The second parity rule states that, if there is no bias in mutation or selection, then within each strand of DNA complementary bases are present at approximately equal frequencies. In bacteria, however, there is commonly an excess of G (over C) and, to a lesser extent, T (over A) in the replicatory leading strand. The low G+C Firmicutes, such as Staphylococcus aureus, are unusual in displaying an excess of A over T on the leading strand. As mutation has been established as a major force in the generation of such skews across various bacterial taxa, this anomaly has been assumed to reflect unusual mutation biases in Firmicute genomes. Here we show that this is not the case and that mutation bias does not explain the atypical AT skew seen in S. aureus. First, recently arisen intergenic SNPs predict the classical replication-derived equilibrium enrichment of T relative to A, contrary to what is observed. Second, sites predicted to be under weak purifying selection display only weak AT skew. Third, AT skew is primarily associated with largely non-synonymous first and second codon sites and is seen with respect to their sense direction, not which replicating strand they lie on. The atypical AT skew we show to be a consequence of the strong bias for genes to be co-oriented with the replicating fork, coupled with the selective avoidance of both stop codons and costly amino acids, which tend to have T-rich codons. That intergenic sequence has more A than T, while at mutational equilibrium a preponderance of T is expected, points to a possible further unresolved selective source of skew.

In mammals sequences that are either late replicating or highly recombining have high rates of evolution at putatively neutral sites. As early replicating domains and highly recombining domains both tend to be GC rich we a priori expect these two variables to covary. If so, the relative contribution of either of these variables to the local neutral substitution rate might have been wrongly estimated owing to covariance with the other. Against our expectations, we find that sex-averaged recombination rates show little or no correlation with replication timing, suggesting that they are independent determinants of substitution rates. However, this result masks significant sex-specific complexity: late replicating domains tend to have high recombination rates in females but low recombination rates in males. That these trends are antagonistic explains why sex-averaged recombination is not correlated with replication timing. This unexpected result has several important implications. First, although both male and female recombination rates covary significantly with intronic substitution rates, the magnitude of this correlation is moderately underestimated for male recombination and slightly overestimated for female recombination, owing to covariance with replicating timing. Second, the result could explain why male recombination is strongly correlated with GC content but female recombination is not. If to explain the correlation between GC content and replication timing we suppose that late replication forces reduced GC content, then GC promotion by biased gene conversion during female recombination is partly countered by the antagonistic effect of later replicating sequence tending increase AT content. Indeed, the strength of the correlation between female recombination rate and local GC content is more than doubled by control for replication timing. Our results underpin the need to consider sex-specific recombination rates and potential covariates in analysis of GC content and rates of evolution.

Beyond selection for optimal protein functioning, coding sequences (CDSs) are under selection at the RNA and DNA levels. Here, we identify a possible signature of "dual-coding," namely extensive adenine (A) enrichment at bacterial CDS fourth sites. In 99.07% of studied bacterial genomes, fourth site A use is greater than expected given genomic A-starting codon use. Arguing for nucleotide level selection, A-starting serine and arginine second codons are heavily utilized when compared with their non-A starting synonyms. Several models have the ability to explain some of this trend. In part, A-enrichment likely reduces 5' mRNA stability, promoting translation initiation. However T/U, which may also reduce stability, is avoided. Further, +1 frameshifts on the initiating ATG encode a stop codon (TGA) provided A is the fourth residue, acting either as a frameshift "catch and destroy" or a frameshift stop and adjust mechanism and hence implicated in translation initiation. Consistent with both, genomes lacking TGA stop codons exhibit weaker fourth site A-enrichment. Sequences lacking a Shine-Dalgarno sequence and those without upstream leader genes, that may be more error prone during initiation, have greater utilization of A, again suggesting a role in initiation. The frameshift correction model is consistent with the notion that many genomic features are error-mitigation factors and provides the first evidence for site-specific out of frame stop codon selection. We conjecture that the NTG universal start codon may have evolved as a consequence of TGA being a stop codon and the ability of NTGA to rapidly terminate or adjust a ribosome.

A major challenge lies in understanding the complexities of gene regulation. Mutation of the transcription factor SOX10 is associated with several human diseases. The disease phenotypes reflect the function of SOX10 in diverse tissues including the neural crest, central nervous system and otic vesicle. As expected, the SOX10 expression pattern is complex and highly dynamic, but little is known of the underlying mechanisms regulating its spatiotemporal pattern. SOX10 expression is highly conserved between all vertebrates characterised.

Theory predicts that haploid-expressed genes should have noisier expression than comparable diploid-expressed ones with the same expression level. However, in mammals there are several classes of gene that are monoallelically expressed, including X-linked genes, imprinted genes and some other autosomal genes. Does it follow that the evolution of X chromosomes in eukaryotes comes at the cost of increased transcriptional noise in the heterogametic sex? Moreover, is escaping X-inactivation in mammalian females associated with an increase in transcriptional variation? To address these questions, we analyze gene expression variation between replicate samples of diverse mammalian cell lines in steady-state using microarray data.

It has recently been suggested that the use of optimal codons limits mistranslation-induced protein misfolding, yet evidence for this remains largely circumstantial. In contrast, molecular chaperones have long been recognized to play crucial roles in misfolding prevention and remedy. We propose that putative error limitation in cis can be elucidated by examining the interaction between codon usage and chaperoning processes. Using Escherichia coli as a model system, we find that codon optimality covaries with dependency on the chaperonin GroEL. Sporadic but not obligate substrates of GroEL exhibit higher average codon adaptation and are conspicuously enriched for optimal codons at structurally sensitive sites. Further, codon optimality of sporadic clients is more conserved in the E. coli clone Shigella dysenteriae. We suggest that highly expressed genes cannot routinely use GroEL for error control so that codon usage has evolved to provide complementary error limitation. These findings provide independent evidence for a role of misfolding in shaping gene evolution and highlight the need to co-characterize adaptations in cis and trans to unravel the workings of integrated molecular systems.

RASSF7, a member of the N-terminal Ras association domain family, has increased expression in various cancers and, on the basis of our previous work in Xenopus embryos, may be a regulator of mitosis. In the present study, we address, for the first time, the role of human RASSF7 in mitosis. We demonstrate that RASSF7 is expressed in a broad range of different cell types and that this expression could be enhanced following exposure to hypoxia. Knocking down RASSF7 in human cell lines inhibited cell growth and induced defects in mitosis, including aberrant spindle formation and a failure in chromosomal congression. In order to understand the molecular basis of the defects in more detail, we analysed the activity of mitotic signalling proteins and found that activation of Aurora B did not occur in cells in which RASSF7 was knocked down. We also show that endogenous RASSF7 protein localizes to the centrosome and demonstrate using microtubule-regrowth assays that RASSF7 is an important regulator of microtubule dynamics. On the basis of these observations, we propose that, owing to its key role in regulating the microtubule cytoskeleton, RASSF7 is required for mitosis in human cells.

A fundamental problem in developmental biology concerns how multipotent precursors choose specific fates. Neural crest cells (NCCs) are multipotent, yet the mechanisms driving specific fate choices remain incompletely understood. Sox10 is required for specification of neural cells and melanocytes from NCCs. Like sox10 mutants, zebrafish shady mutants lack iridophores; we have proposed that sox10 and shady are required for iridophore specification from NCCs. We show using diverse approaches that shady encodes zebrafish leukocyte tyrosine kinase (Ltk). Cell transplantation studies show that Ltk acts cell-autonomously within the iridophore lineage. Consistent with this, ltk is expressed in a subset of NCCs, before becoming restricted to the iridophore lineage. Marker analysis reveals a primary defect in iridophore specification in ltk mutants. We saw no evidence for a fate-shift of neural crest cells into other pigment cell fates and some NCCs were subsequently lost by apoptosis. These features are also characteristic of the neural crest cell phenotype in sox10 mutants, leading us to examine iridophores in sox10 mutants. As expected, sox10 mutants largely lacked iridophore markers at late stages. In addition, sox10 mutants unexpectedly showed more ltk-expressing cells than wild-type siblings. These cells remained in a premigratory position and expressed sox10 but not the earliest neural crest markers and may represent multipotent, but partially-restricted, progenitors. In summary, we have discovered a novel signalling pathway in NCC development and demonstrate fate specification of iridophores as the first identified role for Ltk.

Errors throughout gene expression are likely deleterious, hence genomes are under selection to ameliorate their consequences. Additional stop codons (ASCs) are in-frame nonsense 'codons' downstream of the primary stop which may be read by translational machinery should the primary stop have been accidentally read through. Prior evidence in several eukaryotes suggests that ASCs are selected to prevent potentially-deleterious consequences of read-through. We extend this evidence showing that enrichment of ASCs is common but not universal for single cell eukaryotes. By contrast, there is limited evidence as to whether the same is true in other taxa. Here, we provide the first systematic test of the hypothesis that ASCs act as a fail-safe mechanism in eubacteria, a group with high read-through rates. Contra to the predictions of the hypothesis we find: there is paucity, not enrichment, of ASCs downstream; substitutions that degrade stops are more frequent in-frame than out-of-frame in 3' sequence; highly expressed genes are no more likely to have ASCs than lowly expressed genes; usage of the leakiest primary stop (TGA) in highly expressed genes does not predict ASC enrichment even compared to usage of non-leaky stops (TAA) in lowly expressed genes, beyond downstream codon +1. Any effect at the codon immediately proximal to the primary stop can be accounted for by a preference for a T/U residue immediately following the stop, although if anything, TT- and TC- starting codons are preferred. We conclude that there is no compelling evidence for ASC selection in eubacteria. This presents an unusual case in which the same error could be solved by the same mechanism in eukaryotes and prokaryotes but is not. We discuss two possible explanations: that, owing to the absence of nonsense mediated decay, bacteria may solve read-through via gene truncation and in eukaryotes certain prion states cause raised read-through rates.

Identifying high-risk patients is crucial for effective cardiovascular disease (CVD) prevention. It is not known whether electronic health record (EHR)-based machine-learning (ML) models can improve CVD risk stratification compared with a secondary prevention risk score developed from randomised clinical trials (Thrombolysis in Myocardial Infarction Risk Score for Secondary Prevention, TRS 2°P).

Alternative synonymous codons are often used at unequal frequencies. Classically, studies of such codon usage bias (CUB) attempted to separate the impact of neutral from selective forces by assuming that deviations from a predicted neutral equilibrium capture selection. However, GC-biased gene conversion (gBGC) can also cause deviation from a neutral null. Alternatively, selection has been inferred from CUB in highly expressed genes, but the accuracy of this approach has not been extensively tested, and gBGC can interfere with such extrapolations (e.g., if expression and gene conversion rates covary). It is therefore critical to examine deviations from a mutational null in a species with no gBGC. To achieve this goal, we implement such an analysis in the highly AT rich genome of Dictyostelium discoideum, where we find no evidence of gBGC. We infer neutral CUB under mutational equilibrium to quantify "adaptive codon preference," a nontautologous genome wide quantitative measure of the relative selection strength driving CUB. We observe signatures of purifying selection consistent with selection favoring adaptive codon preference. Preferred codons are not GC rich, underscoring the independence from gBGC. Expression-associated "preference" largely matches adaptive codon preference but does not wholly capture the influence of selection shaping patterns across all genes, suggesting selective constraints associated specifically with high expression. We observe patterns consistent with effects on mRNA translation and stability shaping adaptive codon preference. Thus, our approach to quantifying adaptive codon preference provides a framework for inferring the sources of selection that shape CUB across different contexts within the genome.

Owing to a lag between a deleterious mutation's appearance and its selective removal, gold-standard methods for mutation rate estimation assume no meaningful loss of mutations between parents and offspring. Indeed, from analysis of closely related lineages, in SARS-CoV-2, the Ka/Ks ratio was previously estimated as 1.008, suggesting no within-host selection. By contrast, we find a higher number of observed SNPs at 4-fold degenerate sites than elsewhere and, allowing for the virus's complex mutational and compositional biases, estimate that the mutation rate is at least 49-67% higher than would be estimated based on the rate of appearance of variants in sampled genomes. Given the high Ka/Ks one might assume that the majority of such intrahost selection is the purging of nonsense mutations. However, we estimate that selection against nonsense mutations accounts for only ∼10% of all the "missing" mutations. Instead, classical protein-level selective filters (against chemically disparate amino acids and those predicted to disrupt protein functionality) account for many missing mutations. It is less obvious why for an intracellular parasite, amino acid cost parameters, notably amino acid decay rate, is also significant. Perhaps most surprisingly, we also find evidence for real-time selection against synonymous mutations that move codon usage away from that of humans. We conclude that there is common intrahost selection on SARS-CoV-2 that acts on nonsense, missense, and possibly synonymous mutations. This has implications for methods of mutation rate estimation, for determining times to common ancestry and the potential for intrahost evolution including vaccine escape.

Reprogramming of H3K9me3-dependent heterochromatin is required for early development. How H3K9me3 is involved in early human development remains, however, largely unclear. Here, we resolve the temporal landscape of H3K9me3 during human preimplantation development and its regulation for diverse hominoid-specific retrotransposons. At the 8-cell stage, H3K9me3 reprogramming at hominoid-specific retrotransposons termed SINE-VNTR-Alu (SVA) facilitates interaction between certain promoters and SVA-derived enhancers, promoting the zygotic genome activation. In trophectoderm, de novo H3K9me3 domains prevent pluripotent transcription factors from binding to hominoid-specific retrotransposons-derived regulatory elements for inner cell mass (ICM)-specific genes. H3K9me3 re-establishment at SVA elements in the ICM is associated with higher transcription of DNA repair genes, when compared with naive human pluripotent stem cells. Our data demonstrate that species-specific reorganization of H3K9me3-dependent heterochromatin at hominoid-specific retrotransposons plays important roles during early human development, shedding light on how the epigenetic regulation for early development has evolved in mammals.

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology.

The control of RNA splicing is often modulated by exonic motifs near splice sites. Chief among these are exonic splice enhancers (ESEs). Well-described ESEs in mammals are purine rich and cause predictable skews in codon and amino acid usage toward exonic ends. Looking across species, those with relatively abundant intronic sequence are those with the more profound end of exon skews, indicative of exonization of splice site recognition. To date, the only intron-rich species that have been analyzed are mammals, precluding any conclusions about the likely ancestral condition. Here, we examine the patterns of codon and amino acid usage in the vicinity of exon-intron junctions in the brown alga Ectocarpus siliculosus, a species with abundant large introns, known SR proteins, and classical splice sites. We find that amino acids and codons preferred/avoided at both 3' and 5' ends in Ectocarpus, of which there are many, tend, on average, to also be preferred/avoided at the same exon ends in humans. Moreover, the preferences observed at the 5' ends of exons are largely the same as those at the 3' ends, a symmetry trend only previously observed in animals. We predict putative hexameric ESEs in Ectocarpus and show that these are purine rich and that there are many more of these identified as functional ESEs in humans than expected by chance. These results are consistent with deep phylogenetic conservation of SR protein binding motifs. Assuming codons preferred near boundaries are "splice optimal" codons, in Ectocarpus, unlike Drosophila, splice optimal and translationally optimal codons are not mutually exclusive. The exclusivity of translationally optimal and splice optimal codon sets is thus not universal.

The zebrafish is an ideal model for elucidating the cellular and molecular mechanisms that underlie development of the peripheral nervous system. A transgenic line that selectively labels all the sensory circuits would be a valuable tool for such investigations. In this study, we describe such a line: the enhancer trap zebrafish line Tg(SKIV2L2:gfp)(j1775) which expresses green fluorescent protein (gfp) in the peripheral sensory ganglia. We show that this transgene marks all peripheral ganglia and sensory nerves, beginning at the time when the neurons are first extending their processes, but does not label the efferent nerves. The trapped reporter is inserted just upstream of a previously poorly described gene: lhfpl4 on LG6. The expression pattern of this gene by in situ hybridization reveals a different, but overlapping, pattern of expression compared to that of the transgene. This pattern also does not mimic that of the gene (skiv2l2), which provided the promoter element in the construct. These findings indicate that reporter expression is not dictated by an endogenous enhancer element, but instead arises through an unknown mechanism. Regardless, this reporter line should prove to be a valuable tool in the investigation of peripheral nervous system formation in the zebrafish.

Structural and biochemical studies of mammalian membrane proteins remain hampered by inefficient production of pure protein. We explored codon optimization based on highly expressed Pichia pastoris genes to enhance co-translational folding and production of P-glycoprotein (Pgp), an ATP-dependent drug efflux pump involved in multidrug resistance of cancers.

Both for understanding mechanisms of disease and for the design of transgenes, it is important to understand the determinants of ribosome velocity, as changes in the rate of translation are important for protein folding, error attenuation, and localization. While there is great variation in ribosomal occupancy along even a single transcript, what determines a ribosome's occupancy is unclear. We examine this issue using data from a ribosomal footprinting assay in yeast. While codon usage is classically considered a major determinant, we find no evidence for this. By contrast, we find that positively charged amino acids greatly retard ribosomes downstream from where they are encoded, consistent with the suggestion that positively charged residues interact with the negatively charged ribosomal exit tunnel. Such slowing is independent of and greater than the average effect owing to mRNA folding. The effect of charged amino acids is additive, with ribosomal occupancy well-predicted by a linear fit to the density of positively charged residues. We thus expect that a translated poly-A tail, encoding for positively charged lysines regardless of the reading frame, would act as a sandtrap for the ribosome, consistent with experimental data.

The efficacy and economy of most in vitro human models used in research is limited by the lack of a physiologically-relevant three-dimensional perfused environment and the inability to noninvasively quantify the structural and biochemical characteristics of the tissue. The goal of this project was to develop a perfusion bioreactor system compatible with two-photon imaging to noninvasively assess tissue engineered human adipose tissue structure and function in vitro. Three-dimensional (3D) vascularized human adipose tissues were engineered in vitro, before being introduced to a perfusion environment and tracked over time by automated quantification of endogenous markers of metabolism using two-photon excited fluorescence (TPEF). Depth-resolved image stacks were analyzed for redox ratio metabolic profiling and compared to prior analyses performed on 3D engineered adipose tissue in static culture. Traditional assessments with H&E staining were used to qualitatively measure extracellular matrix generation and cell density with respect to location within the tissue. The distribution of cells within the tissue and average cellular redox ratios were different between static and perfusion cultures, while the trends of decreased redox ratio and increased cellular proliferation with time in both static and perfusion cultures were similar. These results establish a basis for noninvasive optical tracking of tissue structure and function in vitro, which can be applied to future studies to assess tissue development or drug toxicity screening and disease progression.

The analysis of cross-frequency coupling (CFC) has become popular in studies involving intracranial and scalp EEG recordings in humans. It has been argued that some cases where CFC is mathematically present may not reflect an interaction of two distinct yet functionally coupled neural sources with different frequencies. Here we provide two empirical examples from intracranial recordings where CFC can be shown to be driven by the shape of a periodic waveform rather than by a functional interaction between distinct sources. Using simulations, we also present a generalized and realistic scenario where such coupling may arise. This scenario, which we term waveform-dependent CFC, arises when sharp waveforms (e.g., cortical potentials) occur throughout parts of the data, in particular if they occur rhythmically. Since the waveforms contain both low- and high-frequency components, these components can be inherently phase-aligned as long as the waveforms are spaced with appropriate intervals. We submit that such behavior of the data, which seems to be present in various cortical signals, cannot be interpreted as reflecting functional modulation between distinct neural sources without additional evidence. In addition, we show that even low amplitude periodic potentials that cannot be readily observed or controlled for, are sufficient for significant CFC to occur.