Discovering Fragile Clades and Causal Sequences in Phylogenomics by Evolutionary Sparse Learning.

Phylogenomic analyses of long sequences, consisting of many genes and genomic segments, reconstruct organismal relationships with high statistical confidence. But, inferred relationships can be sensitive to excluding just a few sequences. Currently, there is no direct way to identify fragile relationships and the associated individual gene sequences in species. Here, we introduce novel metrics for gene-species sequence concordance and clade probability derived from evolutionary sparse learning models. We validated these metrics using fungi, plant, and animal phylogenomic datasets, highlighting the ability of the new metrics to pinpoint fragile clades and the sequences responsible. The new approach does not necessitate the investigation of alternative phylogenetic hypotheses, substitution models, or repeated data subset analyses. Our methodology offers a streamlined approach to evaluating major inferred clades and identifying sequences that may distort reconstructed phylogenies using large datasets.

Methods for Estimating Personal Disease Risk and Phylogenetic Diversity of Hematopoietic Stem Cells.

An individual's chronological age does not always correspond to the health of different tissues in their body, especially in cases of disease. Therefore, estimating and contrasting the physiological age of tissues with an individual's chronological age may be a useful tool to diagnose disease and its progression. In this study, we present novel metrics to quantify the loss of phylogenetic diversity in hematopoietic stem cells (HSCs), which are precursors to most blood cell types and are associated with many blood-related diseases. These metrics showed an excellent correspondence with an age-related increase in blood cancer incidence, enabling a model to estimate the phylogeny-derived age (phyloAge) of HSCs present in an individual. The HSC phyloAge was generally older than the chronological age of patients suffering from myeloproliferative neoplasms (MPNs). We present a model that relates excess HSC aging with increased MPN risk. It predicted an over 200 times greater risk based on the HSC phylogenies of the youngest MPN patients analyzed. Our new metrics are designed to be robust to sampling biases and do not rely on prior knowledge of driver mutations or physiological assessments. Consequently, they complement conventional biomarker-based methods to estimate physiological age and disease risk.

Plasmodium GPI-anchored micronemal antigen is essential for parasite transmission through the mosquito host.

Plasmodium parasites, the eukaryotic pathogens that cause malaria, feature three distinct invasive forms tailored to the host environment they must navigate and invade for life cycle progression. One conserved feature of these invasive forms is the micronemes, apically oriented secretory organelles involved in egress, motility, adhesion, and invasion. Here we investigate the role of GPI-anchored micronemal antigen (GAMA), which shows a micronemal localization in all zoite forms of the rodent-infecting species Plasmodium berghei. ∆GAMA parasites are severely defective for invasion of the mosquito midgut. Once formed, oocysts develop normally, however, sporozoites are unable to egress and exhibit defective motility. Epitope-tagging of GAMA revealed tight temporal expression late during sporogony and showed that GAMA is shed during sporozoite gliding motility in a similar manner to circumsporozoite protein. Complementation of P. berghei knockout parasites with full-length P. falciparum GAMA partially restored infectivity to mosquitoes, indicating conservation of function across Plasmodium species. A suite of parasites with GAMA expressed under the promoters of CTRP, CAP380, and TRAP, further confirmed the involvement of GAMA in midgut infection, motility, and vertebrate infection. These data show GAMA's involvement in sporozoite motility, egress, and invasion, implicating GAMA as a regulator of microneme function.

Computational Reproducibility of Molecular Phylogenies.

Repeated runs of the same program can generate different molecular phylogenies from identical data sets under the same analytical conditions. This lack of reproducibility of inferred phylogenies casts a long shadow on downstream research employing these phylogenies in areas such as comparative genomics, systematics, and functional biology. We have assessed the relative accuracies and log-likelihoods of alternative phylogenies generated for computer-simulated and empirical data sets. Our findings indicate that these alternative phylogenies reconstruct evolutionary relationships with comparable accuracy. They also have similar log-likelihoods that are not inferior to the log-likelihoods of the true tree. We determined that the direct relationship between irreproducibility and inaccuracy is due to their common dependence on the amount of phylogenetic information in the data. While computational reproducibility can be enhanced through more extensive heuristic searches for the maximum likelihood tree, this does not lead to higher accuracy. We conclude that computational irreproducibility plays a minor role in molecular phylogenetics.

Omics-driven utilization of wild relatives for empowering pre-breeding in pearl millet.

MAIN CONCLUSION: Pearl millet wild relatives harbour novel alleles which could be utilized to broaden genetic base of cultivated species. Genomics-informed pre-breeding is needed to speed up introgression from wild to cultivated gene pool in pearl millet. Rising episodes of intense biotic and abiotic stresses challenge pearl millet production globally. Wild relatives provide a wide spectrum of novel alleles which could address challenges posed by climate change. Pre-breeding holds potential to introgress novel diversity in genetically narrow cultivated Pennisetum glaucum from diverse gene pool. Practical utilization of gene pool diversity remained elusive due to genetic intricacies. Harnessing promising traits from wild pennisetum is limited by lack of information on underlying candidate genes/QTLs. Next-Generation Omics provide vast scope to speed up pre-breeding in pearl millet. Genomic resources generated out of draft genome sequence and improved genome assemblies can be employed to utilize gene bank accessions effectively. The article highlights genetic richness in pearl millet and its utilization with a focus on harnessing next-generation Omics to empower pre-breeding.

Discovering research articles containing evolutionary timetrees by machine learning.

MOTIVATION: Timetrees depict evolutionary relationships between species and the geological times of their divergence. Hundreds of research articles containing timetrees are published in scientific journals every year. The TimeTree (TT) project has been manually locating, curating and synthesizing timetrees from these articles for almost two decades into a TimeTree of Life, delivered through a unique, user-friendly web interface (timetree.org). The manual process of finding articles containing timetrees is becoming increasingly expensive and time-consuming. So, we have explored the effectiveness of text-mining approaches and developed optimizations to find research articles containing timetrees automatically. RESULTS: We have developed an optimized machine learning system to determine if a research article contains an evolutionary timetree appropriate for inclusion in the TT resource. We found that BERT classification fine-tuned on whole-text articles achieved an F1 score of 0.67, which we increased to 0.88 by text-mining article excerpts surrounding the mentioning of figures. The new method is implemented in the TimeTreeFinder (TTF) tool, which automatically processes millions of articles to discover timetree-containing articles. We estimate that the TTF tool would produce twice as many timetree-containing articles as those discovered manually, whose inclusion in the TT database would potentially double the knowledge accessible to a wider community. Manual inspection showed that the precision on out-of-distribution recently published articles is 87%. This automation will speed up the collection and curation of timetrees with much lower human and time costs. AVAILABILITY AND IMPLEMENTATION: https://github.com/marija-stanojevic/time-tree-classification. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Insights into the gut microbiome of vitiligo patients from India.

BACKGROUND: Vitiligo is an autoimmune disease characterized by loss of pigmentation in the skin. It affects 0.4 to 2% of the global population, but the factors that trigger autoimmunity remain elusive. Previous work on several immune-mediated dermatological disorders has illuminated the substantial roles of the gut microbiome in disease pathogenesis. Here, we examined the gut microbiome composition in a cohort of vitiligo patients and healthy controls from India, including patients with a family history of the disease. RESULTS: Our results show significant alterations in the gut microbiome of vitiligo patients compared to healthy controls, affecting taxonomic and functional profiles as well as community structure. We observed a reduction in the abundance of several bacterial taxa commonly associated with a healthy gut microbiome and noted a decrease in the abundance of SCFA (Short Chain Fatty Acids) producing taxa in the vitiligo group. Observation of a higher abundance of genes linked to bacteria-mediated degradation of intestinal mucus suggested a potential compromise of the gut mucus barrier in vitiligo. Functional analysis also revealed a higher abundance of fatty acid and lipid metabolism-related genes in the vitiligo group. Combined analysis with data from a French cohort of vitiligo also led to the identification of common genera differentiating healthy and gut microbiome across populations. CONCLUSION: Our observations, together with available data, strengthen the role of gut microbiome dysbiosis in symptom exacerbation and possibly pathogenesis in vitiligo. The reported microbiome changes also showed similarities with other autoimmune disorders, suggesting common gut microbiome-mediated mechanisms in autoimmune diseases. Further investigation can lead to the exploration of dietary interventions and probiotics for the management of these conditions.

Predictors of brain metastases in patients with oligometastatic solid tumours treated with stereotactic body radiation therapy.

PURPOSE: In patients with oligometastatic disease (OMD) treated with stereotactic body radiation therapy (SBRT), those who develop brain metastases (BrM) may have poor outcomes. We aimed to investigate variables associated with BrM development in this population. METHODS: Patients with ≤ 5 extracranial metastases from solid tumors treated with SBRT from 2008 to 2016 at Sunnybrook Odette Cancer Centre were included. We investigated the association between covariates and CIBrM (cumulative incidence of BrM) using Fine-Gray analysis, and progression-free survival (PFS) and overall survival (OS) using Cox regression. We investigated the association between extracranial progression and CIBrM using time-based conditional analysis. RESULTS: Among 404 patients, the most common primary sites were lung, colorectal, prostate, breast and kidney. Median follow-up was 49 months. Median PFS was 25 months. Median OS was 70 months. 58 patients developed BrM, and 5-year CIBrM was 16%. On multivariable analysis, number of extracranial metastases, location of metastases, total planning target volume (PTV), and time from primary diagnosis to OMD were not associated with CIBrM, although several of these variables were associated with extracranial PFS and OS. Primary site was associated with CIBrM, with colorectal and prostate cancer associated with lower CIBrM compared to lung cancer. Widespread extracranial progression (≥ 5 sites) within 24, 36, 48 and 60 months of OMD diagnosis was independently associated with higher CIBrM. CONCLUSION: In patients with OMD treated with SBRT, baseline variables related to extracranial disease burden and distribution were not associated with BrM development, while primary site and widespread extracranial progression were associated with BrM development.

Acid mine drainage from coal mines in the eastern Himalayan sub-region: Hydrogeochemical processes, seasonal variations and insights from hydrogen and oxygen stable isotopes.

Uncontrolled coal mining using non-scientific methods has presented a major threat to the quality of environment, particularly the water resources in eastern himalayan sub-region of India. Water bodies in the vicinity of mining areas are contaminated by acid mine drainage (AMD) that is released into streams and rivers. This study attempted to assess the impact of AMD, deciphering hydrogeochemical processes, seasonal fluctuations, and stable isotope features of water bodies flowing through and around coal mining areas. Self-organizing maps (SOMs) used to separate and categorize AMD, AMD-impacted and non-AMD impacted water from the different study locations for two sampling seasons revealed four clusters (C), with C1 and C2 impacted by AMD, C3 and C4 showing negligible to no impact of AMD. AMD impacted water was SO42- - Mg2+- Ca2+ hydrochemical type with sulphide oxidation and evaporation dominating water chemistry, followed by silicate weathering during both the sampling seasons. Water with negligible-to-no AMD-impact was Mg2+- Ca2+- SO42- to Ca2+ - HCO3- to mixed hydrochemical type with rock weathering and dissolution, followed by ion exchange as major factors controlling water chemistry during both the sampling seasons. Most of physicochemical parameters of C1 and C2 exceeded the prescribed limits, whereas in C3 and C4 water samples, parameters were found within the prescribed limits. Stable isotopes of hydrogen (δ2H) and oxygen (δ18O) during post-monsoon (PoM) varied between -41.04 ‰ and -29.98 ‰, and -6.60 ‰ to -3.94 ‰; and during pre-monsoon (PrM) varied between -58.18 ‰ and - 33.76 ‰ and -8.60 ‰ to -5.46 ‰. Deuterium excess (d-excess) ranged between 1.57 ‰ and 12.47 ‰ during PoM and 5.70 ‰ to 15.17 ‰ during PrM season. The stable isotopes analysis revealed that evaporation, mineral dissolution and mixing with rainwater are the key factors in study area.

Effect of tropospheric ozone and its protectants on gas exchange parameters, antioxidant enzymes and quality of Garlic (Allium sativum. L).

An experimental study was conducted to assess the detrimental effect of ground-level ozone (O3) on garlic physiology and to find out appropriate control measures against ground-level O3, at TNAU-Horticultural Research farm, Udhagamandalam. Elevated ground ozone levels significantly decreased garlic leaf chlorophyll, photosynthetic rate, stomatal conductance, total soluble solids and pungency. The garlic chlorophyll content was highest in ambient ozone level and lowest in elevated ozone@200 ppb, highest stomatal conductance was recorded in ambient ozone with foliar spray of 3%Panchagavya, and the lowest was observed in elevated ozone@200 ppb. Since the elevated O3 had reduced in garlic photosynthetic rate significantly the lowest was observed in elevated O3@200 ppb and the highest photosynthetic rate was observed in ambient Ozone with foliar spray 3% of panchagavya after a week. The antioxidant enzymes of garlic were increased with increased concentration of tropospheric ozone. The highest catalase (60.97 µg of H2O2/g of leaf) and peroxidase (9.13 ΔA/min/g of leaf) concentration was observed at 200 ppb elevated ozone level. Garlic pungency content was highest in ambient ozone with foliar spray of 0.1% ascorbic acid and the lowest was observed under elevated O3@200 ppb. Highest total soluble solids were observed in ambient ozone with foliar spray of 3%Panchagavya and the lowest observed in elevated ozone@200 ppb. Thus, tropospheric ozone has a detrimental impact on the physiology of crops, which reduced crop growth and yield. Under elevated O3 levels, ascorbic acid performed well followed by panchagavya and neem oil. The antioxidant such as catalase and peroxidase had positive correlation among themselves and had negative correlation with chlorophyll content, stomatal conductance, photosynthetic rate, pungency and TSS. The photosynthetic rate has high positive correlation with chlorophyll content, pungency and TSS. Correlation analysis confirmed the negative effects of tropospheric ozone and garlic gas exchange parameters and clove quality. The ozone protectants will reduce stomatal opening by which the entry of O3 in to the cell will be restricted and other hand they also will alleviate ROS and allied stresses.

Protein dynamics provide mechanistic insights about epistasis among common missense polymorphisms.

Sequencing of the protein coding genome has revealed many different missense mutations of human proteins and different population frequencies of corresponding haplotypes, which consist of different sets of those mutations. Here, we present evidence for pairwise intramolecular epistasis (i.e., nonadditive interactions) between many such mutations through an analysis of protein dynamics. We suggest that functional compensation for conserving protein dynamics is a likely evolutionary mechanism that maintains high-frequency mutations that are individually nonneutral but epistatically compensating within proteins. This analysis is the first of its type to look at human proteins with specific high population frequency mutations and examine the relationship between mutations that make up that observed high-frequency protein haplotype. Importantly, protein dynamics revealed a separation between high and low frequency haplotypes within a target protein cytochrome P450 2A7, with the high-frequency haplotypes showing behavior closer to the wild-type protein. Common protein haplotypes containing two mutations display dynamic compensation in which one mutation can correct for the dynamic effects of the other. We also utilize a dynamics-based metric, EpiScore, that evaluates the epistatic interactions and allows us to see dynamic compensation within many other proteins.

Embracing Green Computing in Molecular Phylogenetics.

Molecular evolutionary analyses require computationally intensive steps such as aligning multiple sequences, optimizing substitution models, inferring evolutionary trees, testing phylogenies by bootstrap analysis, and estimating divergence times. With the rise of large genomic data sets, phylogenomics is imposing a big carbon footprint on the environment with consequences for the planet's health. Electronic waste and energy usage are large environmental issues. Fortunately, innovative methods and heuristics are available to shrink the carbon footprint, presenting researchers with opportunities to lower the environmental costs and greener evolutionary computing. Green computing will also enable greater scientific rigor and encourage broader participation in big data analytics.

Taming the Selection of Optimal Substitution Models in Phylogenomics by Site Subsampling and Upsampling.

The selection of the optimal substitution model of molecular evolution imposes a high computational burden for long sequence alignments in phylogenomics. We discovered that the analysis of multiple tiny subsamples of site patterns from a full sequence alignment recovers the correct optimal substitution model when sites in the subsample are upsampled to match the total number of sites in the full alignment. The computational costs of maximum-likelihood analyses are reduced by orders of magnitude in the subsample-upsample (SU) approach because the upsampled alignment contains only a small fraction of all site patterns. We present an adaptive protocol, ModelTamer, that implements the new SU approach and automatically selects subsamples to estimate optimal models reliably. ModelTamer selects models hundreds to thousands of times faster than the full data analysis while needing megabytes rather than gigabytes of computer memory.

Epistasis Creates Invariant Sites and Modulates the Rate of Molecular Evolution.

Invariant sites are a common feature of amino acid sequence evolution. The presence of invariant sites is frequently attributed to the need to preserve function through site-specific conservation of amino acid residues. Amino acid substitution models without a provision for invariant sites often fit the data significantly worse than those that allow for an excess of invariant sites beyond those predicted by models that only incorporate rate variation among sites (e.g., a Gamma distribution). An alternative is epistasis between sites to preserve residue interactions that can create invariant sites. Through computer-simulated sequence evolution, we evaluated the relative effects of site-specific preferences and site-site couplings in the generation of invariant sites and the modulation of the rate of molecular evolution. In an analysis of ten major families of protein domains with diverse sequence and functional properties, we find that the negative selection imposed by epistasis creates many more invariant sites than site-specific residue preferences alone. Further, epistasis plays an increasingly larger role in creating invariant sites over longer evolutionary periods. Epistasis also dictates rates of domain evolution over time by exerting significant additional purifying selection to preserve site couplings. These patterns illuminate the mechanistic role of epistasis in the processes underlying observed site invariance and evolutionary rates.

Limitations of Phylogenomic Data Can Drive Inferred Speciation Rate Shifts.

Biodiversity analyses of phylogenomic timetrees have produced many high-profile examples of shifts in the rate of speciation across the tree of life. Temporally correlated events in ecology, climate, and biogeography are frequently invoked to explain these rate shifts. In a re-examination of 15 genomic timetrees and 25 major published studies of the pattern of speciation through time, we observed an unexpected correlation between the timing of reported rate shifts and the information content of sequence alignments. Here, we show that the paucity of sequence variation and insufficient species sampling in phylogenomic data sets are the likely drivers of many inferred speciation rate shifts, rather than the proposed biological explanations. Therefore, data limitations can produce predictable but spurious signals of rate shifts even when speciation rates may be similar across taxa and time. Our results suggest that the reliable detection of speciation rate shifts requires the acquisition and assembly of long phylogenomic alignments with near-complete species sampling and accurate estimates of species richness for the clades of study.

TimeTree 5: An Expanded Resource for Species Divergence Times.

We present the fifth edition of the TimeTree of Life resource (TToL5), a product of the timetree of life project that aims to synthesize published molecular timetrees and make evolutionary knowledge easily accessible to all. Using the TToL5 web portal, users can retrieve published studies and divergence times between species, the timeline of a species' evolution beginning with the origin of life, and the timetree for a given evolutionary group at the desired taxonomic rank. TToL5 contains divergence time information on 137,306 species, 41% more than the previous edition. The TToL5 web interface is now ADA-compliant and mobile-friendly, a result of comprehensive source code refactoring. TToL5 also offers programmatic access to species divergence times and timelines through an application programming interface, which is accessible at timetree.temple.edu/api. TToL5 is publicly available at timetree.org.

TopHap: rapid inference of key phylogenetic structures from common haplotypes in large genome collections with limited diversity.

MOTIVATION: Building reliable phylogenies from very large collections of sequences with a limited number of phylogenetically informative sites is challenging because sequencing errors and recurrent/backward mutations interfere with the phylogenetic signal, confounding true evolutionary relationships. Massive global efforts of sequencing genomes and reconstructing the phylogeny of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains exemplify these difficulties since there are only hundreds of phylogenetically informative sites but millions of genomes. For such datasets, we set out to develop a method for building the phylogenetic tree of genomic haplotypes consisting of positions harboring common variants to improve the signal-to-noise ratio for more accurate and fast phylogenetic inference of resolvable phylogenetic features. RESULTS: We present the TopHap approach that determines spatiotemporally common haplotypes of common variants and builds their phylogeny at a fraction of the computational time of traditional methods. We develop a bootstrap strategy that resamples genomes spatiotemporally to assess topological robustness. The application of TopHap to build a phylogeny of 68 057 SARS-CoV-2 genomes (68KG) from the first year of the pandemic produced an evolutionary tree of major SARS-CoV-2 haplotypes. This phylogeny is concordant with the mutation tree inferred using the co-occurrence pattern of mutations and recovers key phylogenetic relationships from more traditional analyses. We also evaluated alternative roots of the SARS-CoV-2 phylogeny and found that the earliest sampled genomes in 2019 likely evolved by four mutations of the most recent common ancestor of all SARS-CoV-2 genomes. An application of TopHap to more than 1 million SARS-CoV-2 genomes reconstructed the most comprehensive evolutionary relationships of major variants, which confirmed the 68KG phylogeny and provided evolutionary origins of major and recent variants of concern. AVAILABILITY AND IMPLEMENTATION: TopHap is available at https://github.com/SayakaMiura/TopHap. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Endotoxin-Stimulated Hepatic Stellate Cells Augment Acetaminophen-Induced Hepatocyte Injury.

Acetaminophen (APAP)-induced liver injury is influenced by inflammatory Gram-negative bacterial endotoxin [lipopolysaccharide (LPS)], mechanisms of which are not completely understood. Because LPS-stimulated perisinusoidal hepatic stellate cells (HSCs) produce cytokines that affect survival of hepatocytes, this study investigated their role in APAP-induced liver injury. Fed (nonstarved) rats were administered 5 mg/kg LPS or phosphate-buffered saline (PBS) vehicle, followed by 200 mg/kg APAP or PBS an hour later, and euthanized at 6 hours. Control rats received PBS at both time points. Both LPS and APAP caused mild hepatocyte injury (apoptosis), as assessed by histopathology, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, and caspase-3 activation. The liver injury was augmented in rats administered LPS + APAP, in association with increased nuclear translocation of interferon-regulatory factor-1 (IRF1). In vitro, APAP augmented LPS/HSC-conditioned medium-induced inhibition of DNA and protein synthesis, apoptosis, and nuclear IRF1 in hepatocytes. LPS-stimulated HSCs produced interferon-ß (IFN-ß), and LPS/HSC + APAP-induced hepatocyte apoptosis was inhibited by anti-IFN-ß antibody. Finally, HSC-depleted mice produced significantly lower IFN-ß and tumor necrosis factor-α, exhibited less oxidative stress, and were protected from excessive injury due to high APAP dose (600 mg/kg), as well as LPS (5 mg/kg overnight) followed by APAP. In co-culture with or without LPS, HSCs increased expression of proinflammatory cytokines by Kupffer cells. These results suggest that HSCs play a critical role in APAP-induced liver injury without or with LPS preconditioning, and it involves INF-ß-IRF1 signaling.

Dynamic coupling of residues within proteins as a mechanistic foundation of many enigmatic pathogenic missense variants.

Many pathogenic missense mutations are found in protein positions that are neither well-conserved nor fall in any known functional domains. Consequently, we lack any mechanistic underpinning of dysfunction caused by such mutations. We explored the disruption of allosteric dynamic coupling between these positions and the known functional sites as a possible mechanism for pathogenesis. In this study, we present an analysis of 591 pathogenic missense variants in 144 human enzymes that suggests that allosteric dynamic coupling of mutated positions with known active sites is a plausible biophysical mechanism and evidence of their functional importance. We illustrate this mechanism in a case study of ß-Glucocerebrosidase (GCase) in which a vast majority of 94 sites harboring Gaucher disease-associated missense variants are located some distance away from the active site. An analysis of the conformational dynamics of GCase suggests that mutations on these distal sites cause changes in the flexibility of active site residues despite their distance, indicating a dynamic communication network throughout the protein. The disruption of the long-distance dynamic coupling caused by missense mutations may provide a plausible general mechanistic explanation for biological dysfunction and disease.