The evolution and mutational robustness of chromatin accessibility in Drosophila.

BACKGROUND: The evolution of genomic regulatory regions plays a critical role in shaping the diversity of life. While this process is primarily sequence-dependent, the enormous complexity of biological systems complicates the understanding of the factors underlying regulation and its evolution. Here, we apply deep neural networks as a tool to investigate the sequence determinants underlying chromatin accessibility in different species and tissues of Drosophila. RESULTS: We train hybrid convolution-attention neural networks to accurately predict ATAC-seq peaks using only local DNA sequences as input. We show that our models generalize well across substantially evolutionarily diverged species of insects, implying that the sequence determinants of accessibility are highly conserved. Using our model to examine species-specific gains in accessibility, we find evidence suggesting that these regions may be ancestrally poised for evolution. Using in silico mutagenesis, we show that accessibility can be accurately predicted from short subsequences in each example. However, in silico knock-out of these sequences does not qualitatively impair classification, implying that accessibility is mutationally robust. Subsequently, we show that accessibility is predicted to be robust to large-scale random mutation even in the absence of selection. Conversely, simulations under strong selection demonstrate that accessibility can be extremely malleable despite its robustness. Finally, we identify motifs predictive of accessibility, recovering both novel and previously known motifs. CONCLUSIONS: These results demonstrate the conservation of the sequence determinants of accessibility and the general robustness of chromatin accessibility, as well as the power of deep neural networks to explore fundamental questions in regulatory genomics and evolution.

The evolution and mutational robustness of chromatin accessibility in Drosophila.

The evolution of regulatory regions in the genome plays a critical role in shaping the diversity of life. While this process is primarily sequence-dependent, the enormous complexity of biological systems has made it difficult to understand the factors underlying regulation and its evolution. Here, we apply deep neural networks as a tool to investigate the sequence determinants underlying chromatin accessibility in different tissues of Drosophila. We train hybrid convolution-attention neural networks to accurately predict ATAC-seq peaks using only local DNA sequences as input. We show that a model trained in one species has nearly identical performance when tested in another species, implying that the sequence determinants of accessibility are highly conserved. Indeed, model performance remains excellent even in distantly-related species. By using our model to examine species-specific gains in chromatin accessibility, we find that their orthologous inaccessible regions in other species have surprisingly similar model outputs, suggesting that these regions may be ancestrally poised for evolution. We then use in silico saturation mutagenesis to reveal evidence of selective constraint acting specifically on inaccessible chromatin regions. We further show that chromatin accessibility can be accurately predicted from short subsequences in each example. However, in silico knock-out of these sequences does not qualitatively impair classification, implying that chromatin accessibility is mutationally robust. Subsequently, we demonstrate that chromatin accessibility is predicted to be robust to large-scale random mutation even in the absence of selection. We also perform in silico evolution experiments under the regime of strong selection and weak mutation (SSWM) and show that chromatin accessibility can be extremely malleable despite its mutational robustness. However, selection acting in different directions in a tissue-specific manner can substantially slow adaptation. Finally, we identify motifs predictive of chromatin accessibility and recover motifs corresponding to known chromatin accessibility activators and repressors. These results demonstrate the conservation of the sequence determinants of accessibility and the general robustness of chromatin accessibility, as well as the power of deep neural networks as tools to answer fundamental questions in regulatory genomics and evolution.

Protein evidence of unannotated ORFs in Drosophila reveals diversity in the evolution and properties of young proteins.

De novo gene origination, where a previously nongenic genomic sequence becomes genic through evolution, is increasingly recognized as an important source of novelty. Many de novo genes have been proposed to be protein-coding, and a few have been experimentally shown to yield protein products. However, the systematic study of de novo proteins has been hampered by doubts regarding their translation without the experimental observation of protein products. Using a systematic, mass-spectrometry-first computational approach, we identify 993 unannotated open reading frames with evidence of translation (utORFs) in Drosophila melanogaster. To quantify the similarity of these utORFs across Drosophila and infer phylostratigraphic age, we develop a synteny-based protein similarity approach. Combining these results with reference datasets ontissue- and life stage-specific transcription and conservation, we identify different properties amongst these utORFs. Contrary to expectations, the fastest-evolving utORFs are not the youngest evolutionarily. We observed more utORFs in the brain than in the testis. Most of the identified utORFs may be of de novo origin, even accounting for the possibility of false-negative similarity detection. Finally, sequence divergence after an inferred de novo origin event remains substantial, suggesting that de novo proteins turn over frequently. Our results suggest that there is substantial unappreciated diversity in de novo protein evolution: many more may exist than previously appreciated; there may be divergent evolutionary trajectories, and they may be gained and lost frequently. All in all, there may not exist a single characteristic model of de novo protein evolution, but instead, there may be diverse evolutionary trajectories.

Sex differences in interindividual gene expression variability across human tissues.

Understanding phenotypic sex differences has long been a goal of biology from both a medical and evolutionary perspective. Although much attention has been paid to mean differences in phenotype between the sexes, little is known about sex differences in phenotypic variability. To gain insight into sex differences in interindividual variability at the molecular level, we analyzed RNA-seq data from 43 tissues from the Genotype-Tissue Expression project (GTEx). Within each tissue, we identified genes that show sex differences in gene expression variability. We found that these sex-differentially variable (SDV) genes are associated with various important biological functions, including sex hormone response, immune response, and other signaling pathways. By analyzing single-cell RNA sequencing data collected from breast epithelial cells, we found that genes with sex differences in gene expression variability in breast tissue tend to be expressed in a cell-type-specific manner. We looked for an association between SDV expression and Graves' disease, a well-known heavily female-biased disease, and found a significant enrichment of Graves' associated genes among genes with higher variability in females in thyroid tissue. This suggests a possible role for SDV expression in sex-biased disease. We then examined the evolutionary constraints acting on genes with sex differences in variability and found that they exhibit evidence of increased selective constraint. Through analysis of sex-biased eQTL data, we found evidence that SDV expression may have a genetic basis. Finally, we propose a simple evolutionary model for the emergence of SDV expression from sex-specific constraints.

Error-prone, stress-induced 3' flap-based Okazaki fragment maturation supports cell survival.

How cells with DNA replication defects acquire mutations that allow them to escape apoptosis under environmental stress is a long-standing question. Here, we report that an error-prone Okazaki fragment maturation (OFM) pathway is activated at restrictive temperatures in rad27Δ yeast cells. Restrictive temperature stress activated Dun1, facilitating transformation of unprocessed 5' flaps into 3' flaps, which were removed by 3' nucleases, including DNA polymerase δ (Polδ). However, at certain regions, 3' flaps formed secondary structures that facilitated 3' end extension rather than degradation, producing alternative duplications with short spacer sequences, such as pol3 internal tandem duplications. Consequently, little 5' flap was formed, suppressing rad27Δ-induced lethality at restrictive temperatures. We define a stress-induced, error-prone OFM pathway that generates mutations that counteract replication defects and drive cellular evolution and survival.

Colonoscopy competence assessment tools: a systematic review of validity evidence.

BACKGROUND: Assessment tools are essential for endoscopy training, being required to support feedback provision, optimize learner capabilities, and document competence. We aimed to evaluate the strength of validity evidence that supports the available colonoscopy direct observation assessment tools using the unified framework of validity. METHODS: We systematically searched five databases for studies investigating colonoscopy direct observation assessment tools from inception until 8 April 2020. We extracted data outlining validity evidence (content, response process, internal structure, relations to other variables, and consequences) from the five sources and graded the degree of evidence, with a maximum score of 15. We assessed educational utility using an Accreditation Council for Graduate Medical Education framework and methodological quality using the Medical Education Research Quality Instrument (MERSQI). RESULTS: From 10 841 records, we identified 27 studies representing 13 assessment tools (10 adult, 2 pediatric, 1 both). All tools assessed technical skills, while 10 each assessed cognitive and integrative skills. Validity evidence scores ranged from 1-15. The Assessment of Competency in Endoscopy (ACE) tool, the Direct Observation of Procedural Skills (DOPS) tool, and the Gastrointestinal Endoscopy Competency Assessment Tool (GiECAT) had the strongest validity evidence, with scores of 13, 15, and 14, respectively. Most tools were easy to use and interpret, and required minimal resources. MERSQI scores ranged from 9.5-11.5 (maximum score 14.5). CONCLUSIONS: The ACE, DOPS, and GiECAT have strong validity evidence compared with other assessments. Future studies should identify barriers to widespread implementation and report on the use of these tools in credentialing examinations.

Outcomes and Costs Following Ommaya Placement with Thrombocytopenia Among U.S. Patients with Cancer.

BACKGROUND: Placement of Ommaya reservoirs for the administration of intrathecal chemotherapy may be complicated by comorbid thrombocytopenia among patients with hematologic or leptomeningeal disease. Aggregated data on risks of Ommaya placement among thrombocytopenic patients are lacking. This study assesses complications, revision rates, and costs associated with Ommaya placement among patients with thrombocytopenia in a large population sample. METHODS: Using a national administrative database, this retrospective study identifies a cohort of adult patients with cancer who underwent Ommaya placement between 2007 and 2016. Preoperative thrombocytopenia was defined as diagnosis of secondary thrombocytopenia, bleeding event, procedure to control bleeding, or platelet transfusion, within 30 days before index admission. Univariate and multivariate analyses were performed to assess costs, 30-day complications, readmissions, and revisions among patients with and without preoperative thrombocytopenia. RESULTS: The analytic cohort included 1652 patients, of whom 29.3% met criteria for preoperative thrombocytopenia. In-hospital mortality rates were 7.7% among patients thrombocytopenia with versus 1.2% among patients without thrombocytopenia (P < 0.001). Preoperative thrombocytopenia was associated with 14.5 times greater hazard of intracranial hemorrhage within 30 days following Ommaya placement, occurring in 25.6% versus 2.0% of patients with and without thrombocytopenia, respectively (P < 0.014). Revision rates did not differ significantly between patients with and without thrombocytopenia. Thrombocytopenia was associated with longer length of stay (7.4 vs. 13.9 days, P < 0.001) and additional $10,000 per patient in costs of index hospitalization (P < 0.001). CONCLUSIONS: This is the largest study to date documenting costs and complication rates of Ommaya placement in patients with and without thrombocytopenia.

Nonreciprocal and Conditional Cooperativity Directs the Pioneer Activity of Pluripotency Transcription Factors.

Cooperative binding of transcription factors (TFs) to chromatin orchestrates gene expression programming and cell fate specification. However, the biophysical principles of TF cooperativity remain incompletely understood. Here we use single-molecule fluorescence microscopy to study the partnership between Sox2 and Oct4, two core members of the pluripotency gene regulatory network. We find that the ability of Sox2 to target DNA inside nucleosomes is strongly affected by the translational and rotational positioning of its binding motif. In contrast, Oct4 can access nucleosomal sites with equal capacities. Furthermore, the Sox2-Oct4 pair displays nonreciprocal cooperativity, with Oct4 modulating interaction of Sox2 with the nucleosome but not vice versa. Such cooperativity is conditional upon the composite motif's residing at specific nucleosomal locations. These results reveal that pioneer factors possess distinct chromatin-binding properties and suggest that the same set of TFs can differentially regulate gene activities on the basis of their motif positions in the nucleosomal context.

The superiority of three-dimensional physical models to two-dimensional computer presentations in anatomy learning.

BACKGROUND: Although several studies (Anat Sci Educ, 8 [6], 525, 2015) have shown that computer-based anatomy programs (three-dimensional visualisation technology [3DVT]) are inferior to ordinary physical models (PMs), the mechanism is not clear. In this study, we explored three mechanisms: haptic feedback, transfer-appropriate processing and stereoscopic vision. METHODS: The test of these hypotheses required nine groups of 20 students: two from a previous study (Anat Sci Educ, 6 [4], 211, 2013) and seven new groups. (i) To explore haptic feedback from physical models, participants in one group were allowed to touch the model during learning; in the other group, they could not; (ii) to test 'transfer-appropriate processing' (TAP), learning ( PM or 3DVT) was crossed with testing (cadaver or two-dimensional display of cadaver); (iii) finally, to examine the role of stereo vision, we tested groups who had the non-dominant eye covered during learning and testing, during learning, or not at all, on both PM and 3DVT. The test was a 15-item short-answer test requiring naming structures on a cadaver pelvis. A list of names was provided. RESULTS: The test of haptic feedback showed a large advantage of the PM over 3DVT regardless of whether or not participants had haptic feedback: 67% correct for the PM with haptic feedback, 69% for PM without haptic feedback, versus 41% for 3DVT (p < 0.0001). In the study of TAP, the PM had an average score of 74% versus 43% for 3DVT (p < 0.0001) regardless of two-dimensional versus three-dimensional test outcome. The third study showed that the large advantage of the PM over 3DVT (28%) with binocular vision nearly disappeared (5%) when the non-dominant eye was covered for both learning and testing. CONCLUSIONS: A physical model is superior to a computer projection, primarily as a consequence of stereoscopic vision with the PM. The results have implications for the use of digital technology in spatial learning.

Genetic mutation analysis at early stages of cell line development using next generation sequencing.

A central goal for most biopharmaceutical companies is to reduce the development timeline to reach clinical proof of concept. This objective requires the development of tools that ensure the quality of biotherapeutic material destined for the clinic. Recent advances in high throughput protein analytics provide confidence in our ability to assess productivity and product quality attributes at early stages of cell line development. However, one quality attribute has, until recently, been absent from the standard battery of analytical tests facilitating informed choices early in cell line selection: genetic sequence confirmation. Techniques historically used for mutation analysis, such as detailed mass spectrometry, have limitations on the sample number and turnaround times making it less attractive at early stages. Thus, we explored the utility of Next-Generation Sequencing (NGS) as a solution to address these limitations. Amplicon sequencing is one such NGS technique that is robust, rapid, sensitive, and amenable to multiplexing, all of which are essential attributes for our purposes. Here we report a NGS method based upon amplicon sequencing that has been successfully incorporated into our cell line development workflow alongside other high-throughput protein analytical assays. The NGS method has demonstrated its value by identifying at least one Chinese hamster ovary (CHO) clone expressing a variant form of the biotherapeutic in each of the four clinical programs in which it has been utilized. We believe this sequence confirmation method is essential to safely accelerating the time to clinical proof of concept of biotherapeutics, and guard against delays related to sequence mutations. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:813-817, 2016.