optimal phenotypes maximizing the organismal fitness (or Pareto front) and inferring the relevant tasks allow testing phenotypic adaptations and help delineate evolutionary constraints, tradeoffs, and critical fitness components, so are of broad interest. It has been proposed that Pareto fronts can be identified from high-dimensional phenotypic data, including molecular phenotypes such as gene expression levels, by fitting polytopes (lines, triangles, tetrahedrons, and so on), and a program named ParTI was recently introduced for this purpose. ParTI has identified Pareto fronts and inferred phenotypes best for individual tasks (or archetypes) from numerous data sets such as the beak morphologies of Darwin's finches and mRNA concentrations in human tumors, implying evolutionary optimizations of the involved traits. Nevertheless, the reliabilities of these findings are unknown. Using real and simulated data that lack evolutionary optimization, we here report extremely high false-positive rates of ParTI. The errors arise from phylogenetic relationships or population structures of the organisms analyzed and the flexibility of data analysis in ParTI that is equivalent to p-hacking. Because these problems are virtually universal, our findings cast doubt on almost all ParTI-based results and suggest that reliably identifying Pareto fronts and archetypes from high-dimensional phenotypic data are currently generally difficult.

Is the Genetic Code Optimized for Resource Conservation?

The causes and consequences of the nonrandom structure of the standard genetic code (SGC) have been of long-standing interest. A recent study reported that mutations in present-day protein-coding sequences are less likely to increase proteomic nitrogen and carbon uses under the SGC than under random genetic codes, concluding that the SGC has been selectively optimized for resource conservation. If true, this finding might offer important information on the environment in which the SGC and some of the earliest life forms evolved. However, we here show that the hypothesis of optimization of a genetic code for resource conservation is theoretically untenable. We discover that the aforementioned study estimated the expected mutational effect by inappropriately excluding mutations lowering resource consumptions and including mutations involving stop codons. After remedying these problems, we find no evidence that the SGC is optimized for nitrogen or carbon conservation.

On the Origin of Frameshift-Robustness of the Standard Genetic Code.

The standard genetic code (SGC) has been extensively analyzed for the biological ramifications of its nonrandom structure. For instance, mismatch errors due to point mutation or mistranslation have an overall smaller effect on the amino acid polar requirement under the SGC than under random genetic codes (RGCs). A similar observation was recently made for frameshift errors, prompting the assertion that the SGC has been shaped by natural selection for frameshift-robustness-conservation of certain amino acid properties upon a frameshift mutation or translational frameshift. However, frameshift-robustness confers no benefit because frameshifts usually create premature stop codons that cause nonsense-mediated mRNA decay or production of nonfunctional truncated proteins. We here propose that the frameshift-robustness of the SGC is a byproduct of its mismatch-robustness. Of 564 amino acid properties considered, the SGC exhibits mismatch-robustness in 93-133 properties and frameshift-robustness in 55 properties, respectively, and that the latter is largely a subset of the former. For each of the 564 real and 564 randomly constructed fake properties of amino acids, there is a positive correlation between mismatch-robustness and frameshift-robustness across one million RGCs; this correlation arises because most amino acid changes resulting from a frameshift are also achievable by a mismatch error. Importantly, the SGC does not show significantly higher frameshift-robustness in any of the 55 properties than RGCs of comparable mismatch-robustness. These findings support that the frameshift-robustness of the SGC need not originate through direct selection and can instead be a site effect of its mismatch-robustness.

Asexual Experimental Evolution of Yeast Does Not Curtail Transposable Elements.

Compared with asexual reproduction, sex facilitates the transmission of transposable elements (TEs) from one genome to another, but boosts the efficacy of selection against deleterious TEs. Thus, theoretically, it is unclear whether sex has a positive net effect on TE's proliferation. An empirical study concluded that sex is at the root of TE's evolutionary success because the yeast TE load was found to decrease rapidly in approximately 1,000 generations of asexual but not sexual experimental evolution. However, this finding contradicts the maintenance of TEs in natural yeast populations where sexual reproduction occurs extremely infrequently. Here, we show that the purported TE load reduction during asexual experimental evolution is likely an artifact of low genomic sequencing coverages. We observe stable TE loads in both sexual and asexual experimental evolution from multiple yeast data sets with sufficient coverages. To understand the evolutionary dynamics of yeast TEs, we turn to asexual mutation accumulation lines that have been under virtually no selection. We find that both TE transposition and excision rates per generation, but not their difference, tend to be higher in environments where yeast grows more slowly. However, the transposition rate is not significantly higher than the excision rate and the variance of the TE number among natural strains is close to its neutral expectation, suggesting that selection against TEs is at best weak in yeast. We conclude that the yeast TE load is maintained largely by a transposition-excision balance and that the influence of sex remains unclear.

Are Nonsynonymous Transversions Generally More Deleterious than Nonsynonymous Transitions?

It has been suggested that, due to the structure of the genetic code, nonsynonymous transitions are less likely than transversions to cause radical changes in amino acid physicochemical properties so are on average less deleterious. This view was supported by some but not all mutagenesis experiments. Because laboratory measures of fitness effects have limited sensitivities and relative frequencies of different mutations in mutagenesis studies may not match those in nature, we here revisit this issue using comparative genomics. We extend the standard codon model of sequence evolution by adding the parameter η that quantifies the ratio of the fixation probability of transitional nonsynonymous mutations to that of transversional nonsynonymous mutations. We then estimate η from the concatenated alignment of all protein-coding DNA sequences of two closely related genomes. Surprisingly, η ranges from 0.13 to 2.0 across 90 species pairs sampled from the tree of life, with 51 incidences of η < 1 and 30 incidences of η >1 that are statistically significant. Hence, whether nonsynonymous transversions are overall more deleterious than nonsynonymous transitions is species-dependent. Because the corresponding groups of amino acid replacements differ between nonsynonymous transitions and transversions, η is influenced by the relative exchangeabilities of amino acid pairs. Indeed, an extensive search reveals that the large variation in η is primarily explainable by the recently reported among-species disparity in amino acid exchangeabilities. These findings demonstrate that genome-wide nucleotide substitution patterns in coding sequences have species-specific features and are more variable among evolutionary lineages than are currently thought.

Environment-dependent pleiotropic effects of mutations on the maximum growth rate r and carrying capacity K of population growth.

Maximum growth rate per individual (r) and carrying capacity (K) are key life-history traits that together characterize the density-dependent population growth and therefore are crucial parameters of many ecological and evolutionary theories such as r/K selection. Although r and K are generally thought to correlate inversely, both r/K tradeoffs and trade-ups have been observed. Nonetheless, neither the conditions under which each of these relationships occur nor the causes of these relationships are fully understood. Here, we address these questions using yeast as a model system. We estimated r and K using the growth curves of over 7,000 yeast recombinants in nine environments and found that the r-K correlation among genotypes changes from 0.53 to -0.52 with the rise of environment quality, measured by the mean r of all genotypes in the environment. We respectively mapped quantitative trait loci (QTLs) for r and K in each environment. Many QTLs simultaneously influence r and K, but the directions of their effects are environment dependent such that QTLs tend to show concordant effects on the two traits in poor environments but antagonistic effects in rich environments. We propose that these contrasting trends are generated by the relative impacts of two factors-the tradeoff between the speed and efficiency of ATP production and the energetic cost of cell maintenance relative to reproduction-and demonstrate an agreement between model predictions and empirical observations. These results reveal and explain the complex environment dependency of the r-K relationship, which bears on many ecological and evolutionary phenomena and has biomedical implications.

Evidence that alternative transcriptional initiation is largely nonadaptive.

Alternative transcriptional initiation (ATI) refers to the frequent observation that one gene has multiple transcription start sites (TSSs). Although this phenomenon is thought to be adaptive, the specific advantage is rarely known. Here, we propose that each gene has one optimal TSS and that ATI arises primarily from imprecise transcriptional initiation that could be deleterious. This error hypothesis predicts that (i) the TSS diversity of a gene reduces with its expression level; (ii) the fractional use of the major TSS increases, but that of each minor TSS decreases, with the gene expression level; and (iii) cis-elements for major TSSs are selectively constrained, while those for minor TSSs are not. By contrast, the adaptive hypothesis does not make these predictions a priori. Our analysis of human and mouse transcriptomes confirms each of the three predictions. These and other findings strongly suggest that ATI predominantly results from molecular errors, requiring a major revision of our understanding of the precision and regulation of transcription.